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Epigenetic Modifications in Pediatric Acute Lymphoblastic Leukemia

Frontiers in pediatrics

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INTRODUCTION in oncogenesis (7, 8). The methylation occurs at cytosine (C) bases located 5′ to guanosine (G) in a CpG dinucleotide and often in regions rich in repetitive CpGs known as CpG islands. The methyl groups are transferred to the CpG dinucleotide via DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) and serve to transcriptionally silence genes downstream of the methy- lated promoter. When aberrant methylation occurs in a cancer cell, it typically results in hypermethylation of tumor suppres- sor genes. This can lead to disruption of key molecular pathways such as apoptosis, DNA repair pathways, cell cycle checkpoints, and cell differentiation as well as result in activation of metas- tasis/invasion pathways, drug resistance, and proliferation signal transduction (9). Epigenetics is the study of biochemical modifications of chromatin (1) and have been implicated in the pathogenesis of cancer (2). Epigenetic modifications to DNA are not secondary to changes to the nucleotide sequence itself but rather heritable changes affect- ing the activity of genes and their cellular expression. Examples include DNA methylation, histone modification, and alterations in non-coding microRNAs (miRNAs). Each of these mechanisms can alter how genes are expressed or silenced without modifying the DNA sequence. If these epigenetic modifications lead to silenc- ing of tumor suppressor genes or activation of oncogenes then it is easy to conceptualize how leukemogenesis can occur. Unlike chromosomal translocations or gene mutations, which are permanent,hypermethylation of gene promoters is a reversible event that could be targeted with therapeutic agents designed to alter aberrant epigenetic events. Incorporating epigenetic mod- ifying agents into the treatment of pediatric ALL is an exciting approach that theoretically could have a significant impact in the treatment of this disease. This would be particularly true for relapse ALL, which is highly hypermethylated (3–5), and accounts for more deaths than any other pediatric disease and remains the fifth most common pediatric cancer overall (6). Various groups have used DNA methylation studies to investi- gatetheunderlyingepigeneticmechanismsinchildhoodleukemia. In a large cohort of 137 B-lineage and 30 T-lineage pediatric ALL cases, distinct DNA methylation signatures with significant concordant correlation of gene expression were found to be char- acteristic of various cytogenetic sub-types (10). In fact, a core set of epigenetically deregulated genes, common to all cases, was identified; suggesting their central role in leukemia initiation and maintenance. INTRODUCTION Likewise, DNA methylation interrogation of 69 pediatric B-ALL and 42 non-leukemic control samples revealed 325 genes hypermethylated and down regulated, and 45 genes hypomethylated and up-regulated across all the samples, irre- spective of subtype (11). Furthermore, gene ontology analysis of these epigenetically deregulated genes highlighted the role of genes involved in cell signaling, cellular development, cell sur- vival, and apoptosis. Another study investigating 764 cases of newly diagnosed ALL and 27 cases of relapse, identified 9406 predominantly hypermethylated CpG sites, independent of cyto- genetic background, with each cytogenetic subtype displaying a unique set of hyper- and hypomethylated sites (12). These dif- ferentially hypermethylated CpG sites were enriched for genes in In this brief review, we will focus on the three main areas of epigenetics, which have been implicated in the leukemogenesis of pediatric ALL; DNA hypermethylation, histone modification, and microRNA alterations. As we continue to gain better under- standing of the driving mechanisms for pediatric ALL at both diagnosis and relapse, therapeutic interventions directed toward these pathways and mechanisms can be harnessed and introduced into clinical trials. DNA HYPERMETHYLATION Michael J. Burke1* andTeena Bhatla2 Michael J. Burke andTeena Bhatla 1 Division of Pediatric Hematology-Oncology, Medical College of Wisconsin, Milwaukee, WI, USA 2 Division of Pediatric Hematology-Oncology, New York University Langone Medical Center, New York, NY, USA 1 Division of Pediatric Hematology-Oncology, Medical College of Wisconsin, Milwaukee, WI, USA 1 Division of Pediatric Hematology-Oncology, Medical College of Wisconsin, Milwaukee, WI, USA 2 Division of Pediatric Hematology-Oncology, New York University Langone Medical Center, New York, NY, USA gy gy, g , , , 2 Division of Pediatric Hematology-Oncology, New York University Langone Medical Center, New York, N Aberrant epigenetic modifications are well-recognized drivers for oncogenesis. Pediatric acute lymphoblastic leukemia (ALL) is no exception and serves as a model toward the sig- nificant impact these heritable alterations can have in leukemogenesis. In this brief review, we will focus on the main aspects of epigenetics, which control leukemogenesis in pedi- atric ALL, mainly DNA methylation, histone modification, and microRNA alterations. As we continue to gain better understanding of the driving mechanisms for pediatric ALL at both diagnosis and relapse, therapeutic interventions directed toward these pathways and mechanisms can be harnessed and introduced into clinical trials for pediatric ALL. PEDIATRICS REVIEW ARTICLE published: 14 May 2014 doi: 10.3389/fped.2014.00042 Epigenetic modifications in pediatric acute lymphoblastic leukemia REVIEW ARTICLE published: 14 May 2014 doi: 10.3389/fped.2014.00042 PEDIATRICS *Correspondence: *Correspondence: Michael J. Burke, Medical College of Wisconsin, MACC Fund Research Center, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA e-mail: [email protected] Keywords: epigenetics, methylation, histone, pediatric, leukemia, ALL Keywords: epigenetics, methylation, histone, pediatric, leukemia, ALL Edited by: Alan Wayne, Children’s Hospital Los Angeles, USA Reviewed by: Patrick Brown, Johns Hopkins University, USA Weili Sun, University of Southern California, USA Reviewed by: Patrick Brown, Johns Hopkins University, USA Weili Sun, University of Southern California, USA Edited by: Edited by: Alan Wayne, Children’s Hospital Los Angeles, USA Edited by: Alan Wayne, Children’s Hospital Los Angeles, USA DNA HYPERMETHYLATION These mutations can result in a gain or loss of function of key genes known to regulate histone marks. Jaffe and colleagues, in pediatric ALL cell lines, have used global chromatin profiling, a tandem mass spectrometry strategy, to measure levels of histone modifications on bulk chromatin (29). In this work, a novel cluster of cell lines with a specific epigenetic signature was identified, charac- terized by increased dimethylation of histone H3 at lysine 36 (H3K36me2) and decreased unmodified H3K36. Approximately half of the cell lines in this cluster harbored the t(4;14) translo- cation, which is known to induce overexpression of NSD2 (24, 32, 33). NSD2 is a member of the HKMTs that catalyze the con- version of unmodified H3K36 to mono- and dimethylated forms (28). Upon targeted sequencing in an extensive patient sample set, NSD2 mutations were found to be enriched in ETV6-RUNX1 and TCF3-PBX1 sub-types of pediatric B-ALL, while no muta- tions were identified in 30 adult ALL samples. These were gain- of-function mutations and their overexpression led to a global increase in H3K36me2, with concomitant decrease in H3K27me3. Similar results were reported by others (34), showing these muta- tions affect expression of a number of genes involved in normal lymphoid development. py y Relapsed ALL is a highly aggressive disease marked predomi- nantly by drug resistance (20). Efforts are currently being under- taken to identify the role of epigenetic mechanisms in driving relapse and chemoresistance (3). Genome-wide DNA methyla- tion profiling performed on 33 matched relapse-diagnosis pairs demonstrated that the relapsed genome was distinctly more hyper- methylated compared to matched samples at diagnosis (3). In this study, 1147 CpG sites corresponding to 905 genes were differen- tially hypermethylated at relapse. About a third of these genes exhibited concordant down-regulation of mRNA expression. Many of the known regulators of the Wnt pathway were hyper- methylated and down regulated at relapse, including inhibitors of the β-catenin/TCF/LEF activity, as well as APC, WT1, cadherins (CDH1, CDH11), and SOX genes (SOX2, SOX8, SOX11, SOX21). Interestingly, PTPRO, a negative feedback inhibitor of the Wnt pathway that binds to Wnt and blocks its association with other receptors(21),wasalso hypermethylatedand downregulated. This suggests that the Wnt pathway is over-activated at relapse and that aberrant DNA methylation may play a significant role in the acti- vation of this pathway in relapsed ALL (3). DNA HYPERMETHYLATION Gains of DNA methylation tend to occur in the gene promoter region and are one of the most studied epigenetic abnormalities May 2014 | Volume 2 | Article 42 | 1 www.frontiersin.org Epigenetic modifications in ALL Burke and Bhatla environment influences the “on–off” transcriptional states of a gene depending on the post-translational modifications of the histone proteins (22). Numerous covalent histone tail modifi- cations, the most prominent being methylation and acetylation, can directly affect gene transcription (23). These modifications are highly specific for the particular amino acid position on the N-terminal tails of the histones. For example, H3K4me3, H3K9 acetylation, H3K14 acetylation, and H3K79me2 are associated with open chromatin structures and linked with transcriptional activation, while H3K9me3 and H3K27me3 are associated with closed chromatin, and hence transcriptional repression. These histone marks are regulated by the balance between competing enzymes such as the histone lysine methyltransferases (HKMTs) and histone demethylases (HKDMs), and the histone acetyltrans- ferases (HATs) and histone deacetylases (HDACs) (24). Moreover, multiple histone modifications can be associated with critical reg- ulatory elements of transcription such as enhancers, which can determine cell fate and differentiation (23, 25). the transcriptional regulatory network such as NANOG, OCT4, SOX2, and REST. These genes are known to be regulated by a polycomb group of proteins and have been identified as targets for hypermethylation in solid tumors (13), leukemia (14), and lymphoma (15). MLL-rearranged infant leukemia is one specific ALL subtype that has been shown to exhibit distinct promoter hypermethyla- tion (16–19). Stumpel and colleagues identified a distinct DNA methylation pattern dependent on the presence and type of MLL-fusion partner in a cohort of 57 newly diagnosed infant ALL patients (19). In addition, the degree of hypermethylation appeared to correlate with a higher risk of relapse among infants carrying t(4;11) or t(11;19) translocations. In another study of 5 MLL-rearranged infant ALL samples, genes known to be involved inoncogenesisandtumorprogression(DAPK1,CCR6,HRK,LIFR, and FHIT) were differentially methylated suggesting a role in the leukemogenesis of MLL-rearranged ALL (17). As well, four of five genes that were hypermethylated and silenced were able to be re-expressed in vitro when exposed to DNMTi and regain their functional roles, thus pointing to the clinical potential epigenetic therapy may have in the treatment of infant leukemia. Mutations in epigenetic modifying genes are common in hema- tologic malignancies, including ALL (26–31). DNA HYPERMETHYLATION Re-expression of these hypermethylated and down regulated genes was observed when leukemia cell lines were treated with decitabine. As well, enhanced chemosensitivity was observed when ALL cell lines and primary patient ALL samples were pretreated with decitabine followed by conventional cytotoxic chemotherapy (4). Accumulating evidence suggests that histone modification is an important aspect of MLL-fusion mediated transformation and leukemogenesis (35, 36). It has been shown that wild type MLL SETdomainisamethyltransferase,modifyinghistoneH3onlysine 4 (H3K4), and positively regulating gene expression of multiple Hox genes (37). In addition,MLL mediated transcriptional regula- tion involves recruitment of HAT,such as CBP (38) and MOF (39). Furthermore,DOT1L,a histone methyltransferase that methylates lysine 79 on histone H3 (H3K79), has been associated with mul- tiple MLL-fusion partners such as AF9, AF10, AF17, and ENL (40–42), and has emerged as an attractive therapeutic target (36). Several groups have used small molecule inhibitors to demonstrate the feasibility of pharmacological inhibition of DOT1L enzymatic activity in preclinical models of MLL-rearranged leukemia (43– 45) and are now under clinical investigation in a phase I study for adults with advanced hematologic malignancies, including acute In summary, DNA hypermethylation appears to play a signifi- cant role in the leukemogenesis of ALL and may be an important contributor toward relapse. As more studies interrogate the spe- cific genes and or pathways influenced by hypermethylation in pediatricALL,wewillgainfurtherinsighttowardstrategiestother- apeutically target these aberrant epigenetic changes and hopefully begin to make a greater impact in the treatment of this disease. MicroRNA ALTERATIONS MicroRNAs are a class of small endogenous single stranded non-coding ribonucleic acids (RNA) composed of roughly 22 nucleotides that are primarily involved in post-transcriptional gene regulation. miRNAs play a critical regulatory role in target- ing mRNAs for cleavage or translational repression, with greater than 1,000 miRNAs currently identified in the human genome (56). MicroRNA genes are preferentially localized to CpG islands, which leads to the plausible mechanism that they can be controlled through aberrant epigenetic regulation (e.g., hypermethylation, histone modification) (57). Altered expression of miRNAs has been implicated in leukemo- genesis and appears to have the ability to influence critical growth regulatory pathways in ALL (58–61). An example of the func- tional impact miRNA can have in B-cell ALL was reported with the restoration of miR-196b expression, which led to significant down-regulation of c-myc and its effector genes fhTERT,Bcl-2,and AATF, suggesting a tumor suppressor function role for miR-196b (62). Some specific miRNAs that have been implicated in pediatric ALL include miRNA (miR) miR-34, miR-128, miR-142, and miR- 181, all reported to be over expressed (58, 63, 64) and miR-100 and miR-196b, both under expressed (59, 63). Schotte and col- leagues investigated 397 miRNAs using qRT-PCR in 81 pediatric ALL cases in comparison to 17 normal CD34+ stem cell con- trols (65). Unique miRNA signatures were identified for various ALL sub-types including ETV6-RUNX1, MLL-rearranged, T-ALL, hyperdiploidy,and E2A-PBX1. Overall,expression of miR-143 and miR-140 were found to be 70- and 140-fold lower in the B-ALL samples compared to controls (pFDR = 0.0007 and pFDR = 0.001, respectively). Hyperdiploid samples showed a clustering of high expression of miR-98, miR-222, miR-223, and miR-511 and the ETV6-RUNX1 cases had a 5- to 1700-fold increase expression in miR-99a, miR-100, miR-125b, and miR-383 compared to controls (pFDR < 0.001). Together these findings lend support for epige- netic alterations involving miRNAs in the leukemogenesis of some of the more common variants of pediatric ALL. Epigenetic alterations are not only restricted to B-ALL,but are a notable feature of T-ALL, particularly the aggressive subtype early T-cell precursor (ETP)ALL.Whole genome sequencing of 12 cases of ETP ALL identified mutations in genes encoding components of the polycomb repressor complex 2 (PRC2), including deletions and sequence mutations of EZH2, SUZ12, and EED (47). Loss of function mutations and deletions of EZH2 and SUZ12 genes have also been found in T-ALL,where authors implicate the tumor suppressor role of the PRC2 complex (48). HISTONE MODIFICATIONS HISTONE MODIFICATIONS Histones are small basic proteins involved in the spatial organization of DNA within the nucleus. The chromatin May 2014 | Volume 2 | Article 42 | 2 Frontiers in Pediatrics | Pediatric Oncology Epigenetic modifications in ALL Burke and Bhatla leukemia with rearrangement of the MLL gene (NCT01684150). One inhibitor in particular, EPZ-5676, has shown potent activity in its ability to selectively inhibit the DOT1L histone methyltrans- ferase, resulting in cell death of acute leukemia cell lines har- boring MLL translocations as well as complete tumor regression in a rat xenograft model of MLL-rearranged leukemia following continuous iv infusion of EPZ-5676 (45). at relapse. Bachmann and colleagues have reported glucocorti- coid resistance associated with epigenetic silencing of the BIM gene in pediatric ALL and showed synergistic effect of vorinos- tat with dexamethasone in both in vitro and in vivo models (54). The potential importance of these changes is highlighted by the promising activity of several other drugs from the same class that target epigenetic alterations (55). In order to identify novel mutations in relapsedALL,Mullighan and colleagues performed targeted resequencing of 300 genes in 23 matched relapse-diagnosis B-ALL pairs (30). The authors identified novel mutations in CREBBP, a gene encoding the tran- scriptional coactivator CREB binding protein with HAT activity. The overall frequencies of these sequence and/or deletional muta- tions were 18.3% in relapse cases (30). However, particularly high incidences of somatic CREBBP alterations (63%) were found in the high hyperdiploidy relapse cases. Of note, the majority of these mutations occurred in the HAT domain (27). Although less common, mutations in other important epigenetic regula- tors were also seen such as NCoR1 (Nuclear corepressor complex), EP300 (a paralog of CREBBP), EZH2 (histone methyltransferase gene),andCTCF (zincfingerproteininvolvedinhistonemodifica- tions) (30). Additionally, transcriptome sequencing has identified relapse-specific mutations in CBX3 (encoding heterochromatin protein), PRMT2 (gene encoding protein arginine methyltrans- ferase 2), and MIER3 (involved in chromatin binding); providing further evidence of aberrant epigenetic mechanisms that play a role at relapse (46). In summary, similar to the influence DNA hypermethylation has in pediatric ALL leukemogenesis, maintenance, and relapse, aberrant epigenetic changes involving histones have been associ- ated with disease progression and relapse in ALL. With growing experience using HDACi in hematologic malignancies, includ- ing pediatric trials (NCT01483690, NCT01321346), the impact of these agents will become clearer as well as their role in future relapse and upfront ALL studies. MicroRNA ALTERATIONS MicroRNA ALTERATIONS No maximum tolerated dose (MTD) was identified and 5/15 patients reported grade 3/4 cytopenias (anemia, throm- bocytopenia, and leukopenia) that were possibly related to the study drug. Similar to the DNMTi, HDACi (e.g., vorinostat, panobinostat) have been studied in the treatment of acute leukemia, primarily as single agents and almost exclusively in adults (74, 75). The COG completed a phase I study investigating vorinostat in combination with 13 cis-retinoic acid in children with refractory/recurrent solid tumors and vorinostat alone for patients with refractory leukemia (76). Six patients with refractory leukemia were enrolled with 2 DLTs reported at the solid tumor MTD (230 mg/m2/day) includ- ing an elevated AST (n = 1), hyperbilirubinemia (n = 1), elevated GGT (n = 1), and hypokalemia (n = 1). As the solid tumor MTD for vorinostat did not appear tolerable for patients with hemato- logic malignancies, there was no further dose finding attempt in this study. Currently, there is a phase I study of panobinostat in children with refractory hematologic malignancies open through the therapeutic advances in childhood leukemia and lymphoma (TACL) Consortium (NCT01321346). g g In a report of 18 matched-pair diagnosis and relapse (n = 8) or diagnosis and remission (n = 10) pediatric ALL samples, data was summarized for the most differentially expressed miRNAs (66). Down-regulation of miR-23a and miR-223 was observed at time of relapse compared to remission whereas miR130b, -181, and -708 were over expressed at relapse. Specifically, the expression of miR-708 was greater in relapse samples and lower in remis- sion samples when compared to diagnosis whereas miR-223 was up-regulated in remission samples compared to diagnosis and confirmed with qRT-PCR. These two miRNAs at diagnosis along with miR-27a were shown to correlate significantly with 3-year relapse-free survival (p = 0.0483, 0.0079, and 0.0024, respectively) and thus could potentially be used as prognostic biomarkers for newly diagnosed patients. The functional impact these miRNAs hadongeneexpressionwasdescribedaswellwithtargetsidentified for BMI1, transcription factor necessary for hematopoietic stem cell and leukemia stem cell self-renewal,in miR-27a and miR-128b as well as E2F1, master cell cycle regulator, a target of miR-223. The variations in miRNA expression that exist between diagnostic, remission, and relapse samples identified by Han and colleagues suggest that critical epigenetic mechanisms exist through these non-coding miRNAs that may assist in driving leukemogenesis and disease recurrence. MicroRNA ALTERATIONS The first study incorporating a DNMTi and HDACi followed by chemotherapy for children and adults with relapsed/refractory ALL was recently completed (72). In this phase II trial, decitabine (15 mg/m2/day) and vorinostat (230 mg/m2 divided BID) were given over four consecutive days prior to re-induction chemother- apy (vincristine, prednisone, PEG-asparaginase, doxorubicin) (NCT00882206) (72). Thirteen eligible patients enrolled with a median age of 16 (range, 3–54) years. There was a single toxic death occurring on study attributed to the chemotherapy regi- men, which included a grade five hemorrhage/bleeding (n = 1). A second patient experiencing grade five hypoxia/acute respira- tory distress died on day 4 of study attributed to disease pro- gression (n = 1). There were an additional 14 grade 3/4 serious adverse events,which were at least possibly attributed to decitabine or vorinostat, the most common being fever with neutropenia (n = 2) and infection (blood) with neutropenia (n = 5). Results of the eight patients evaluable for response, identified a CR rate of 50% (n = 4/8) (95% CI 15.7–84.3%) and an overall response rate (CR + PR) of 75% (n = 6/8) (95% CI 34.9–96.8%). As well, minimal residual disease (MRD) negativity by flow cytometry was observed in 4/8 patients (50%, CI: 15.7–84.3%). Five of the eight patients who completed the study proceeded to allogeneic hematopoietic cell transplantation (four in second CR and one in third CR). Three patients succumbed to transplant related deaths without evidence of leukemia while the remaining two patients remain alive with no evidence of disease. Based on the results of this study, a pediatric trial for relapse/refractory ALL combin- ing decitabine and vorinostat with re-induction chemotherapy is currently open through the TACL Consortium (NCT01483690; R21CA161688-01). In an analysis of 353 diagnostic bone marrow samples from patients withALL (<15 years of age,n = 179),65% had at least one of 13 previously identified miRNAs hypermethylated (67). These 13 miRNAs were found to be regulated by methylation and histone modification and associated with a closed chromatin conforma- tion of 11 CpG islands close to where the 13 miRNAs resided. The hypermethylation was associated with miRNA under expression but could be reversed with decitabine. In summary, aberrant miRNA expression, particularly sec- ondary to methylation, is a common finding in ALL. These data support that epigenetic modifications of specific miRNAs are asso- ciated with chemotherapy resistance and clinical outcomes. MicroRNA ALTERATIONS In addition to the discovery of somatic mutations in epige- netic machinery in ALL, mRNA expression of HDACs has been shown to be dysregulated. Higher mRNA expression of HDAC7 and HDAC9 in a study of 94 childhood ALL cases was shown to correlate with poor prognosis (49). Similarly, another group identified the correlation of HDAC4 overexpression with pred- nisone poor response, T-ALL phenotype, and a high initial WBC (50). Given the compelling evidence of HDAC’s involvement in tumor development and progression, inhibitors of HDACs have emerged as an attractive therapeutic option in hematologic malig- nancies (4,51). Through a connectivity map search (52) for agents, which could potentially reverse the characteristic gene expression signature specific for relapse ALL (3, 53) and potentially endow chemosensitivity, vorinostat (HDACi) was identified as the most promising candidate (4). In fact, vorinostat not only modulated the gene expression signature characteristic of relapse in ALL cell lines and patient samples, but showed a synergistic effect when given sequentially with chemotherapy (4). The fact that vorino- stat showed significant alteration of gene expression correlating with histone modifications, indicates that the perturbation of histone marks may have a key role in aberrant gene regulation Aberrant miRNA expression has been implicated in leukemia drug resistance and lower event-free survival (EFS). Schotte and colleagues identified a lower expression of miR-454 (1.9- fold lower) in leukemia blasts with l-asparaginase resistance (pFDR = 0.017) and patient samples resistant to vincristine and May 2014 | Volume 2 | Article 42 | 3 www.frontiersin.org www.frontiersin.org Epigenetic modifications in ALL Burke and Bhatla daunorubicin were found to have over expression of miR- 99a, miR-100, and miR-125b (14- to 25-fold) (pFDR ≤0.002 and pFDR < 0.05, respectively) (65). In terms of EFS, six miR- NAs (miR-33, -215, -369-5p, -496, -518d, and -599) were asso- ciated with worse survival (HR 1.3–1.52, 95% CI 1.01–2.04; 0.003 ≤p ≤0.046) and another eight (miR-10a, -134, -214, -484, -572, -580, -624, and -627) with greater EFS (HR 0.59–0.82, 95% CI 0.41–0.99, 0.004 ≤p ≤0.045) (65). The authors concluded that the miRNAs associated with a more favorable outcome likely had tumor suppressor activity through their signaling of apoptosis (miR-10a), inhibition of proliferation (miR-10a and miR-214), and oncogene SOX2 down-regulation (miR-134). investigating decitabine (10 mg/m2/day × 5 days/week × 2 weeks) in children with relapsed/refractory acute leukemia that closed prematurely due to low patient accrual (NCT00042796, unpub- lished). MicroRNA ALTERATIONS As these modifications can be secondary to DNA hypermethylation (65, 68–71), exposure to agents such as DNMTi could reverse the aberrant expression, normalize miRNA levels, and ultimately lead to improved clinical outcomes. REFERENCES 1. Garcia-Manero G, Yang H, Kuang SQ, O’Brien S, Thomas D, Kantarjian H. Epigenetics of acute lymphocytic leukemia. Semin Hematol (2009) 46:24–32. doi:10.1053/j.seminhematol.2008.09.008 1. Garcia-Manero G, Yang H, Kuang SQ, O’Brien S, Thomas D, Kantarjian H. 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CLINICAL TRIALS INVESTIGATING EPIGENETIC MODIFYING THERAPIES IN PEDIATRIC ALL SUMMARY The majority of clinical experience using epigenetic modifying agents in the treatment of acute leukemia has been in adults (72, 73).TheChildren’sOncologyGroup(COG)pilotedaphaseIstudy Underlying epigenetic alterations in pediatric ALL are com- mon events, which appear to be more common at relapse than May 2014 | Volume 2 | Article 42 | 4 Frontiers in Pediatrics | Pediatric Oncology Epigenetic modifications in ALL Burke and Bhatla diagnosis. Thus children with relapse ALL may be an ideal popu- lation for clinical trials incorporating epigenetic modifying agents aimed at reversing these aberrant signatures. Whether such trials will lead to improved clinical outcomes has yet to be determined but early findings in studies incorporating these agents have been encouraging. new insights into the mechanisms underlying silencing of B cell-specific genes. Leukemia (2012) 26:185–8. doi:10.1038/leu.2011.194 new insights into the mechanisms underlying silencing of B cell-specific genes. Leukemia (2012) 26:185–8. doi:10.1038/leu.2011.194 16. Stumpel DJ, Schotte D, Lange-Turenhout EA, Schneider P, Seslija L, de Menezes RX, et al. Hypermethylation of specific microRNA genes in MLL-rearranged infant acute lymphoblastic leukemia: major matters at a micro scale. Leukemia (2011) 25:429–39. doi:10.1038/leu.2010.282 17. Schafer E, Irizarry R, Negi S, McIntyre E, Small D, Figueroa ME, et al. Promoter hypermethylation in MLL-r infant acute lymphoblastic leukemia: biology and therapeutic targeting. Blood (2010) 115:4798–809. doi:10.1182/blood-2009-09- 243634 g g In conclusion,leukemogenesis of pediatric ALL is heavily influ- enced by epigenetics, particularly DNA hypermethylation, histone modification, and alterations in miRNA expression. Epigenetic modifying agents such as DNMTi and HDACi as well as newer therapies(e.g.,histonemethyltransferaseinhibitors)arenowbeing incorporated into early phase clinical trials for relapse leukemia. As more trials for children with relapse ALL, incorporating epige- netic therapies into standard and/or novel salvage regimens, are developed and completed, we will have a better understanding as to which patients might benefit the most using this approach and ultimately where these agents may be best served in treating pediatric ALL. 18. Nishi M, Eguchi-Ishimae M, Wu Z, Gao W, Iwabuki H, Kawakami S, et al. Sup- pression of the let-7b microRNA pathway by DNA hypermethylation in infant acute lymphoblastic leukemia with MLL gene rearrangements. Leukemia (2013) 27:389–97. doi:10.1038/leu.2012.242 19. Stumpel DJ, Schneider P, van Roon EH, Boer JM, de Lorenzo P, Valsecchi MG, et al. Specific promoter methylation identifies different subgroups of MLL- rearranged infant acute lymphoblastic leukemia, influences clinical outcome, and provides therapeutic options. Blood (2009) 114:5490–8. doi:10.1182/blood- 2009-06-227660 20. REFERENCES Roman-Gomez J, Castillejo JA, Jimenez A, Barrios M, Heiniger A, Torres A. The role of DNA hypermethylation in the pathogenesis and prognosis of acutelymphoblasticleukemia.LeukLymphoma (2003)44:1855–64.doi:10.1080/ 1042819031000116689 29. Jaffe JD, Wang Y, Chan HM, Zhang J, Huether R, Kryukov GV, et al. Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia. Nat Genet (2013) 45:1386–91. doi:10.1038/ng.2777 10. Figueroa ME,Chen SC,AnderssonAK,Phillips LA,LiY,Sotzen J,et al. Integrated genetic and epigenetic analysis of childhood acute lymphoblastic leukemia. J Clin Invest (2013) 123:3099–111. doi:10.1172/JCI66203 30. Mullighan CG, Zhang J, Kasper LH, Lerach S, Payne-Turner D, Phillips LA, et al. 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J Clin Oncol (2009) 27:1316–22. doi:10.1200/JCO.2008.19. 3441 52. Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al. The Connectivity Map: using gene-expression signatures to connect small mol- ecules, genes, and disease. Science (2006) 313:1929–35. doi:10.1126/science. 1132939 53. Bhojwani D, Kang H, Moskowitz NP, Min DJ, Lee H, Potter JW, et al. Biologic pathways associated with relapse in childhood acute lymphoblastic leukemia: a Children’s Oncology Group study. Blood (2006) 108:711–7. doi:10.1182/blood- 2006-02-002824 72. Burke MJ, Lamba J, Weigel B, Bachanova V, Verneris MR, Miller JS. REFERENCES Array-based DNA methylation analysis in classical Hodgkin lymphoma reveals May 2014 | Volume 2 | Article 42 | 5 www.frontiersin.org Epigenetic modifications in ALL Burke and Bhatla lymphoblastic leukemia, and its reversal by histone deacetylase inhibition. Blood (2010) 116:3013–22. doi:10.1182/blood-2010-05-284968 lymphoblastic leukemia, and its reversal by histone deacetylase inhibition. Blood (2010) 116:3013–22. doi:10.1182/blood-2010-05-284968 35. Krivtsov AV, Feng Z, Lemieux ME, Faber J, Vempati S, Sinha AU, et al. H3K79 methylation profiles define murine and human MLL-AF4 leukemias. Cancer Cell (2008) 14:355–68. doi:10.1016/j.ccr.2008.10.001 55. Jones PA, Baylin SB. The epigenomics of cancer. Cell (2007) 128:683–92. doi:10.1016/j.cell.2007.01.029 36. Bernt KM, Armstrong SA. Targeting epigenetic programs in MLL-rearranged leukemias. Hematology Am Soc Hematol Educ Program (2011) 2011:354–60. doi:10.1182/asheducation-2011.1.354 56. de Oliveira JC, Brassesco MS, Scrideli CA, Tone LG, Narendran A. MicroRNA expression and activity in pediatric acute lymphoblastic leukemia (ALL). Pediatr Blood Cancer (2012) 59:599–604. doi:10.1002/pbc.24167 37. MilneTA,BriggsSD,BrockHW,MartinME,GibbsD,AllisCD,et al.SETdomain methyltransferase activity to Hox gene promoters. Mol Cell (2002) 10:1107–17. doi:10.1016/S1097-2765(02)00741-4 Blood Cancer (2012) 59:599–604. doi:10.1002/pbc.24167 57. Weber B, Stresemann C, Brueckner B, Lyko F. Methylation of human microRNA genes in normal and neoplastic cells. Cell Cycle (2007) 6:1001–5. doi:10.4161/ cc.6.9.4209 38. Ernst P, Wang J, Huang M, Goodman RH, Korsmeyer SJ. MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein. Mol Cell Biol (2001) 21:2249–58. doi:10.1128/MCB.21.7.2249-2258.2001 58. Ju X, Li D, Shi Q, Hou H, Sun N, Shen B. Differential microRNA expression in childhood B-cell precursor acute lymphoblastic leukemia. Pediatr Hematol Oncol (2009) 26:1–10. doi:10.1080/08880010802378338 39. Dou Y, Milne TA, Tackett AJ, Smith ER, Fukuda A, Wysocka J, et al. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell (2005) 121:873–85. doi:10.1016/j.cell. 2005.04.031 59. Schotte D, Chau JC, Sylvester G, Liu G, Chen C, van der Velden VH, et al. Iden- tification of new microRNA genes and aberrant microRNA profiles in child- hood acute lymphoblastic leukemia. Leukemia (2009) 23:313–22. doi:10.1038/ leu.2008.286 40. Okada Y, Feng Q, Lin Y, Jiang Q, Li Y, Coffield VM, et al. hDOT1L links histone methylation to leukemogenesis. Cell (2005) 121:167–78. doi:10.1016/j.cell.2005. 05.021 60. Mi S, Lu J, Sun M, Li Z, Zhang H, Neilly MB, et al. MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia. Proc Natl Acad Sci USA (2007) 104:19971–6. doi:10.1073/ pnas.0709313104 41. Mueller D, Bach C, Zeisig D, Garcia-Cuellar M-P, Monroe S, Sreekumar A, et al. 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Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell (2011) 20:53–65. doi:10.1016/ j.ccr.2011.06.009 63. de Oliveira JC, Scrideli CA, Brassesco MS, Morales AG, Pezuk JA, Queiroz Rde P, et al. Differential miRNA expression in childhood acute lymphoblastic leukemia and association with clinical and biological features. Leuk Res (2012) 36:293–8. doi:10.1016/j.leukres.2011.10.005 44. Chen L, Deshpande AJ, Banka D, Bernt KM, Dias S, Buske C, et al. Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inac- tivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia (2013) 27:813–22. doi:10.1038/leu.2012.327 64. Zhang H,Luo XQ,Zhang P,Huang LB,ZhengYS,Wu J,et al. MicroRNA patterns associated with clinical prognostic parameters and CNS relapse prediction in pediatric acute leukemia. PLoS One (2009) 4:e7826. doi:10.1371/journal.pone. 0007826 45. Daigle SR, Olhava EJ, Therkelsen CA, Basavapathruni A, Jin L, Boriack-Sjodin PA, et al. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood (2013) 122:1017–25. doi:10.1182/blood-2013-04-497644 65. Schotte D, De Menezes RX, Akbari Moqadam F, Khankahdani LM, Lange- Turenhout E, Chen C, et al. MicroRNA characterize genetic diversity and drug resistance in pediatric acute lymphoblastic leukemia. Haematologica (2011) 96:703–11. doi:10.3324/haematol.2010.026138 46. Meyer JA,Wang J, Hogan LE,Yang JJ, Dandekar S, Patel JP, et al. Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia. Nat Genet (2013) 45:290–4. doi:10.1038/ng.2558 66. Han BW, Feng DD, Li ZG, Luo XQ, Zhang H, Li XJ, et al. A set of miRNAs that involve in the pathways of drug resistance and leukemic stem-cell differentiation is associated with the risk of relapse and glucocorticoid response in childhood ALL. Hum Mol Genet (2011) 20:4903–15. doi:10.1093/hmg/ddr428 47. 76. Fouladi M, Park JR, Stewart CF, Gilbertson RJ, Schaiquevich P, Sun J, et al. Pediatric phase I trial and pharmacokinetic study of vorinostat: a Children’s Oncology Group phase I consortium report. J Clin Oncol (2010) 28:3623–9. doi:10.1200/JCO.2009.25.9119 REFERENCES A phase II trial of decitabine and vorinostat in combination with chemother- apy for relapsed/refractory acute lymphoblastic leukemia. ASH Annual Meeting Abstracts (2012) 120:4307. 54. Bachmann PS, Piazza RG, Janes ME, Wong NC, Davies C, Mogavero A, et al. Epigenetic silencing of BIM in glucocorticoid poor-responsive pediatric acute 73. Garcia-Manero G, Thomas D, Rytting M, Zweidler-McKay P, Estrov Z, Brown DL, et al. Phase I study of 5-aza-2’-deoxycitidine, alone or in combination with May 2014 | Volume 2 | Article 42 | 6 Frontiers in Pediatrics | Pediatric Oncology Burke and Bhatla Epigenetic modifications in ALL hyper-CVAD, in relapsed or refractory acute lymphocytic leukemia (ALL). ASH Annual Meeting Abstracts (2007) 110:2826. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 74. Giles F, Fischer T, Cortes J, Garcia-Manero G, Beck J, Ravandi F, et al. A phase I study of intravenous LBH589, a novel cinnamic hydroxamic acid analogue his- tone deacetylase inhibitor,in patients with refractory hematologic malignancies. Clin Cancer Res (2006) 12:4628–35. doi:10.1158/1078-0432.CCR-06-0511 Received: 30 January 2014; accepted: 29 April 2014; published online: 14 May 2014. Citation: Burke MJ and Bhatla T (2014) Epigenetic modifications in pediatric acute lymphoblastic leukemia. Front. Pediatr. 2:42. doi: 10.3389/fped.2014.00042 This article was submitted to Pediatric Oncology, a section of the journal Frontiers in Pediatrics. 75. Garcia-Manero G, Yang H, Bueso-Ramos C, Ferrajoli A, Cortes J, Wierda WG, et al. Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes. Blood (2008) 111:1060–6. doi:10.1182/blood- 2007-06-098061 Copyright © 2014 Burke and Bhatla. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 76. Fouladi M, Park JR, Stewart CF, Gilbertson RJ, Schaiquevich P, Sun J, et al. Pediatric phase I trial and pharmacokinetic study of vorinostat: a Children’s Oncology Group phase I consortium report. J Clin Oncol (2010) 28:3623–9. doi:10.1200/JCO.2009.25.9119 May 2014 | Volume 2 | Article 42 | 7 www.frontiersin.org

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EFFECTIVE ANTI-PIRACY IN VIETNAM: A JOURNEY THROUGH SITE BLOCKING

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50 50 University of California, Los Angeles & Loyola Law School. EFFECTIVE ANTI-PIRACY IN VIETNAM: A JOURNEY THROUGH SITE BLOCKING  Jonathan Lee Xue Han Abstract: The dawn of the Internet has created significant challenges to protecting copyrighted materials from piracy. In the past, copyright protection was mainly concerned with the threat of infringement from the sale of counterfeit goods, like pirated CDs and optical media. However, the Internet has now allowed for online piracy where infringing materials can be accessed online or copied with even greater ease. Vietnam has recently become a jurisdiction that is a hotbed for online piracy and copyright infringement globally. This is largely because Vietnam's current statutory and regulatory regime fail to effectively create an effective anti- piracy regime in accordance with its WTO TRIPS obligations. Furthermore, Vietnam's recent accession of various treaties like the CPTPP and WCT further galvanizes a need for change in Vietnam's intellectual property regime. Thus far, Vietnam's attempts at fashioning an effective site-blocking regime have not succeeded. This paper will look at other effective site-blocking regimes, namely Singapore, India, and France, to look prospectively at what may be possible in Vietnam. Keywords: Vietnam, Copyright, TRIPS, CPTPP, Site-blocking 51 Table of Contents Table of Contents Table of Contents Introduction ............................................................................................................................ 52 I. Site Blocking: an Overview.......................................................................................... 54 II. A Statutory Framework: Site Blocking in Singapore ............................................... 56 A. Overview of Statutory Site Blocking in Singapore ............................................. 56 B. Disney Enterprises, Inc V M1 Ltd: Statutory Site Blocking in Practice and Dynamic Injunctions ...................................................................................................... 58 III. Coming to the Same Conclusion: Common Law Site Blocking in India ................. 59 A. Overview of Site Blocking in India ...................................................................... 60 B. Utv Software Communication Ltd. ... V 1337x: Common Law Site Blocking and Dynamic Injunctions .............................................................................................. 61 IV. A Civil Law Approach: Site Blocking in France ....................................................... 64 V. Clearing the Fog: the Need for Effective Site Blocking in Vietnam ........................ 66 A. Copyright Protection in Vietnam: a Lack of Clarity and Efficacy ................... 66 1. Vietnam’s Law on Intellectual Property ............................................................. 67 2. Circular 07 .......................................................................................................... 67 3. Structural Problems ............................................................................................. 68 B. Site Blocking in Vietnam: Possible Approaches ................................................. 69 1. A Statutory Approach ......................................................................................... 69 2. Administrative Approach .................................................................................... 70 Conclusion .............................................................................................................................. 71 Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 52 1 Advisory Committee on Enforcement, Study on IP Enforcement Measures, Especially Anti-piracy Measures in the Digital Environment, WIPO, July 23, 2019. 2 Id. at 1. 6 Nigel Cory, The Normalization of Website Blocking Around the World in the Fight Against Piracy Online, June 12, 2018, https://itif.org/publications/2018/06/12/normalization-website-blocking-around- world-fight-against-piracy-online/. Id. at 1. 4 Justin Hughes, Copyright Law in Foreign Jurisdictions: How are other countries handling digital piracy Hearing Before the United States Senate Judiciary Committee Subcommittee on Intellectual Property, 10 March 2020. 5 d 4 5 Id. at 4. 7 New survey shows Vietnam among highest in online piracy in Southeast Asia., AVIA, May 17, 2021, https://avia.org/new-survey-shows-vietnam-among-highest-in-online-piracy-in-southeast-asia/. 9 USTR, 2021 Special 301 Report, USTR, 84; IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106; See also USTR, USTR Releases Annual Special 301 Report on Intellectual Property Protection and Review of Notorious Markets for Counterfeiting and Piracy, 29 April 2020, https://ustr.gov/about-us/policy-offices/press-office/press-releases/2020/april/ustr- releases-annual-special-301-report-intellectual-property-protection-and-review-notorious (“The Special 301 Report identifies trading partners that do not adequately or effectively protect and enforce intellectual property (IP) rights or otherwise deny market access to U.S. innovators and creators that rely on protection of their IP rights… These trading partners will be the subject of increased bilateral engagement with USTR to address IP concerns. Over the coming weeks, USTR will review the developments against the benchmarks established in the Special 301 action plans for those countries. For countries failing to address U.S. concerns, USTR will take appropriate actions, which may include enforcement actions under Section 301 of the Trade Act or pursuant to World Trade Organization (WTO) or other trade agreement dispute settlement procedures.) 7 New survey shows Vietnam among highest in online piracy in Southeast Asia., AVIA, May 17, 2021, https://avia.org/new-survey-shows-vietnam-among-highest-in-online-piracy-in-southeast-asia/. 8 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, IIPA,106 (January 28, 2021). INTRODUCTION The dawn of the internet has created significant challenges to the protection of copyrighted materials from piracy. 1 In the past, copyright protection was mostly concerned with the threat of infringement from the sale of counterfeit goods, like pirated CDs and optical media.2 However, the internet has now allowed for a form of online piracy where infringing materials can be accessed online or copied with an easy click of a button or tap of a screen.3 One of the most promising attempts at addressing online piracy is the practice of site blocking.4 While not always the case, numerous nations have outlined a framework for site blocking as a part of safe harbor provisions.5 Broadly, these safe harbor provisions broker a compromise between internet service providers (ISPs) and rights holders, by indemnifying ISPs in exchange for allowing rights-holders to get ISPs to block domestic access to sites that are known to host or distribute large amounts of pirated material.6 While effective, many of these measures have still have not reached many jurisdictions in Asia which have growing numbers of their population joining the internet. There is no place where this is truer than Vietnam. Vietnam has one of the fastest growing online piracy cultures in Asia and already has many online users that openly admit to frequently using infringing sites.7 Moreover, it seems that the combination of unclear laws, poor enforcement mechanisms, and restricted market access has resulted in a weak copyright regime that is susceptible to rampant online infringement.8 The Office of the United States Trade Representative (USTR), recognizing the threat that Vietnam poses to intellectual property (IP) protection and enforcement among U.S. trading partners, elevated Vietnam to the watch list. 9 1 Advisory Committee on Enforcement, Study on IP Enforcement Measures, Especially Anti-piracy Measures in the Digital Environment, WIPO, July 23, 2019. 2 Id. at 1. 4 Justin Hughes, Copyright Law in Foreign Jurisdictions: How are other countries handling digital piracy? Hearing Before the United States Senate Judiciary Committee Subcommittee on Intellectual Property, 10 March 2020. 5 Id t 4 6 Nigel Cory, The Normalization of Website Blocking Around the World in the Fight Against Piracy Online, June 12, 2018, https://itif.org/publications/2018/06/12/normalization-website-blocking-around- world-fight-against-piracy-online/. 1 Advisory Committee on Enforcement, Study on IP Enforcement Measures, Especially Anti-piracy Measures in the Digital Environment, WIPO, July 23, 2019. 2 Id. at 1. 3 Id. at 1. 4 Justin Hughes, Copyright Law in Foreign Jurisdictions: How are other countries handling digital piracy? Hearing Before the United States Senate Judiciary Committee Subcommittee on Intellectual Property, 10 March 2020. 5 Id. at 4. 6 Nigel Cory, The Normalization of Website Blocking Around the World in the Fight Against Piracy Online, June 12, 2018, https://itif.org/publications/2018/06/12/normalization-website-blocking-around- world-fight-against-piracy-online/. 7 New survey shows Vietnam among highest in online piracy in Southeast Asia., AVIA, May 17, 2021, https://avia.org/new-survey-shows-vietnam-among-highest-in-online-piracy-in-southeast-asia/. 8 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, IIPA,106 (January 28, 2021). 9 USTR, 2021 Special 301 Report, USTR, 84; IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106; See also USTR, USTR Releases Annual Special 301 Report on Intellectual Property Protection and Review of Notorious Markets for Counterfeiting and Piracy, 29 April 2020, https://ustr.gov/about-us/policy-offices/press-office/press-releases/2020/april/ustr- releases-annual-special-301-report-intellectual-property-protection-and-review-notorious (“The Special 301 Report identifies trading partners that do not adequately or effectively protect and enforce intellectual property (IP) rights or otherwise deny market access to U.S. innovators and creators that rely on protection of their IP rights… These trading partners will be the subject of increased bilateral engagement with USTR to address IP concerns. Over the coming weeks, USTR will review the developments against the benchmarks established in the Special 301 action plans for those countries. For countries failing to address U.S. concerns, USTR will take appropriate actions, which may include enforcement actions under Section 301 of the Trade Act or pursuant to World Trade Organization (WTO) or other trade agreement dispute settlement procedures.) https://avia.org/new-survey-shows-vietnam-among-highest-in-online-piracy-in-southeast-asia/. 8 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, IIPA,106 (January 28, 2021). 8 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, IIPA,106 (January 28, 2021). 3 Id. at 1. 10 World Trade Organization, Members and Observers, https://www.wto.org/english/thewto_e/whatis_e/tif_e/org6_e.htm/. 11 World Trade Organization, Frequently asked questions about TRIPS [trade-related aspects of intellectual property rights] in the WTO, https://www.wto.org/english/tratop_e/trips_e/tripfq_e.htm#Who'sSigned/. 12 TRIPS Agreement, Part III, Section 5, Article 61, https://www.wto.org/english/docs_e/legal_e/31bis_trips_05_e.htm#5/. 13 TRIPS Agreement, Part III, Section 1, Article 41, https://www.wto.org/english/docs_e/legal_e/31bis_trips_05_e.htm#1/. 14 WIPO Lex, WIPO Copyright Treaty, Article 14(2). 15 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, IIPA,110, 112 (January 28, 2021). 16 Government of New Zealand, Viet Nam seventh nation to ratify CPTPP, 15 November 2018, https://www.beehive.govt.nz/release/viet-nam-seventh-nation-ratify-cptpp/. 17 Vietnam becomes signatory to WIPO Copyright Treaty, People’s Army Newspaper, 25 November 2021, https://en.qdnd.vn/foreign-affairs/bilateral-relations/vietnam-becomes-signatory-to-wipo-copyright- treaty-536158/; See also WIPO Lex, Contracting Parties > WIPO Copyright Treaty, https://wipolex.wipo.int/en/treaties/ShowResults?search_what=C&treaty_id=16/. 18 See CPTPP, Chapter 18, Article 18.74, page 44-47, https://www.mfat.govt.nz/assets/Trade- agreements/TPP/Text-ENGLISH/18.-Intellectual-Property-Chapter.pdf/; WIPO Lex, WIPO Copyright Treaty, Article 14(2). 19 Nigel Cory, supra note 6; See Australia, Copyright Amendment (Online Infringement) Act 2015, Section 115A; United Kingdom, Copyright, Designs and Patents Act 1988, Section 97A; EU 2001 Information Society Directive, Article 8(3); Singapore, Copyright Act (Chapter 63 of Singapore Laws), Article 193DDA(1)(b) (revised 31st January 2006). See also Hugh Stephens, Disabling Access to Large- Scale Pirate Sites (Site Blocking)—It Works!, Hugh Stephens Blog, 18 April 2017, https://hughstephensblog.net/2017/04/18/disabling-access-to-large-scale-pirate-sites-site-blocking-it- works/; INTRODUCTION 9 USTR, 2021 Special 301 Report, USTR, 84; IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106; See also USTR, USTR Releases Annual Special 301 Report on Intellectual Property Protection and Review of Notorious Markets for Counterfeiting and Piracy, 29 April 2020, https://ustr.gov/about-us/policy-offices/press-office/press-releases/2020/april/ustr- releases-annual-special-301-report-intellectual-property-protection-and-review-notorious (“The Special 301 Report identifies trading partners that do not adequately or effectively protect and enforce intellectual property (IP) rights or otherwise deny market access to U.S. innovators and creators that rely on protection of their IP rights… These trading partners will be the subject of increased bilateral engagement with USTR to address IP concerns. Over the coming weeks, USTR will review the developments against the benchmarks established in the Special 301 action plans for those countries. For countries failing to address U.S. concerns, USTR will take appropriate actions, which may include enforcement actions under Section 301 of the Trade Act or pursuant to World Trade Organization (WTO) or other trade agreement dispute settlement procedures.) Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 53 Moreover, Vietnam is a member of the World Trade Organization (WTO) 10 and a signatory to the WTO Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS).11 The TRIPS agreement requires Vietnam to “criminalize copyright piracy on a commercial scale” 12 and “make available to right holders civil judicial procedures concerning the enforcement of any intellectual property right”.13 This means that Vietnam is legally obligated to ensure that there are available enforcement procedures for effective action against all forms of copyright infringement covered in the treaty14, including the kind of piracy happening online in Vietnam daily. However, it seems like Vietnam has yet to do so.15 Further, Vietnam’s recent accession of the Comprehensive and Progressive Agreement for Trans-Pacific Partnership (CPTPP) 16 and the World Intellectual Property Organization Copyright Treaty (WCT) 17 further galvanize and impose obligations for effective copyright enforcement on Vietnam.18 Hence, the topic of effective Vietnamese measures against online piracy is becoming increasingly relevant. The international experience has shown that many nations with a variety of legal traditions have managed to create effective copyright regimes that have aspects of site blocking.19 g https://www.wto.org/english/thewto_e/whatis_e/tif_e/org6_e.htm/. p g g g 11 World Trade Organization, Frequently asked questions about TRIPS [trade-related aspects of intellectual property rights] in the WTO, p p y g https://www.wto.org/english/tratop_e/trips_e/tripfq_e.htm#Who'sSigned/. ttps://www.wto.org/english/docs_e/legal_e/31bis_trips_05_e.htm#5 g https://www.wto.org/english/docs_e/legal_e/31bis_trips_05_e.htm#1/. World Trade Organization, Members and Observers 20 Indian courts have been ordering ISPs to block pirate websites to protect new releases of Indian films for many years. See Delhi HC restrains 30 torrent sites from hosting copyrighted content, orders ISPs to block them, FINANCIAL EXPRESS, April 11, 2019, https://www.financialexpress.com/india-news/delhi- hc-restrains-30-torrent-sites-from-hosting-copyrighted-content-orders-isps-to-block-them/1545480/; Bill Toulas, ISPs in India Ordered to Block Pirate Bay, Torrentz2, YTS, and 1337x, TECHNADU, April 12, 2019, https://www.technadu.com/isps-india-ordered-block-pirate-bay-torrentz2-yts-1337x/64592/; Javed Anwer, 830 more websites blocked in India, many torrent links in list, INDIA TODAY, August 25, 2016 (“Blocking of hundreds of URLs at the behest of film producers is not new in India. It has become almost routine to for film producers to approach court before release of a film and take John Doe orders, leading to the blocking of the websites. Not only torrent sites have been blocked under such orders but also image hosts, file hosts and websites that share URLs”), https://www.indiatoday.in/technology/news/story/830- more-websites-blocked-in-india-many-torrent-links-in-list-337177-2016-08-25; Anupam Saxena, ISP Wise List Of Blocked Sites #IndiaBlocks, MEDIANAMA, May 17, 2012, INTRODUCTION 14 WIPO Lex, WIPO Copyright Treaty, Article 14(2). 15 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, IIPA,110, 112 (January 28, 2021). , , ( y , ) 16 Government of New Zealand, Viet Nam seventh nation to ratify CPTPP, 15 November 2018, 16 Government of New Zealand, Viet Nam seventh nation to ratify CPTPP, 1 https://www.beehive.govt.nz/release/viet-nam-seventh-nation-ratify-cptpp/. , y , https://www.beehive.govt.nz/release/viet-nam-seventh-nation-ratify-cptpp/. p g y p pp Vietnam becomes signatory to WIPO Copyright Treaty, People’s Army Newspaper, 25 November 21, https://en.qdnd.vn/foreign-affairs/bilateral-relations/vietnam-becomes-signatory-to-wipo-copyright- aty-536158/; See also WIPO Lex, Contracting Parties > WIPO Copyright Treaty, p p p _ y_ 18 See CPTPP, Chapter 18, Article 18.74, page 44-47, https://www.mfat.govt.nz/assets/Trade- agreements/TPP/Text-ENGLISH/18.-Intellectual-Property-Chapter.pdf/; WIPO Lex, WIPO Copyright Treaty, Article 14(2). 19 Nigel Cory, supra note 6; See Australia, Copyright Amendment (Online Infringement) Act 2015, Section 115A; United Kingdom, Copyright, Designs and Patents Act 1988, Section 97A; EU 2001 Information Society Directive, Article 8(3); Singapore, Copyright Act (Chapter 63 of Singapore Laws), Article 193DDA(1)(b) (revised 31st January 2006). See also Hugh Stephens, Disabling Access to Large- Scale Pirate Sites (Site Blocking)—It Works!, Hugh Stephens Blog, 18 April 2017, https://hughstephensblog.net/2017/04/18/disabling-access-to-large-scale-pirate-sites-site-blocking-it- works/; Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 54 Furthermore, the case of India20 and Singapore21, show that even in Asia site blocking is a viable and effective means of containing online piracy. Thus, this paper through the study and analysis of site blocking regimes in Singapore, India, and France shall show that a clear and effective Vietnamese site blocking regime which solves online piracy and fulfils treaty obligations, is possible. Part II of this paper describes the practice of site blocking generally. Part III of this paper analyzes and looks at Singapore’s site blocking regime as an example of how a country in the same region as Vietnam has tackled the issue of site blocking via statute. Part IV of the paper will look at India’s common law site blocking regime and discusses commonalities in various site blocking regimes. Part V of the paper looks at the French civil law site blocking regime given Vietnam’s civil law system. Finally, Part VI will look at what an effective site blocking regime might look like in Vietnam. g y p 26 European Union Intellectual Property Office, Study on Dynamic Blocking Injunctions in the European Union, 16, March 2021. , , y , , https://www.medianama.com/2012/05/223-isp-wise-list-of-blocked-sites-indiablocks/. 20 Indian courts have been ordering ISPs to block pirate websites to protect new releases of Indian films for many years. See Delhi HC restrains 30 torrent sites from hosting copyrighted content, orders ISPs to block them, FINANCIAL EXPRESS, April 11, 2019, https://www.financialexpress.com/india-news/delhi- hc-restrains-30-torrent-sites-from-hosting-copyrighted-content-orders-isps-to-block-them/1545480/; Bill Toulas, ISPs in India Ordered to Block Pirate Bay, Torrentz2, YTS, and 1337x, TECHNADU, April 12, 2019, https://www.technadu.com/isps-india-ordered-block-pirate-bay-torrentz2-yts-1337x/64592/; Javed Anwer, 830 more websites blocked in India, many torrent links in list, INDIA TODAY, August 25, 2016 (“Blocking of hundreds of URLs at the behest of film producers is not new in India. It has become almost routine to for film producers to approach court before release of a film and take John Doe orders, leading to the blocking of the websites. Not only torrent sites have been blocked under such orders but also image hosts, file hosts and websites that share URLs”), https://www.indiatoday.in/technology/news/story/830- more-websites-blocked-in-india-many-torrent-links-in-list-337177-2016-08-25; Anupam Saxena, ISP Wise List Of Blocked Sites #IndiaBlocks, MEDIANAMA, May 17, 2012, https://www.medianama.com/2012/05/223-isp-wise-list-of-blocked-sites-indiablocks/. 21 Irene Tham, Solarmovie.ph is first piracy website to be blocked under amended Copyright Act, The Straits Times, 16 February 2016, https://www.straitstimes.com/singapore/solarmovieph-is-first-piracy- website-to-be-blocked-under-amended-copyright-act/ ; See also Motion Picture Association Asia Pacific, Singapore allows dynamic site blocking in landmark court ruling – Any Web address linking to blocked piracy sites can now be blocked as well, MPA APAC In The News, 19 July 2018, (“A spokesman for the Intellectual Property Office of Singapore said: “We are glad to see rights holders utili[z]ing the legal framework that we have put in place to protect their copyright works.””), https://www.mpa- apac.org/in_the_news/singapore-allows-dynamic-site-blocking-in-landmark-court-ruling-any-web- address-linking-to-blocked-piracy-sites-can-now-be-blocked-as-well/. 22 Nigel Cory, supra note 6. 23 Id. at 22. 24 Id. at 22. 25 Nigel Cory, supra note 6. 26 European Union Intellectual Property Office, Study on Dynamic Blocking Injunctions in the European Union, 16, March 2021. I. SITE BLOCKING: AN OVERVIEW The age of the internet has enabled electronic copying and sharing of content across many geographic and jurisdictional borders. 22 This often can include protected copyrighted material. 23 However, IP laws and regulations are usually limited jurisdictionally to the countries in which they are enacted, while the domain of the internet is borderless.24 Thus, site blocking is generally seen as a response to limitations faced by domestic copyright regimes against a borderless internet.25 Site blocking operates by allowing rights-holders to get internet service providers (ISPs) to block domestic access to sites that are known to host or distribute large amounts of pirated or infringing material.26 Research has generally shown that efficient and prolific site blocking leads to 21 Irene Tham, Solarmovie.ph is first piracy website to be blocked under amended Copyright Act, The Straits Times, 16 February 2016, https://www.straitstimes.com/singapore/solarmovieph-is-first-piracy- website-to-be-blocked-under-amended-copyright-act/ ; See also Motion Picture Association Asia Pacific, Singapore allows dynamic site blocking in landmark court ruling – Any Web address linking to blocked piracy sites can now be blocked as well, MPA APAC In The News, 19 July 2018, (“A spokesman for the Intellectual Property Office of Singapore said: “We are glad to see rights holders utili[z]ing the legal framework that we have put in place to protect their copyright works.””), https://www.mpa- apac.org/in_the_news/singapore-allows-dynamic-site-blocking-in-landmark-court-ruling-any-web- dd li ki t bl k d i it b bl k d ll/ 21 Irene Tham, Solarmovie.ph is first piracy website to be blocked under amended Copyright Act, The Straits Times, 16 February 2016, https://www.straitstimes.com/singapore/solarmovieph-is-first-piracy- website-to-be-blocked-under-amended-copyright-act/ ; See also Motion Picture Association Asia Pacific, Singapore allows dynamic site blocking in landmark court ruling – Any Web address linking to blocked piracy sites can now be blocked as well, MPA APAC In The News, 19 July 2018, (“A spokesman for the Intellectual Property Office of Singapore said: “We are glad to see rights holders utili[z]ing the legal framework that we have put in place to protect their copyright works.””), https://www.mpa- apac.org/in_the_news/singapore-allows-dynamic-site-blocking-in-landmark-court-ruling-any-web- address linking to blocked piracy sites can now be blocked as well/ 22 Nigel Cory, supra note 6. 23 Id. at 22. 24 Id. at 22. 25 Nigel Cory, supra note 6. 27 Brett Danaher et al., Website Blocking Revisited: The Effect of the UK November 2014 Blocks on Consumer Behavior, 16-19, 18 April 2016. https://papers.ssrn.com/sol3/Delivery.cfm/SSRN_ID2766795_code986726.pdf?abstractid=2766795&miri d=1/; Motion Picture Association, Measuring the Effect of Piracy Website Blocking in Australia on Consumer Behavior: December 2018, 6-8, January 2020, https://www.mpa-apac.org/wp- content/uploads/2020/02/Australia-Site-Blocking-Summary-January-2020.pdf/. 28 Justin Hughes, In response to questions from Senators Tillis, Coons, and Blumenthal, Senate Judiciary Committee / Intellectual Property Subcommittee, 7, 14 April 2020. 29 34 Peter Carstairs, supra note 30 at 305; Brett Danaher et al., supra note 27 at 16-19; Motion Picture Association, Measuring the Effect of Piracy Website Blocking in Australia on Consumer Behavior: December 2018, supra note 27 at 6-8. g y 37 Peter Carstairs, supra note 30 at 287. 32 Justin Hughes, supra note 28 at 11-12. 31 Victor Loh, Court order makes it easier for copyright owners to curb access to piracy websites, https://www.todayonline.com/singapore/court-order-makes-it-easier-copyright-owners-curb-access- piracy-websites/. 32 27 Brett Danaher et al., Website Blocking Revisited: The Effect of the UK November 2014 Blocks on Consumer Behavior, 16-19, 18 April 2016. https://papers.ssrn.com/sol3/Delivery.cfm/SSRN_ID2766795_code986726.pdf?abstractid=2766795&mi d=1/; Motion Picture Association, Measuring the Effect of Piracy Website Blocking in Australia on Consumer Behavior: December 2018, 6-8, January 2020, https://www.mpa-apac.org/wp- content/uploads/2020/02/Australia-Site-Blocking-Summary-January-2020.pdf/. 28 Justin Hughes, In response to questions from Senators Tillis, Coons, and Blumenthal, Senate Judiciary Committee / Intellectual Property Subcommittee, 7, 14 April 2020. 29 Justin Hughes, supra note 28 at 9. 30 Peter Carstairs, The Inevitable Actors: An Analysis of Australia’s Recent Anti-piracy Website Blocking Laws, Their Balancing of Rights and Overall Effectiveness, (2021) 31 AIPJ 280, 286. 31 Victor Loh, Court order makes it easier for copyright owners to curb access to piracy websites, https://www.todayonline.com/singapore/court-order-makes-it-easier-copyright-owners-curb-access- piracy-websites/. 32 Justin Hughes, supra note 28 at 11-12. 33 Id. at 32. 34 Peter Carstairs, supra note 30 at 305; Brett Danaher et al., supra note 27 at 16-19; Motion Picture Association, Measuring the Effect of Piracy Website Blocking in Australia on Consumer Behavior: December 2018, supra note 27 at 6-8. 35 Peter Carstairs, supra note 30 at 300; Grace Espinosa, Internet Piracy: Is Protecting Intellectual Property Worth Government Censorship?, 18 Tex. Wesleyan L. Rev. 309, 332-34 (2011). 36 [CS(COMM) 724/2017 & I.As. 12269/2017, 12271/2017, 6985/2018, 8949/2018 AND 16781/2018], Decision of 10 April 2019 at ¶55-56; See also Nigel Cory, India and Website Blocking: Courts Allow Dynamic Injunctions to Fight Digital Piracy, May 29, 2019, https://itif.org/publications/2019/05/29/india and-website-blocking-courts-allow-dynamic-injunctions-fight-digital/. 37 Peter Carstairs, supra note 30 at 287. p y p , y , ( ) 36 [CS(COMM) 724/2017 & I.As. 12269/2017, 12271/2017, 6985/2018, 8949/2018 AND 16781/2018], Decision of 10 April 2019 at ¶55-56; See also Nigel Cory, India and Website Blocking: Courts Allow Dynamic Injunctions to Fight Digital Piracy, May 29, 2019, https://itif.org/publications/2019/05/29/india and-website-blocking-courts-allow-dynamic-injunctions-fight-digital/. 35 Peter Carstairs, supra note 30 at 300; Grace Espinosa, Internet Piracy: Is Protecting Intellectual Property Worth Government Censorship?, 18 Tex. Wesleyan L. Rev. 309, 332-34 (2011). g p 30 Peter Carstairs, The Inevitable Actors: An Analysis of Australia’s Recent Anti-piracy Website Blocking Laws, Their Balancing of Rights and Overall Effectiveness, (2021) 31 AIPJ 280, 286. p y 29 Justin Hughes, supra note 28 at 9. II. A STATUTORY FRAMEWORK: SITE BLOCKING IN SINGAPORE Less than a three-hour plane ride away from Vietnam’s capital Hanoi lies Singapore. Singapore inherited its common law tradition from the British and shares membership in the Association of South-East Asian Nations (ASEAN) with Vietnam.38 Besides geographic proximity, Singapore also happens to be a party to many of the treaties that Vietnam has signed or is planning to sign, including the WCT39 and the CPTPP40. These treaties are relevant because they stipulate that signatory nations have a responsibility to ensure effective enforcement mechanisms around all forms of copyright infringement covered by the WCT, which likely includes online piracy.41 Further, both Singapore and Vietnam have been parties to numerous ASEAN treaties, including the ASEAN Framework Agreement on Intellectual Property Cooperation.42 The ASEAN Framework Agreement on Intellectual Property Cooperation requires signatories to cooperate in areas around intellectual property legislation, particularly where it involves the implementation of international intellectual property treaties like the WCT.43 Thus, Singapore’s current site blocking regime is likely going to be relevant when Vietnam’s government considers what should be implemented in Vietnam. In short, because of Singapore’s geographic proximity and many shared multilateral treaties, a look at Singapore’s site blocking regime can be informative and useful. I. SITE BLOCKING: AN OVERVIEW Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 55 real world decreases in total online piracy and increases in the use of paid legal streaming services.27 Therefore, it should not be surprising that the creation of a domestic site blocking regime has been embraced in many jurisdictions, including Australia, many parts of the EU, Singapore, India, and the United Kingdom.28 However, the creation of a site blocking regime is not always so simple. Firstly, countries looking to implement such a regime must ensure that it only targets sites that can be identified as embracing online piracy or infringement.29 This is difficult because infringing content is being posted online everyday by end users on a myriad of legitimate and illegitimate sites. If one does not adequately identify which sites should be blocked for embracing piracy, you run the risk of blocking even legitimate sites and stifling the internet, i.e. “over blocking”.30 Secondly, the reality of online practice is that when infringing sites are blocked or taken down, infringing sites attempt to circumvent such orders by changing domain names, redirecting traffic, or having dynamic IP addresses.31 This creates a situation where the original blocking order is essentially rendered useless and rights holders would have to begin the process all over again for every slight change in domain name or IP address. Hence, to ensure that site blocking regimes remain effective, courts in the France, Singapore, and India, have allowed for the creation of dynamic injunctions.32 These dynamic injunctions allow for the blocking of IP addresses and multiple domain names to account for the common practice of redirecting or changing domain names. 33 These two issues have created similarities among countries with effective site blocking regimes around clear legal standards targeting online piracy and responsive enforcement measures to keep up with infringers. While site blocking measures are effective at reducing traffic to infringing sites34, numerous concerns in various jurisdictions have been raised about site blocking’s ability to stifle free speech 35, strangle internet freedom 36, and the proportionality of such responses to internet piracy. 37 Hence, understanding how other Asian site blocking p y p , y , ( ) 36 [CS(COMM) 724/2017 & I.As. 12269/2017, 12271/2017, 6985/2018, 8949/2018 AND 16781/2018], Decision of 10 April 2019 at ¶55-56; See also Nigel Cory, India and Website Blocking: Courts Allow Dynamic Injunctions to Fight Digital Piracy, May 29, 2019, https://itif.org/publications/2019/05/29/india- and-website-blocking-courts-allow-dynamic-injunctions-fight-digital/. I. SITE BLOCKING: AN OVERVIEW Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 56 regimes, like India and Singapore, have attempted to create an effective site blocking regime and balance the associated concerns can be informative in ascertaining what site blocking might need to look like in Vietnam. ASEAN, ASEAN Member States, https://asean.org/about asean/member states/. 39 WIPO Lex, WIPO-Administered Treaties, Contracting Parties WIPO Copyright Treaty, https://wipolex.wipo.int/en/treaties/ShowResults?search_what=C&treaty_id=16/ . 40 Singapore Ministry of Trade and Industry, Singapore ratified the Comprehensive and Progressive Agreement for Trans-Pacific Partnership, 19 July 2018, https://www.mti.gov.sg/-/media/MTI/improving- trade/multilateral-and-regional-forums/CPTPP/cptpp-ratification---19-july-2018.pdf/. 41 WIPO Lex, WIPO Copyright Treaty, Article 14(2). 42ASEAN, ASEAN Framework Agreement on Intellectual Property Cooperation, 15 December 1995, 38 ASEAN, ASEAN Member States, https://asean.org/about-asean/member-states/. 39 WIPO Lex, WIPO-Administered Treaties, Contracting Parties WIPO Copyright Treaty, https://wipolex.wipo.int/en/treaties/ShowResults?search_what=C&treaty_id=16/ . 40 Singapore Ministry of Trade and Industry, Singapore ratified the Comprehensive and Progressive Agreement for Trans-Pacific Partnership, 19 July 2018, https://www.mti.gov.sg/-/media/MTI/improving- trade/multilateral-and-regional-forums/CPTPP/cptpp-ratification---19-july-2018.pdf/. 41 WIPO Lex, WIPO Copyright Treaty, Article 14(2). 42ASEAN, ASEAN Framework Agreement on Intellectual Property Cooperation, 15 December 1995, https://www.aseanip.org/Portals/0/PDF/ASEANFrameworkAgreementonIntellectualPropertyCooperation. pdf/. 43 ASEAN, supra note 42 at Article 3(3) 44 There was little to no debate recorded in parliament surrounding the amendments to the Copyright Act creating the site blocking regime. See Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, 7 July 2014, http://sprs.parl.gov.sg/search/sprs3topic?reportid=bill- 100/.Parliament of Singapore, Third Reading of Copyright (Amendment) Bill 2014, 8 July 2014, http://sprs.parl.gov.sg/search/sprs3topic?reportid=bill-337/. 45 Ashley Chia, Amendments to Copyright Act aim to stop online piracy, Today Online, July 08, 2014, https://www.todayonline.com/singapore/amendments-copyright-act-aim-stop-online-piracy/. 46 Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, supra note 44, (Senior Minister of State for Law (Ms Indranee Rajah): “The prevalence of online piracy in Singapore turns customers away from legitimate content and adversely affects Singapore's creative sector. It can also undermine our reputation as a society that respects the protection of intellectual property… We, therefore, need to take stronger measures against online piracy.”). p 43 ASEAN, supra note 42 at Article 3(3) ASEAN, supra note 42 at Article 3(3) 44 There was little to no debate recorded in parliament surrounding the amendments to the Copyright Act creating the site blocking regime. See Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, 7 July 2014, http://sprs.parl.gov.sg/search/sprs3topic?reportid=bill- 100/.Parliament of Singapore, Third Reading of Copyright (Amendment) Bill 2014, 8 July 2014, http://sprs.parl.gov.sg/search/sprs3topic?reportid=bill-337/. 5 , py g y, ( ) 42ASEAN, ASEAN Framework Agreement on Intellectual Property Cooperation, 15 December 1995, 42ASEAN, ASEAN Framework Agreement on Intellectual Property Cooperation, 15 December 1995, https://www.aseanip.org/Portals/0/PDF/ASEANFrameworkAgreementonIntellectualPropertyCooperatio pdf/. creating the site blocking regime. See Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, 7 July 2014, http://sprs.parl.gov.sg/search/sprs3topic?reportid=bill- 100/.Parliament of Singapore, Third Reading of Copyright (Amendment) Bill 2014, 8 July 2014, http://sprs.parl.gov.sg/search/sprs3topic?reportid=bill-337/. 45 Ashley Chia, Amendments to Copyright Act aim to stop online piracy, Today Online, July 08, 2014, https://www.todayonline.com/singapore/amendments-copyright-act-aim-stop-online-piracy/. 46 Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, supra note 44, (Senior Minister of State for Law (Ms Indranee Rajah): “The prevalence of online piracy in Singapore turns t f l iti t t t d d l ff t Si ' ti t It l http://sprs.parl.gov.sg/search/sprs3topic?reportid=bill-337/. 45 Ashley Chia, Amendments to Copyright Act aim to stop online piracy, Today Online, July 08, 2014, https://www.todayonline.com/singapore/amendments-copyright-act-aim-stop-online-piracy/. 38 ASEAN, ASEAN Member States, https://asean.org/about-asean/member-states/. g p pp WIPO Lex, WIPO Copyright Treaty, Article 14(2). g p pp j y p 41 WIPO Lex, WIPO Copyright Treaty, Article 14(2). 42ASEAN, ASEAN Framework Agreement on Intellectual Property Cooperation, 15 December 1995, https://www.aseanip.org/Portals/0/PDF/ASEANFrameworkAgreementonIntellectualPropertyCooperation. pdf/. 43 ASEAN, supra note 42 at Article 3(3) 44 There was little to no debate recorded in parliament surrounding the amendments to the Copyright Act creating the site blocking regime See Parliament of Singapore Second Reading of Copyright A. Overview of Statutory Site Blocking in Singapore The amendment to Singapore’s Copyright Act which instituted its site blocking regime was passed without much opposition44 in 2014.45 The goal of Singapore’s site blocking regime is to actively combat online piracy46 and “empower rights owners to p y g p py g p p y 46 Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, supra note 44, (Senior Minister of State for Law (Ms Indranee Rajah): “The prevalence of online piracy in Singapore turns customers away from legitimate content and adversely affects Singapore's creative sector. It can also undermine our reputation as a society that respects the protection of intellectual property… We, therefore, need to take stronger measures against online piracy.”). Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 57 more effectively disable access to sites that flagrantly infringe copyright”.47 The main statutory provision instituting Singapore’s site blocking is Article 193DDA. It works by allowing for a copyright holder or exclusive licensee to petition the court for a “blocking order” or injunction directing an ISP to block access to a “flagrantly infringing online location” (FIOL).48 To obtain a site blocking order the copyright holder or exclusive licensee would have to prove that 1) the website has been used or is being used to commit or facilitate copyright infringement and 2) the website is a “flagrantly infringing online location”.49 In Singapore, a “flagrantly infringing online location” is defined as a website which “flagrantly infringes or facilitates infringement of copyright materials”. 50 In determining whether an online location is a “flagrantly infringing online location”, courts consider non‑exhaustive factors as set out in the statute:51 (a) whether the primary purpose of the online location is to commit or facilitate copyright infringement; (b) whether the online location makes available or contains directories, indexes or categories of the means to commit or facilitate copyright infringement; (c) whether the owner or operator of the online location demonstrates a disregard for copyright generally; (d) whether access to the online location has been disabled by orders from any court of another country or territory on the ground of or related to copyright infringement; (e) whether the online location contains guides or instructions to circumvent measures, or any order of any court, that disables access to the online location on the ground of or related to copyright infringement; 52 (f) the volume of traffic at or frequency of access to the online location. , p g g g pp 49 Article 193DDA(1)(b), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st Januar 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, supra note 48. 47 Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, supra note 44. 54 Peter Carstairs, supra note 30 at 284-85. See also Australia, Copyright Amendment (Online Infringement) Act (2018), Section 115A; Justin Hughes, supra note 28 at 11 & 42 (noting the similarities in the Australian and Singaporean site blocking statutes). 52 Article 193DDA(2), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). 53 g p , g py g ( ) , p 48 Article 193DDA, Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January g p g 55 Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, supra note 48. 53 David Tan, Copyright reform and what it means for your wedding photos, Straits Times, 17 Septembe 2021, https://www.straitstimes.com/opinion/copyright-reform-and-what-it-means-for-your-wedding- photos/. 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, https://sso.agc.gov.sg/Bills-Supp/16-2014/Published/20140529?DocDate=20140529#xn-. , g p py g ( p g p ) ( y 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill p 50 Parliament of Singapore, supra note 49. 52 Article 193DDA(2), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). 53 David Tan, Copyright reform and what it means for your wedding photos, Straits Times, 17 September 2021 https://www straitstimes com/opinion/copyright reform and what it means for your wedding Article 193DDA(2), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). 53 David Tan, Copyright reform and what it means for your wedding photos, Straits Times, 17 September 2021, https://www.straitstimes.com/opinion/copyright-reform-and-what-it-means-for-your-wedding- photos/. 54 Peter Carstairs supra note 30 at 284-85 See also Australia Copyright Amendment (Online 47 Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, supra note 44. 48 Article 193DDA, Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, https://sso.agc.gov.sg/Bills-Supp/16-2014/Published/20140529?DocDate=20140529#xn-. 49 Article 193DDA(1)(b), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, supra note 48. 50 Parliament of Singapore supra note 49 Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, supra note 44. 48 Article 193DDA, Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, https://sso.agc.gov.sg/Bills-Supp/16-2014/Published/20140529?DocDate=20140529#xn-. 49 47 Parliament of Singapore, Second Reading of Copyright (Amendment) Bill 2014, supra note 44. 48 Article 193DDA, Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, https://sso.agc.gov.sg/Bills-Supp/16-2014/Published/20140529?DocDate=20140529#xn-. 49 Article 193DDA(1)(b), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, supra note 48. 50 Parliament of Singapore, supra note 49. 51 Id. at 50. 52 Article 193DDA(2), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). 53 David Tan, Copyright reform and what it means for your wedding photos, Straits Times, 17 September 2021, https://www.straitstimes.com/opinion/copyright-reform-and-what-it-means-for-your-wedding- photos/. 54 Peter Carstairs, supra note 30 at 284-85. See also Australia, Copyright Amendment (Online Infringement) Act (2018), Section 115A; Justin Hughes, supra note 28 at 11 & 42 (noting the similarities in the Australian and Singaporean site blocking statutes). 55 Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, supra note 48. p 57 Peter Carstairs, supra note 30 at 291-92 (stating that the primary purpose language in the Australian statute creates a high threshold inevitably leading to a site blocking regime that is specific and targeted at flagrant infringing sites). See also Parliament of Australia, Explanatory Statement for the Copyright Amendment (Online Infringement) Bill 2018, ¶ 10; Australian Department of Communication and the Arts, Regulation Impact Statement (2018), ¶83. 62 US DOJ Office of Public Affairs, U.S. Authorities Charge Owner of Most-Visited Illegal File-Sharing Website with Copyright Infringement, 20 July 2016, https://www.justice.gov/opa/pr/us-authorities-charge- owner-most-visited-illegal-file-sharing-website-copyright-infringement/. See also Nick Statt, KickassTorrents domains seized after alleged owner is arrested in Poland, 20 July 2016, The Verge, https://www.theverge.com/2016/7/20/12243592/kickass-torrents-artem-vaulin-founder-arrested-domains- seized/. A. Overview of Statutory Site Blocking in Singapore 52 Considering Singapore’s Copyright Act has historically taken inspiration from the Australian and UK’s Copyright Acts,53 it should not be surprising that the factors listed and the process of obtaining a site blocking order is similar to the Australian site blocking regime, which follows a similar two-step criterion and factor test.54 The factors stated above help determine if a site is a FIOL.55 They ensure that sites largely operated for Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 58 legitimate purposes are excluded56 and ensure that the site blocking regime is specific and targeted at sites that “flagrantly” disregard copyright.57 In essence, sites with a legitimate purpose with only incidental infringing content or piracy are not the targets of the law.58 The factors also ensure that courts consider the proportionality of site blocking and ask, particularly in the case of the last factor around traffic/access, if blocking is appropriate given the circumstances and in the public interest.59 While seemingly well crafted, the statute still does not directly address how the site blocking orders would remain flexible amidst the common online practice of redirects, dynamic IP addresses, and changing domain names. To understand how Singapore came to have an effective site blocking regime that not only clearly defines infringing online locations but also allows for responsive enforcement, we look to the case of Disney Enterprises, Inc v M1 Ltd. 60 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 1-4. 61 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 24. 56 Peter Carstairs, supra note 30 at 280, 291. 63 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 23- (“I was satisfied based on a consideration of all of the factors listed under s 193DDA (2) that the 53 websites were FIOLs. Hence, the requirement under s 193DDA(1)(b) was met. All of the 53 websites were one of the following: …(b) A streaming target website: a website which allows end-users to directly stream copyrighted content. These sites directly make available the films to the public and thereby both infringe and facilitate infringement of copyright”) 64 64 Victor Loh, supra note 31. KickassTorrents domains seized after alleged owner is arrested in Poland, 20 July 2016, The Verge, https://www.theverge.com/2016/7/20/12243592/kickass-torrents-artem-vaulin-founder-arrested-domain seized/. 58 Peter Carstairs, supra note 30 at 291. 59 Peter Carstairs, supra note 30 at 293. 62 US DOJ Office of Public Affairs, U.S. Authorities Charge Owner of Most-Visited Illegal File-Sharing Website with Copyright Infringement, 20 July 2016, https://www.justice.gov/opa/pr/us-authorities-charg owner-most-visited-illegal-file-sharing-website-copyright-infringement/. See also Nick Statt, Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 59 In his decision, Justice Lee stated that a “dynamic injunction anticipates and seeks to counteract circumventive measures that may be taken by owners or operators of the FIOLs.”65 These measures include changes to domain names, IP addresses, or URL redirects.66 To illustrate this Justice Lee pointed to how multiple domain names, URLs, and IP addresses could be associated with one FIOL and showed how over the course of the litigation some FIOLs had even changed their domain names.67 Hence, Justice Lee stated that “the dynamic injunction is necessary to ensure that the [original] injunction operated effectively to reduce further harm to the plaintiffs”.68 He went on to further state that “Without a continuing obligation to block additional domain names, URLs and/or IP addresses upon being informed of such sites, it is unlikely that there would be effective disabling of access to the 53 FIOLs”.69 In short, the court found that dynamic injunctions should be a natural extension of any existing statutory site blocking regime70 and are necessary to ensure the effectiveness of site blocking.71 This ruling also established the practice and precedent for dynamic site blocking.72 Following the ruling, plaintiffs filing for blocking orders may file additional affidavits stating why a new website or domain name falls within the scope of an existing blocking order; the additional affidavits are then forwarded to ISPs, who can either extend the blocking order or dispute the merits of extending the blocking order.73 This system creates a structure that allows for a responsive system of injunctions that can keep pace with the circumventive methods of the internet. Hence, this ruling coupled with the clarity offered by Article 193DDA of Singapore’s Copyright Act, provides for an effective site blocking regime that is both clear and responsive. As shown later, Singapore’s rather simple yet effective site blocking regime leaves much to be admired and inspired other jurisdictions in implementing effective site blocking regimes. 74 India Copyright Act, 1957, No. 14, § 55, Acts of Parliament, 1957 (India), India Copyright Act, 1957, No. 14, §§ 63, 63A, Acts of Parliament, 1957 (India). See also Arpan Banerjee, Copyright Piracy and the Indian Film Industry: A "Realist" Assessment, 34 Cardozo Arts & Ent. L.J. 609, 661 (2016). y p 66 Id. at 65, (“This would include measures taken to change the domain name, URL and/or IP address providing access to the FIOL”). 68 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 42. 65 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 35. B. Disney Enterprises, Inc v M1 Ltd: Statutory Site Blocking in Practice and Dynamic Injunctions In Disney Enterprises, Inc v M1 Ltd, Disney, Paramount, and other rights holders sued major Singaporean ISPs seeking site blocking orders under Article 193DDA.60 The plaintiff sought blocking orders concerning 53 sites or online locations which were eventually found to be “fragrantly infringing online locations” (FIOLs).61 Among these 53 sites included notorious piracy site Kickass Torrents, which has also been subject to robust US enforcement.62 Notably, by applying the factors stated above, Justice Lee found the mere making available of infringing content for streaming was sufficient to classify a site as a FIOL.63 Furthermore, because the plaintiffs in the case sought a blocking order that would require ISPs to also block later discovered domain names and IP addresses that provide access to the same FIOLs, the court effectively established the need for dynamic injunctions as a part of a robust site blocking regime.64 64 Victor Loh, supra note 31. 69 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 42. 70 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 38. III. COMING TO THE SAME CONCLUSION: COMMON LAW SITE BLOCKING IN INDIA While Indian copyright legislation does provide for civil and criminal penalties like many other advanced nations74, site blocking in India is largely an operation of common 65 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 35. p g ) 67 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 35-6 (“Owners or operators of FIOLs are able to take measures which circumvent existing blocking orders since it is possible for a single FIOL to be accessed via multiple domain names, URLs and/or IP addresses. As an illustrations of the schedule to the plaintiffs’ application sought to block the FQDNs which provide access to the FIOL known as “series9”. Multiple domain names, URLs and IP addresses were associated with the “series9” FIOL… For example, the primary domain name for the FIOL “xmovies8” has since been changed from “xmovies8.es” to “xmovies8.nu”. As the domain name “xmovies8.nu” did not exist at the time of the application and was not listed under the plaintiffs’ schedule”) p ) 68 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 42. 69 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 42. 70 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 38. 71 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 42. 72 Justin Hughes, supra note 28 at 11; See also Victor Loh, supra note 31. 73 stin Hughes, supra note 28 at 11; See also Victor Loh 73 Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 45. See also Justin Hughes, supra note 28 at 11- 12. 74 74 India Copyright Act, 1957, No. 14, § 55, Acts of Parliament, 1957 (India), India Copyright Act, 1957, No. 14, §§ 63, 63A, Acts of Parliament, 1957 (India). See also Arpan Banerjee, Copyright Piracy and the Indian Film Industry: A "Realist" Assessment, 34 Cardozo Arts & Ent. L.J. 609, 661 (2016). 74 India Copyright Act, 1957, No. 14, § 55, Acts of Parliament, 1957 (India), India Copyright Act, 1957, No. 14, §§ 63, 63A, Acts of Parliament, 1957 (India). See also Arpan Banerjee, Copyright Piracy and the Indian Film Industry: A "Realist" Assessment, 34 Cardozo Arts & Ent. L.J. 609, 661 (2016). 75 Justin Hughes, supra note 28 at 10. 76 FE Bureau, India Box Office collections: Regional cinema led by Telugu, Tamil movies overtakes Bollywood, Financial Express, July 11, 2020, https://www.financialexpress.com/entertainment/bollywoods-big-but-regional-cinema-is-also-raking-in- the-moolah/2020134/; PTI, Indian film industry's gross box office earnings may reach $3.7 billion by 2020: Report, DNA INDIA, September 26, 2016, https://www.dnaindia.com/entertainment/report-indian- film-industry-s-gross-box-office-earnings-may-reach-37-billion-by-2020-report-2258789/. 77 Ni l C t 36 III. COMING TO THE SAME CONCLUSION: COMMON LAW SITE BLOCKING IN INDIA Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 60 law.75 India possesses a billion-dollar film industry76 that is dependent on the protection of intellectual property to grow and remain profitable.77 The existence of a site blocking regime has been recognized as an area of success for copyright law in India, where there seems to be a prevalent culture of online piracy.78 Indian courts have been issuing site blocking orders for years and become a part of addressing online piracy.79 However, unlike Singapore, India did not arrive at its site blocking regime instantly. It was only through numerous judgments did India largely feel its way towards clear legal standards which identify infringing online locations and subsequently responsive enforcement mechanisms. Throughout this process, various Indian courts have had to address concerns around site blocking.80 Therefore, India’s experience in common law site blocking can prove instructive in addressing concerns around site blocking and understanding what makes an effective site blocking regime. 79 See, e.g., Reliance v. Jyoti Cable, (2011) Civil Suit No. 1724 of 2011 (Del. H.C.) (Jul. 20, 2011) (India); Fox v. Macpuler, (2015) Civil Suit No. 2066 of 2011 (Delhi H.C.) (May 14, 2015) (India), Vodafone v. R.K. Productions (2013) 54 P.T.C. (Mad. H.C.) 149, (India), Yash Raj Films v. Bharat Sanchar Nigam, Civil Suit No. 692 of 2016 (Bom. H.C. July 4, 2016); UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018 (Del. H.C.) (Apr. 10, 2019) (India). See Also Javed Anwer, supra note 20; Anupam Saxena, supra note 20. ) ( ) ( ) 85 Peter Carstairs, supra note 30 at 286. 78 Arpan Banerjee, supra note 74 at 609, 672. 77 Nigel Cory, supra note 36. 75 Justin Hughes, supra note 28 at 10. 76 FE Bureau, India Box Office collections: Regional cinema led by Telugu, Tamil movies overtakes Bollywood, Financial Express, July 11, 2020, https://www.financialexpress.com/entertainment/bollywoods-big-but-regional-cinema-is-also-raking-in- the-moolah/2020134/; PTI, Indian film industry's gross box office earnings may reach $3.7 billion by 2020: Report, DNA INDIA, September 26, 2016, https://www.dnaindia.com/entertainment/report-indian- film-industry-s-gross-box-office-earnings-may-reach-37-billion-by-2020-report-2258789/. 77 Nigel Cory, supra note 36. 78 Arpan Banerjee, supra note 74 at 609, 672. 79 See, e.g., Reliance v. Jyoti Cable, (2011) Civil Suit No. 1724 of 2011 (Del. H.C.) (Jul. 20, 2011) (India); Fox v. Macpuler, (2015) Civil Suit No. 2066 of 2011 (Delhi H.C.) (May 14, 2015) (India), Vodafone v. R.K. Productions (2013) 54 P.T.C. (Mad. H.C.) 149, (India), Yash Raj Films v. Bharat Sanchar Nigam, Civil Suit No. 692 of 2016 (Bom. H.C. July 4, 2016); UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018 (Del. H.C.) (Apr. 10, 2019) (India). See Also Javed Anwer, supra note 20; Anupam Saxena, supra note 20. 80 See e.g UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 21 (concerns around the proportionality of site blocking), 50 (concerns that online infringement should be treated differently than physical infringement), 55 (concerns around maintaining a free and open internet) (Del. H.C.) (Apr. 10, 2019) (India). 81 Arpan Banerjee, supra note 74 at 609, 669. 82 Arpan Banerjee, supra note 74 at 609, 666. 83 Justin Hughes, supra note 28 at 10. 84 Arpan Banerjee, supra note 74 at 609, 667; See also Kunal Dua, Confusion Reigns as Indian ISPs Block Vimeo, Torrent Websites, NDTV (May 17, 2012), http://gadgets.ndtv.com/internet/news/confusion- reigns-as-indian-isps-block-vimeo-torrent-websites-223340; Nikhil Pawa, Update: Files Sharing Sites Blocked In India Because Reliance BIG Pictures Got A Court Order, MEDIANAMA (July 21, 2011), http://www.medianama.com/2011/07/223-files-sharing-sites-blocked-in-india-because-reliancebig- picturesgot-a-court-order; See also e.g., Reliance v. Jyoti Cable, (2011) Civil Suit No. 1724 of 2011 (Del. H.C.) (Jul. 20, 2011) (India) 85 Peter Carstairs, supra note 30 at 286. 75 Justin Hughes, supra note 28 at 10. A. Overview of Site Blocking in India Website blocking orders from Indian courts have become a common and reliable means of copyright enforcement.81 In fact in certain industries, like the film industry, there has been a noticeable trend towards pre-emptive site blocking injunctions against infringing sites since 2011.82 Because India’s common law site blocking regime was largely developed through various judgements,83 the evolution of India’s site blocking regime has been mostly a patchwork process. Initially, the broad wording of some site blocking orders led to ISPs blocking entire websites84 and some “over-blocking”.85 This was eventually corrected by another case where the court limited site blocking orders to , p ; p , p 80 See e.g UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 21 (concerns around the proportionality of site blocking), 50 (concerns that online infringement should be treated differently than physical infringement), 55 (concerns around maintaining a free and open internet) (Del. H.C.) (Apr. 10, 2019) (India). 81 Arpan Banerjee, supra note 74 at 609, 669. 82 Arpan Banerjee, supra note 74 at 609, 666. 83 Justin Hughes, supra note 28 at 10. g p 84 Arpan Banerjee, supra note 74 at 609, 667; See also Kunal Dua, Confusion Reigns as Indian ISPs Block Vimeo, Torrent Websites, NDTV (May 17, 2012), http://gadgets.ndtv.com/internet/news/confusion- reigns-as-indian-isps-block-vimeo-torrent-websites-223340; Nikhil Pawa, Update: Files Sharing Sites Blocked In India Because Reliance BIG Pictures Got A Court Order, MEDIANAMA (July 21, 2011), http://www.medianama.com/2011/07/223-files-sharing-sites-blocked-in-india-because-reliancebig- picturesgot a court order; See also e g Reliance v Jyoti Cable (2011) Civil Suit No 1724 of 2011 (Del 84 Arpan Banerjee, supra note 74 at 609, 667; See also Kunal Dua, Confusion Reigns as Indian ISPs Block Vimeo, Torrent Websites, NDTV (May 17, 2012), http://gadgets.ndtv.com/internet/news/confusio reigns-as-indian-isps-block-vimeo-torrent-websites-223340; Nikhil Pawa, Update: Files Sharing Sites Arpan Banerjee, supra note 74 at 609, 667; See also Kunal Dua, Confusion Reigns as Indian ISPs Block Vimeo, Torrent Websites, NDTV (May 17, 2012), http://gadgets.ndtv.com/internet/news/confusion- reigns-as-indian-isps-block-vimeo-torrent-websites-223340; Nikhil Pawa, Update: Files Sharing Sites Blocked In India Because Reliance BIG Pictures Got A Court Order, MEDIANAMA (July 21, 2011), http://www.medianama.com/2011/07/223-files-sharing-sites-blocked-in-india-because-reliancebig- picturesgot-a-court-order; See also e.g., Reliance v. Jyoti Cable, (2011) Civil Suit No. 1724 of 2011 (Del. H.C.) (Jul. 20, 2011) (India) 85 Blocked In India Because Reliance BIG Pictures Got A Court Order, MEDIANAMA (July 21, 2011), http://www.medianama.com/2011/07/223-files-sharing-sites-blocked-in-india-because-reliancebig- picturesgot-a-court-order; See also e.g., Reliance v. Jyoti Cable, (2011) Civil Suit No. 1724 of 2011 (Del. H.C.) (Jul. 83 Justin Hughes, supra note 28 at 10. 82 Arpan Banerjee, supra note 74 at 609, 666. p g H.C.) (Jul. 20, 2011) (India) 86 Vodafone v. R.K. Productions (2013) 54 P.T.C. (Mad. H.C.) 149, ¶ 4 (India) (quoting an earlier order where the court had stated that “the interim injunction is granted only in respect of a particular URL where the infringing movie is kept and not in respect of the entire website.”). 8 A. Overview of Site Blocking in India 20, 2011) (India) 85 Peter Carstairs, supra note 30 at 286. Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 61 only the URLs that were specifically hosting infringing content.86 However, by narrowly tailoring site blocking orders to specific URLs, it became difficult to enforce them when infringers simply changed their URL or domain names.87 The sometimes overbroad, inadequate, or contradictory site blocking orders, and corollary international developments in site blocking eventually led to the case of UTV Software Communication Ltd. V 1337X.88 94 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 57 (Del. H.C.) (Apr. 10, 2019) (India). 5 95 Peter Carstairs, supra note 30 at 291-93. g g p p ) 87 Arpan Banerjee, supra note 74 at 609, 668-9; See also Deity v. Star, Review Petition in First Appeal B. Utv Software Communication Ltd. ... v 1337X: Common Law Site Blocking and Dynamic Injunctions 92 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 55 (Del. H.C.) (Apr. 10, 2019) (India) (“just as supporting bans on the import of ivory or cross-border human trafficking does not make one a protectionist, supporting website blocking for sites dedicated to piracy does not make one an opponent of a free and open Internet. Consequently, this Court is of the opinion that advocating limits on accessing illegal content online does not violate open Internet principles.”). 93 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 53 (Del. H.C.) (Apr. 10, 2019) (India) (“should an infringer of the copyright on the Internet be treated differently from an infringer in the physical world? If the view of the aforesaid Internet exceptionalists school of thought is accepted, then all infringers would shift to the e-world and claim immunity! A world without law is a lawless world. In fact, this Court is of the view that there is no logical reason why a crime in the physical world is not a crime in the digital world especially when the Copyright Act does not make any such distinction”). 92 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 55 (Del. H.C.) (Apr. 10, 2019) (India) (“just as supporting bans on the import of ivory or cross-border human trafficking does not make one a protectionist, supporting website blocking for sites dedicated to piracy does not make one an opponent of a free and open Internet. Consequently, this Court is of the opinion that advocating limits on accessing illegal content online does not violate open Internet principles.”). 92 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 55 (Del. H.C.) (Apr. 10, 2019) (India) (“just as supporting bans on the import of ivory or cross-border human trafficking does not make one a protectionist, supporting website blocking for sites dedicated to piracy does not make one an opponent of a free and open Internet. Consequently, this Court is of the opinion that advocating limits on accessing illegal content online does not violate open Internet principles.”). limits on accessing illegal content online does not violate open Internet principles. ). 93 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 53 (Del. H.C.) (Apr. Arpan Banerjee, supra note 74 at 609, 668 9; See also Deity v. Star, Review Petition in First Appeal Order No. 57 of 2015, ¶ 14 (Del. H.C. July 29, 2016), available at B. Utv Software Communication Ltd. ... v 1337X: Common Law Site Blocking and Dynamic Injunctions B. Utv Software Communication Ltd. ... v 1337X: Common Law Site Blocking and Dynamic Injunctions In the 2019 case of UTV Software Communication Ltd. v. 1337X, companies that were in the business of creating content, producing, and distributing films in India sued a host of defendants, including infringing websites, ISPs, and relevant Indian government agencies.89The plaintiffs sought an order directing ISPs to block access to a number of infringing websites like “1337.to” and “yts.am”.90 These websites were eventually found to be “rogue sites” or FIOLs.91 Addressing the “threat” a site blocking regime poses to internet freedom, Justice Manmohan stated that a site blocking regime was not inconsistent with a free and open internet.92 He also further iterated the need for the law to address online infringement no differently from offline infringement.93 The opinion then proceeded to distinguish between accidental and intentional piracy.94 Doing so requires effectively defining the scope of what a “rogue website” or “flagrantly infringing online location” (FIOLs) is. 95 This involves considering the proportionality of granting a site blocking order and creating a means of evaluating online http://lobis.nic.in/ddir/dhc/PNJ/judgement/29 07 2016/PNJ29072016REVIEWPET1312016.pdf/. 88 The case cites and points to various international developments in site blocking that it uses to decide the issue. See UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 11, 88- 93, 97-98 (Del. H.C.) (Apr. 10, 2019) (India). http://lobis.nic.in/ddir/dhc/PNJ/judgement/29 07 2016/PNJ29072016REVIEWPET1312016.pdf/. 88 The case cites and points to various international developments in site blocking that it uses to decide the issue. See UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 11, 88- 93, 97-98 (Del. H.C.) (Apr. 10, 2019) (India). , ( ) ( p , ) ( ) 89 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 2-4 (Del. H.C.) (Apr. 10, 2019) (India). ( ) ( p ) ( ) 89 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 2-4 (Del. H.C.) (Apr. 10, 2019) (India). ( p ) ( ) 90 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 2-4 (Del. H.C.) (Apr. 10, 2019) (India). 91 Ni l C 36 90 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 2-4 (Del. H.C.) (Apr. 10, 2019) (India). (Apr. 10, 2019) (India). 91 Nigel Cory, supra note 36. 86 Vodafone v. R.K. Productions (2013) 54 P.T.C. (Mad. H.C.) 149, ¶ 4 (India) (quoting an earlier order where the court had stated that “the interim injunction is granted only in respect of a particular URL where the infringing movie is kept and not in respect of the entire website.”). 87 Arpan Banerjee, supra note 74 at 609, 668-9; See also Deity v. Star, Review Petition in First Appeal Order No. 57 of 2015, ¶ 14 (Del. H.C. July 29, 2016), available at http://lobis.nic.in/ddir/dhc/PNJ/judgement/29-07-2016/PNJ29072016REVIEWPET1312016.pdf/. 88 The case cites and points to various international developments in site blocking that it uses to decide the issue. See UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 11, 88- 93, 97-98 (Del. H.C.) (Apr. 10, 2019) (India). 89 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 2-4 (Del. H.C.) (Apr 10 2019) (India) p j g p 88 The case cites and points to various international developments in site blocking that it uses to decide the issue. See UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 11, 8 93, 97-98 (Del. H.C.) (Apr. 10, 2019) (India). Arpan Banerjee, supra note 74 at 609, 668-9; See also Deity v. Star, Review Petition in First Appeal der No. 57 of 2015, ¶ 14 (Del. H.C. July 29, 2016), available at Order No. 57 of 2015, ¶ 14 (Del. H.C. July 29, 2016), available at http://lobis.nic.in/ddir/dhc/PNJ/judgement/29-07-2016/PNJ29072016REVIEWPET1312016.pdf/. B. Utv Software Communication Ltd. ... v 1337X: Common Law Site Blocking and Dynamic Injunctions 10, 2019) (India) (“should an infringer of the copyright on the Internet be treated differently from an infringer in the physical world? If the view of the aforesaid Internet exceptionalists school of thought is accepted, then all infringers would shift to the e-world and claim immunity! A world without law is a lawless world. In fact, this Court is of the view that there is no logical reason why a crime in the physical world is not a crime in the digital world especially when the Copyright Act does not make any such distinction”). 93 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 53 (Del. H.C.) (Apr. 10, 2019) (India) (“should an infringer of the copyright on the Internet be treated differently from an infringer in the physical world? If the view of the aforesaid Internet exceptionalists school of thought is accepted, then all infringers would shift to the e-world and claim immunity! A world without law is a lawless world. In fact, this Court is of the view that there is no logical reason why a crime in the physical world is not a crime in the digital world especially when the Copyright Act does not make any such distinction”). 93 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 53 (Del. H.C.) (Apr. 10, 2019) (India) (“should an infringer of the copyright on the Internet be treated differently from an infringer in the physical world? If the view of the aforesaid Internet exceptionalists school of thought is accepted, then all infringers would shift to the e-world and claim immunity! A world without law is a lawless world. In fact, this Court is of the view that there is no logical reason why a crime in the physical world is not a crime in the digital world especially when the Copyright Act does not make any such distinction”). Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 62 behavior.96 After an exhaustive review of site blocking regimes internationally, including Singapore,97 the court eventually arrived at a non-exhaustive list of factors to determine if a site is “rogue” or a FIOL. 98 The list of factors are presented below alongside Singapore’s aforementioned statutory factors: UTX v. 1337X Factors99 Singapore Statutory Factors100 (a) Primary Purpose of the website 1. 96 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶¶ 57, 75-82 (Del. H.C.) (Apr. 10, 2019) (India). 97 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 11 (Del. H.C.) (Apr. 10, 2019) (India). See also Article 193DDA, Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). See also Parliament of Singapore, Explanatory Statement for the Copyright (Amendment) Bill 2014, supra note 48; Australia, Copyright Amendment (Online Infringement) Act (2018), Section 115A; Justin Hughes, supra note 28 at 9-11. 98 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 59 (Del. H.C.) (Apr. 10, 2019) (India). 99 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 59 (Del. H.C.) (Apr. 10, 2019) (India). 100 Article 193DDA(2), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006). B. Utv Software Communication Ltd. ... v 1337X: Common Law Site Blocking and Dynamic Injunctions Primary Purpose of the website is to commit or facilitate copyright infringement (b) Flagrancy of infringement or facilitation of infringement (c) There is no traceable or personal detail of the person who registered the website (d) Silence or Inaction by the website after receipt of take down notices for copyright infringement (e) The website makes available or contains directories, indexes or categorizes means to infringe or facilitates infringement 2. The website makes available or contains directories, indexes or categorizes means to infringe or facilitates infringement (f) The owner or operator of the site displays a disregard for copyright generally 3. The owner or operator of the site displays a disregard for copyright generally (g) Access to the website has been disabled by orders from other jurisdictions for copyright infringement 4. Access to the website has been disabled by orders from other jurisdictions for copyright infringement (h) The website contains guides or instructions to circumvent measures or 5. The website contains guides or instructions to circumvent measures or Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 63 orders of any court that blocks the site due to copyright infringement orders of any court that blocks the site due to copyright infringement (i) Volume of traffic or frequency of access to the website 6. Volume of traffic or frequency of access to the website (j) Any other relevant matter The similarity of these factors reflects a consensus around clear and effective standards which define a “rogue website” or FIOL. Furthermore, the factors have also been regarded in other jurisdictions as a means to ensure that sites largely operated for legitimate purposes are excluded and ensure that a site blocking regime is specifically targeted at sites that “flagrantly” disregard copyright. 101 They reflect a basic understanding that one of the first issues when designing a site blocking regime is to ensure that it only applies to FIOLs or “rogue websites” and to avoid the practice of “over- blocking”. One of the best ways to do that is to have a proportionate criterion in making that determination. 101 Peter Carstairs, supra note 30 at 291-92 (stating that the primary purpose language in the Australian statute creates a high threshold inevitably leading to a site blocking regime that is specific and targeted at flagrant infringing sites). See also Article 193DDA(2), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006); Parliament of Australia, Explanatory Statement for the Copyright Amendment (Online Infringement) Bill 2018, ¶ 10; Australian Department of Communication and the Arts, Regulation Impact Statement (2018), ¶83. Compare MGM Studios, Inc. v. Grokster, Ltd., 545 U.S. 913, 919 (2005) (“We hold that one who distributes a device with the object of promoting its use to infringe copyright, as shown by clear expression or other affirmative steps taken to foster infringement, is liable for the resulting acts of infringement by third parties”). 102 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 94-95 (Del. H.C.) (Apr. 10, 2019) (India). 103 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 100 (Del. H.C.) (Apr. 10, 2019) (India). See also Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 35, 38, 42 (Singapore H.C.) (Singapore) 104 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 101 (Del. H.C.) (Apr. 10, 2019) (India); See also Disney Enterprise v. Ml Ltd., (2018) SGHC 206 at ¶ 45 (Singapore H.C.) (Singapore) 105 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 61 (Del. H.C.) (Apr. 10, 2019) (India). 101 Peter Carstairs, supra note 30 at 291-92 (stating that the primary purpose language in the Australian statute creates a high threshold inevitably leading to a site blocking regime that is specific and targeted at flagrant infringing sites). See also Article 193DDA(2), Singapore Copyright Act (Chapter 63 of Singapore Laws) (revised 31st January 2006); Parliament of Australia, Explanatory Statement for the Copyright Amendment (Online Infringement) Bill 2018, ¶ 10; Australian Department of Communication and the Arts, Regulation Impact Statement (2018), ¶83. Compare MGM Studios, Inc. v. Grokster, Ltd., 545 U.S. 913, 919 (2005) (“We hold that one who distributes a device with the object of promoting its use to infringe copyright, as shown by clear expression or other affirmative steps taken to foster infringement, is liable for the resulting acts of infringement by third parties”). B. Utv Software Communication Ltd. ... v 1337X: Common Law Site Blocking and Dynamic Injunctions Justice Manmohan Singh then also addresses the question of how to make site blocking effective against the practice of “hydra headed rogue websites” resurfacing under mirror websites, changed domain names, or dynamic IP addresses.102 Explicitly drawing lessons from the Singapore High Court’s judgment in Disney Enterprises, Inc v M1 Ltd, the court similarly established a practice of dynamic injunctions to ensure that site blocking orders were effective. 103 Unsurprisingly, the court implemented a similar procedure for the administration of dynamic injunctions allowing for plaintiffs to submit affidavits asserting with evidence that a website is merely a mirror, redirect, or changed IP address of an already blocked site.104 The reason for dynamic injunctions is a natural extension of the court’s qualitative determination of “rogue websites”. When considering a blocking order against a site, a court evaluates the site’s primary purpose qualitatively to determine if the actions of the site are infringing.105 This is different from considering it quantitatively, which will limit the court to considering specific URLs or domain names in isolation and blocking orders will lack the breadth necessary to combat the evasive Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 64 practices of “rogue websites”.106 Indeed, the court also suggests that a site changing URLs and domain names to evade a court order in effect shows that the site is “rogue” because it displays a blatant disregard for copyright and the site blocking order.107 Therefore, the court in India has found dynamic injunctions as a natural approach towards maintaining the effectiveness of a site blocking regime. 106 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 63 (Del. H.C.) (Apr. 10, 2019) (India). 107 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 67 (Del. H.C.) (Apr. 10, 2019) (India). 108 Carol V. Rose, The "New" Law and Development Movement in the Post-Cold War Era: A Vietnam Case Study, Law & Society Review, Vol. 32, No. 1. 93, 96-100, (1998). 109 BUI NGOC SON, Contextualizing the Global Constitution-Making Process: The Case of Vietnam, 64 Am. J. Comp. L. 931, 945-47 (2016). 110 The World Bank, Key Features of Common Law or Civil Law Systems, https://ppp.worldbank.org/public-private-partnership/legislation-regulation/framework-assessment/legal- systems/common-vs-civil-law 111 Thomas H. Reynolds (1998). "Introduction to Foreign and Comparative Law". In Rehberg, Jeanne; Popa, Radu D (eds.). Accidental Tourist on the New Frontier: An Introductory Guide to Global Legal Research, 47& 58. 112 Alain Levasseur, Code Napoleon or Code Portalis?, 43 Tul. L. Rev. 762, 769 (1969) (The role of legislation is to set, by taking a broad approach, the general propositions of the law, to establish principles which will be fertile in application, and not to get down to the details.) 113 Most of the EU countries named including France, Germany, and Greece are civil law jurisdictions that have issued site blocking injunctions, see Justin Hughes, supra note 28 at 7. 114 Directive 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the Harmonisation of Certain Aspects of Copyright and Related Rights in the Information Society, art. 8, 2001 O.J. (L 167); Justin Hughes, supra note 28 at 7. 111 Thomas H. Reynolds (1998). "Introduction to Foreign and Comparative Law". In Rehberg, Jeanne; Popa, Radu D (eds.). Accidental Tourist on the New Frontier: An Introductory Guide to Global Legal Research, 47& 58. 106 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 63 (Del. H.C.) (Apr. 10, 2019) (India). 107 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 67 (Del. H.C.) (Apr. 10, 2019) (India). 108 Carol V. Rose, The "New" Law and Development Movement in the Post-Cold War Era: A Vietnam Case Study, Law & Society Review, Vol. 32, No. 1. 93, 96-100, (1998). 109 BUI NGOC SON, Contextualizing the Global Constitution-Making Process: The Case of Vietnam, 64 Am. J. Comp. L. 931, 945-47 (2016). 110 The World Bank, Key Features of Common Law or Civil Law Systems, https://ppp.worldbank.org/public-private-partnership/legislation-regulation/framework-assessment/legal- systems/common-vs-civil-law 111 Thomas H. Reynolds (1998). "Introduction to Foreign and Comparative Law". In Rehberg, Jeanne; Popa, Radu D (eds.). Accidental Tourist on the New Frontier: An Introductory Guide to Global Legal Research, 47& 58. 112 Alain Levasseur Code Napoleon or Code Portalis? 43 Tul L Rev 762 769 (1969) (The role of 112 Alain Levasseur, Code Napoleon or Code Portalis?, 43 Tul. L. Rev. 762, 769 (1969) (The role of legislation is to set, by taking a broad approach, the general propositions of the law, to establish principles which will be fertile in application, and not to get down to the details.) https://ppp.worldbank.org/public-private-partnership/legislation-regulation/framework-assessment/legal- systems/common-vs-civil-law 106 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 63 (Del. H.C.) (Apr. 10, 2019) (India). 107 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 67 (Del. H.C.) (Apr. 10, 2019) (India). 108 Carol V. Rose, The "New" Law and Development Movement in the Post-Cold War Era: A Vietnam Case Study, Law & Society Review, Vol. 32, No. 1. 93, 96-100, (1998). 109 BUI NGOC SON, Contextualizing the Global Constitution-Making Process: The Case of Vietnam, 64 Am. J. Comp. L. 931, 945-47 (2016). 110 The World Bank, Key Features of Common Law or Civil Law Systems, IV. A CIVIL LAW APPROACH: SITE BLOCKING IN FRANCE It is important to note that unlike Singapore, India, or the United States, Vietnam is a code-based civil law system rather than a common law system. This is largely a feature of Vietnam’s colonial history and geography. As a former French colony and communist country, it should not be surprising that Vietnam’s legal system is strongly influenced by the historical French code system, Chinese law, and later communist Soviet law.108 In fact, in its most recent constitutional redrafting, both French and Chinese law were used in various areas as points of reference.109 In broad strokes, civil or code law jurisdictions regard the legal code as the primary source of law.110 The cases that arise out of the code are reviewed in isolation on a case-by-case basis without any precedential value.111 The role of civil code legislation is to be as broad as possible to anticipate the wide variety of potential scenarios.112 While the differences between Vietnam’s code-based system and the common law jurisdictions covered above seem like an obstacle to implementing site blocking in Vietnam, other code-based nations’ experiences in site blocking show it is possible.113 Because of its historical influence on Vietnamese law, it is helpful to also look at how France’s code-based civil law system has implemented an effective site blocking regime, based on similar principles of clear legal standards targeting online piracy and responsive enforcement measures. Within the EU, site blocking begins with the 2001 Information Society Directive. Article 8(3) of the directive provides for the ability for copyright owners to obtain “no fault” injunctions against ISPs to block pirated websites.114 The French legislature has codified this in the French intellectual property code and its laws for the digital 112 Alain Levasseur, Code Napoleon or Code Portalis?, 43 Tul. L. Rev. 762, 769 (1969) (The role of legislation is to set, by taking a broad approach, the general propositions of the law, to establish principle which will be fertile in application, and not to get down to the details.) pp g 113 Most of the EU countries named including France, Germany, and Greece are civil law jurisdictions that have issued site blocking injunctions, see Justin Hughes, supra note 28 at 7. g j g p 114 Directive 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the Harmonisation of Certain Aspects of Copyright and Related Rights in the Information Society, art. IV. A CIVIL LAW APPROACH: SITE BLOCKING IN FRANCE 8, 2001 O.J. (L 167); Justin Hughes, supra note 28 at 7. 114 Directive 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the Harmonisation of Certain Aspects of Copyright and Related Rights in the Information Society, art. 8, 2001 O.J. (L 167); Justin Hughes, supra note 28 at 7. Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 65 economy.115 In applying these laws, French courts have issued site blocking orders not only in cases of copyright infringement116 but also trademark issues.117 Furthermore, France has recently been considering administrative measures implementing site blocking like a blacklist of piracy sites.118 Various European courts have also blocked sites in cases where infringing link without the right holder’s permission was hosted119 and in cases where video sites were found to be “fully dedicated or virtually dedicated” to copyright infringement.120 The relevant legislation and cases suggest that even in a code-based system, it is possible to have a site blocking regime that is clear and responsive. French civil courts have also been open to ordering dynamic injunctions against ISPs to address evasive measures taken by some infringing websites.121 In a case brought by various scientific publishing companies against ISPs seeking dynamic injunctions against infringing sites, the French court applying the aforementioned laws found dynamic injunctions appropriate.122 This decision was based on similar concerns surrounding the evasive measures taken by some infringing sites in changing domain names and access paths.123 Similar decisions can be found in many other EU member states with civil law 115 Article 6 I 8° of the Law for confidence in the digital economy, (“The judicial authority may prescribe, in summary proceedings or on application, to any person mentioned in 2 (host) or, failing that, to any person mentioned in 1 (ISP), any measures to prevent damage or to put an end to damage caused by the content of a communication service to the public online”); Article L. IV. A CIVIL LAW APPROACH: SITE BLOCKING IN FRANCE 336-2 of the French Intellectual Property Code, (“In the event of an infringement of copyright or a related right caused by the content of a communication to the public online service, the president of the judicial court ruling according to the accelerated procedure on the merits may order, at the request of the owner of rights in protected works and objects, their successors in title, collective management bodies governed by Title II of Book III or professional defense bodies referred to in Article L.”. 331-1, any measures to prevent or stop such infringement of copyright or a related right, against any person likely to contribute to remedying it. The request may also be made by the Centre national du cinéma et de l’image animée”). See also Alya Bloum, French Supreme Court: Internet intermediaries must pay for blocking measures against illegal streaming websites, August 3, 2017, Hogan Lovells Global Media and Communications Watch, g g https://www.hlmediacomms.com/2017/08/03/french-supreme-court-internet-intermediaries-must-pay-for- blocking-measures-against-illegal-streaming-websites/. g g https://www.hlmediacomms.com/2017/08/03/french-supreme-court-internet-intermediaries-must-pay-for- blocking-measures-against-illegal-streaming-websites/. 116 Clara Hainsdorf & Bertrand Liard, French Courts Ordered to Block and Delist 16 Streaming Websites, JD SUPRA, Jan. 13, 2014, https://www.jdsupra.com/legalnews/french-courts-ordered-to-block-and-delis- 08092/. 117 Anne-Marie Pecoraro, Rodolphe Boissau, Trademark law: counterfeiting. A look back at two trademark court decisions allowing site-blocking of massively infringing sites in France., Dec. 11, 2020 17 Anne-Marie Pecoraro, Rodolphe Boissau, Trademark law: counterfeiting. A look back at two , p , g trademark court decisions allowing site-blocking of massively infringing sites in France., Dec. 11, 2020, https://www.uggc.com/en/trademark-law-counterfeiting-a-look-back-at-two-trademark-court-decisions- allowing-site-blocking-of-massively-infringing-sites-in-france/. 118 Nigel Cory, supra note 6; Ernesto Van der Sar, French Minister of Culture Calls For Pirate Streaming Blacklist, April 23, 2018, https://torrentfreak.com/french-minister-of-culture-calls-for-pirate-streaming- blacklist-180423/. 119 UPC Telekabel Wien v. Constantin Film Verleih, Court of Justice of the European Union, Case C- 314/12, ¶ 25, March 27, 2014 (“it must be stated that an act of making protected subject-matter available to the public on a website without the rightholders’ consent infringes copyright and related rights”); See also UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 9 (Del. H.C.) (Apr. 10, 2019) (India). 121 European Union Intellectual Property Office, supra note 26 at 35 (“Dynamic blocking injunctions are available – and have been granted – in most SMS, including Denmark, France, Ireland, Italy, the Netherlands, Spain, Sweden, and the UK”). It could be argued that France imposes a heavier burden on ISPs to police evasive measures undertaken by infringing sites through dynamic injunctions. 122 Tribunal de grande instance de Paris, 7 March 2019, 18/14194 (France). See also, supra note 26 at 77 78. 123 Id. at 122. ( p ) ( ) 120 Clara Hainsdorf & Bertrand Liard, supra note 116. V. CLEARING THE FOG: THE NEED FOR EFFECTIVE SITE BLOCKING IN VIETNAM Vietnam has a rapidly growing online marketplace and a vibrant online infringement culture.125 Sixty percent of all internet consumers in Vietnam openly admit to streaming on piracy sites.126 While there are laws that attempt to address the problem of online piracy,127 the reality is that Vietnam’s current framework allows for a fair amount of online piracy.128 However, it is long overdue for Vietnam to address these issues. 129With Vietnam’s accession to both the CPTPP130 in 2018 and the WCT at the end of 2021,131 this has not been more pressing. The previously discussed cases and jurisdictions show that Vietnam has plenty of good examples to follow in establishing an effective site blocking regime with clear legal standards targeting online piracy and responsive enforcement measures to keep up with infringers. They also provide significant guidance on how to consider the potential issues that arise when establishing an effective site blocking regime. However, a lack of clarity and efficacy in Vietnam’s intellectual property laws as well as structural issues in Vietnam’s economic system pose challenges to the direct application of the previously discussed models. IV. A CIVIL LAW APPROACH: SITE BLOCKING IN FRANCE See Supreme Court of France (Cour De Cassation), 6 July 2017, 16-17.217 (France); Tribunal de grande instance de Paris, 23 May 2019, RG 19/001744 (France). 121 European Union Intellectual Property Office, supra note 26 at 35 (“Dynamic blocking injunctions are available – and have been granted – in most SMS, including Denmark, France, Ireland, Italy, the Netherlands, Spain, Sweden, and the UK”). It could be argued that France imposes a heavier burden on ISPs to police evasive measures undertaken by infringing sites through dynamic injunctions. See Supreme Court of France (Cour De Cassation), 6 July 2017, 16-17.217 (France); Tribunal de grande instance de Paris, 23 May 2019, RG 19/001744 (France). , y , ( ) 122 Tribunal de grande instance de Paris, 7 March 2019, 18/14194 (France). See also, supra note 26 at 77- 78. 123 Id. at 122. y ( ) 122 Tribunal de grande instance de Paris, 7 March 2019, 18/14194 (France). See also, supra note 26 at 77- 78. 123 Id. at 122. Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 66 systems.124 Therefore, when taken together, the French experience and that of many other EU civil law regimes show that a clear and effective site blocking regime is possible in a civil law jurisdiction. Vietnam should be no exception. p 126 New survey shows Vietnam among highest in online piracy in Southeast Asia., supra note 7. 127 See Law No 50/2005/QH11 of November 29 2005 on Intellectual Property; Joint Circular No 124 European Union Intellectual Property Office, Study on Dynamic Blocking Injunctions in the European Union, supra note 26 at 110-132; See also e.g. Maritime and Commercial Court (Sø- & Handelsretten), Case Number A-51-17, 21 February 2018, Fritz Hansen A/S and Others (represented by Rettighedsalliancen SMF.) v Telia Danmark A/S and Dominidesign Furniture LTD (Denmark); Sony Music Entertainment (Ireland) & Ors v UPC; Communications Ireland Limited [2016] IECA 231 (Ireland); Amsterdam Court of Appeal, Brein v. Ziggo and XS4ALL, 2 June 2020, ECLI:NL:GHAMS:2020:1421 (Netherlands); Court of Milan, Ordinanza N.42163/2019 R.G. of 5 October , y , g ( y) 125 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 108. y y 2021. See CPTPP, supra note 17 at 18-5, 18-64. 124 European Union Intellectual Property Office, Study on Dynamic Blocking Injunctions in the European Union, supra note 26 at 110-132; See also e.g. Maritime and Commercial Court (Sø- & Handelsretten), Case Number A-51-17, 21 February 2018, Fritz Hansen A/S and Others (represented by Rettighedsalliancen SMF.) v Telia Danmark A/S and Dominidesign Furniture LTD (Denmark); Sony Music Entertainment (Ireland) & Ors v UPC; Communications Ireland Limited [2016] IECA 231 (Ireland); Amsterdam Court of Appeal, Brein v. Ziggo and XS4ALL, 2 June 2020, ECLI:NL:GHAMS:2020:1421 (Netherlands); Court of Milan, Ordinanza N.42163/2019 R.G. of 5 October 2020, Sky Italia, Lega Serie A V Cloudflare and Others (Italy). 125 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 108. 126 New survey shows Vietnam among highest in online piracy in Southeast Asia., supra note 7. 127 See Law No. 50/2005/QH11 of November 29, 2005, on Intellectual Property; Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127. 128 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106. 129 See supra where Vietnam is failing their TRIPS obligations. See also IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 110. 130 Government of New Zealand, supra note 16. 131 In short, when Vietnam signed the CPTPP, it took out a reservation on accession to the WCT for three years. This means that Vietnam was allowed to sign the treaty as long as it acceded to the WCT within 3 years. Since the treaty went into effect on December 31, 2018, three years from then is December 31, 2021. See CPTPP, supra note 17 at 18-5, 18-64. , p In short, when Vietnam signed the CPTPP, it took out a reservation on accession to the WCT for three ars. This means that Vietnam was allowed to sign the treaty as long as it acceded to the WCT within 3 Q , , p y; 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communication and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127. y g y g years. Since the treaty went into effect on December 31, 2018, three years from then is December 31, 128 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106. This means that Vietnam was allowed to sign the treaty as long as it acceded to the WCT within 3 Since the treaty went into effect on December 31, 2018, three years from then is December 31, See CPTPP supra note 17 at 18-5 18-64 years. This means that Vietnam was allowed to sign the treaty as long as it acceded to the WCT within 3 years. Since the treaty went into effect on December 31, 2018, three years from then is December 31, ECLI:NL:GHAMS:2020:1421 (Netherlands); Court of Milan, Ordinanza N.42163/2019 R.G. of 5 Octob 2020, Sky Italia, Lega Serie A V Cloudflare and Others (Italy). 132 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106-7. 133 Law No. 50/2005/QH11 of November 29, 2005, on Intellectual Property, supra note 127. 134 I cannot prove a negative but the law will be cited so people can see for themselves. Law No. 50/2005/QH11 of November 29, 2005, on Intellectual Property, supra note 127; See also IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 135 3 Nimmer on Copyright § 12.04 (2021). 136 Notice these statutes both allow for the holding of third parties to the infringement accountable, Article 6 I 8° of the Law for confidence in the digital economy, see supra note 115; Article L. 336-2 of the French Intellectual Property Code, supra note 115. 137 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5. 138 Id. at 137. 139 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5; IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 1. Vietnam’s Law on Intellectual Property Because Vietnam is a civil code jurisdiction, understanding the copyright protections in Vietnam begins with its intellectual property code. 133 While the law provides for the possibility of administrative sanction and certain remedies for copyright infringement, it does not currently state any form of secondary liability.134 This is highly problematic when considered in the context of online piracy because most infringers are not ISPs hosting infringing content but are end-users who use an ISP’s services to access infringing sites. The lack of secondary liability leaves rights holders with little recourse against online piracy. The problem is further exacerbated when one recalls that secondary liability is largely an operation of common law. 135 Without statutes like the aforementioned French provisions which allow courts to hold third-parties accountable,136 it is unclear how much authority Vietnamese courts have over ISPs. With such a lack of clarity over the law, it would be difficult to hold ISPs accountable much less ask them to block sites. Thus, the lack of ISP accountability has allowed for purveyors of online piracy and their end-users to effectively escape any form of consequences. A. Copyright Protection in Vietnam: A Lack of Clarity and Efficacy If a clear and responsive site blocking regime is possible, there is a natural question around why Vietnam’s current system is inadequate. While the existence of the Vietnamese Law on Intellectual Property and its associated regulations seem to suggest some form of copyright protection that can be used to combat online piracy, Vietnam 129 See supra where Vietnam is failing their TRIPS obligations. See also IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 110. 130 Government of New Zealand, supra note 16. y y 2021. See CPTPP, supra note 17 at 18-5, 18-64. Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 67 suffers from an absence of law and accountability necessary for its already poor enforcement mechanisms.132 1. Vietnam’s Law on Intellectual Property 139 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5; IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 132 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106-7. 137 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5. 138 Id. at 137. 142 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 107; See also Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127. 143 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 1; IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 144 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 2. Circular 07 Interestingly, in 2012, the Ministry of Information and Communications in Vietnam released Joint Circular 07/2012/TTLT- BTTTT – BVHTTDL, stipulating the duties of enterprises providing intermediary service, like ISPs, in the protection of copyright and related rights on the Internet and in the telecommunication networks environment.137 It imposed a duty on ISPs and intermediaries to take down infringing content and terminate services under certain circumstances, 138 and only under state direction.139 Article 5 of Circular 07 does this by stipulating that ISPs have a duty to remove and delete “content of digital information which violates copyright and related rights, cutting, stopping and suspension of the Internet line, telecommunication line as receiving request in written of the inspector of the Ministry of Information and Communications or inspector of the Ministry of Culture, Sports and Tourism or other competent State agencies as prescribed Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 68 by law”.140 Despite the existence of such regulation, this authority has been used in practice only in very narrow circumstances where online services and websites are directly infringing.141 This is likely because Circular 07 does not spell out clear penalties against ISPs for violating such a duty and a general lack of enforcement by the Vietnamese government.142 Further, the type of blocking provided in Circular 07 may only apply to websites that use the "internet services of a Vietnam company," i.e., if an infringing website uses a host that is provided by a Vietnamese company, registered a domain name with a Vietnamese Company (Vietnamese registrar), or uses an IP address that is managed by a Vietnamese company. 143 If this is correct, the effectiveness of the website blocking provision will be greatly different and even reduced144, as it does not account for the borderless nature of online piracy. Moreover, it does not seem like Circular 07 allows for dynamic injunctions or blocking against ISPs. When placed together with Vietnam’s Law on Intellectual Property, it is hard to see any concrete avenues for rights holders to hold ISPs accountable for the rampant online piracy that is occurring in Vietnam. The main issue with Circular 07 is not that it does not create some form of site blocking in Vietnam, but rather it creates a form of site blocking so toothless that it is ineffective. 140 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5. 141 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 140 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5. 141 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 142 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 107; See also Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127. 143 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 1; IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 144 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 145 Nguyen Huy Hoang, Ho Thi Le Tra, Market access conditions applied to foreign investors under Decree No. 31/2021/nd-cp, Lexology, 20 September 2021, https://www.lexology.com/library/detail.aspx?g=35eb4d79-ad70-4865-8fc6-6a03760f6a6e/; See also IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 146 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 147 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 106. 148 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 108-9. 149 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 109. 150 Id. at 148. 151 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 53 (Del. H.C.) (Apr. 10, 2019) (India). 152 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 108. B. Site Blocking in Vietnam: Possible Approaches While a complex array of overlapping factors seems to be the reason behind Vietnam’s currently lackluster copyright regime, the main obstacle seems is a lack of effective ISP regulation. When ISPs are not compelled to move against infringement it creates an environment where the online world is insulated from laws. This creates a flight of infringers online to escape the reach of the law. 151 This is happening online in Vietnam.152 Thus, any solution to online piracy in Vietnam must be able to bring the law into the online world. Effective site blocking arises as a means for bringing physical legal consequences into the online sphere. By enacting a clear, and responsive site blocking regime, Vietnam can begin to hold ISPs accountable for the infringing activity that they facilitate and prevent infringers from accessing those sites. However, if there are any lessons to be learned from Vietnam’s experience with Circular 07 and other countries’ experiences, site blocking regimes cannot be limited by the geographic location or domain names. They must be allowed to act on any site based on the qualitative nature of the site towards copyright infringement. The enactment of such a regime can happen in Vietnam in many ways, this note looks proposes two possible approaches that are feasible and considers their effect on online piracy in Vietnam. 3. Structural Problems Moreover, there are issues with Vietnam’s legal and market system which contribute to a culture of online piracy. Vietnam has and continues to have restrictions preventing foreign companies from setting up subsidiaries to distribute “cultural products” and has entry barriers around the importation and distribution of copyrighted works.145 These restrictions on market access fosters a demand for pirated content, which inevitably pushes Vietnamese consumers towards their illegal alternatives.146 145 Nguyen Huy Hoang, Ho Thi Le Tra, Market access conditions applied to foreign investors under Decree No. 31/2021/nd-cp, Lexology, 20 September 2021, https://www.lexology.com/library/detail.aspx?g=35eb4d79-ad70-4865-8fc6-6a03760f6a6e/; See also IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 146 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 112. 69 Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking Another issue is that no John Doe suits are allowed for the enforcement of copyright.147 This means that, unlike in India, in order to sue for infringement rights holders are required to acquire evidence of each infringer. The lack of John Doe suits and the impossibility of investigation, when placed together with the fact that the Vietnamese have been known to impose onerous and detailed requirements around the identification of infringers 148 are all indicative of an enforcement system that is not necessarily functioning. It should come as no surprise that to date there has never been any criminal proceeding brought for online infringement.149 More significantly, they pose problems for rights holders who may eventually wish to seek redress via a site blocking order but cannot because they lack the ability to ascertain if there is infringement or who is infringing. Further, Vietnam has laws around foreigners conducting investigations which prevents rights holders from effectively discerning if their works are being infringed or gathering evidence to meet Vietnam’s already amorphous yet onerous standards around online piracy and copyright enforcement.150 153 Id, at 112. 154 Directive 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the Harmonisation of Certain Aspects of Copyright and Related Rights in the Information Society, art. 8, 2001 O.J. (L 167) 155 Peter Carstairs, supra note 30 at 284-85. See also Australia, Copyright Amendment (Online Infringement) Act (2018), Section 115A; Professor Justin Hughes, response to questions from Senators Tillis, Coons, and Blumenthal, Senate Judiciary Committee / Intellectual Property Subcommittee, 11 & 42, 14 April 2020 (noting the similarities in the Australian and Singaporean site blocking statutes). 156 Id. at 148. 157 IIPA 2021 Special 301 Report on Copyright Protection and Enforcement submitted to the USTR, supra note 9 at 113. 158 Id, at 148. 159 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5(3). 1. A Statutory Approach One approach Vietnam could take to institute a site blocking regime would be to draw from the Singaporean or French experiences and introduce statutory measures which Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 70 directly enact a Vietnamese site blocking regime that reflects current international norms. This could happen in two ways. Vietnam could, drawing from their French tradition, embrace the broad approach that is traditional to civil law jurisdictions153 and enact a broad statute allowing for rights holders to apply for injunctions when ISP services are being used to infringe copyright. A broad statute establishing such a site blocking regime is likely to look like Article 8(3) of the EU, 2001 Information Society Directive or the other French statutes mentioned above154. This would largely leave effective enforcement to Vietnam’s courts. Alternatively, Vietnam could look to its neighbor Singapore and enact a rather tailored statute, which clearly identifies the scope of the site blocking regime. Such a statute would not look very different from those in Australia.155 In light of Vietnam’s existing judicial and statutory system, this approach does not seem wise. Given the onerous evidentiary requirements and inability of foreign rights holders to investigate any infringement156, it is unlikely that such an approach would be able to successfully change the status quo. In fact, this becomes all the more obvious when there has been a conversation around court reform in Vietnam, particularly in the intellectual property space.157 Furthermore, while a tailored statutory framework may provide more guidance to Vietnam’s courts in their implementation of a site blocking regime, it still does not solve the fact that cases will have to be brought within Vietnam’s onerous evidentiary and investigatory laws.158 The above analysis shows that any statutory approach altering Vietnam’s legal code towards establishing a site blocking regime would likely require secondary legal reforms to even remotely be able to operate. This makes it unlikely that such an approach will have much success in helping Vietnam achieve a more robust copyright regime. , 159 Joint Circular No. 07/2012/TTLT-BTTTT-BVHTTDL of June 19, 2012 of the Ministry of Information and Communications and the Ministry of Culture, Sports and Tourism Stipulating Duty of Enterprises Providing Intermediary Service in Protection of Copyright and Related Rights in the Internet and Telecommunication Networks Environment, supra note 127 at Article 5(3). 2. Administrative Approach A more promising approach towards introducing an effective site blocking regime in Vietnam is via administrative law. This approach largely relies on the fact that Circular 07 has already given Vietnamese inspectors at the Ministry of Information and Communications or the Ministry of Culture, Sports, and Tourism the authority to impose a duty on ISPs to takedown infringing content and stop access. 159 Given that those ministries already have the power to impose site blocking orders on ISPs, one possibility would be the creation of more regulations outlining how rights holders can petition the Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 71 various ministries for relief. This approach has the benefit of allowing the Vietnamese government the flexibility to leave most of their current legal framework entirely intact and simply shift its approach to online piracy within its governmental bureaucracy. This is because a regulatory approach via administrative law allows the Vietnamese ministries to simply stipulate whatever it wishes to consider when asking an ISP to block a site. This would bypass any conflicts with existing issues concerning onerous evidentiary requirements and help lessen the effect of restrictive foreign investigation restrictions. However, as we have observed from the international experience with site blocking, effective site blocking regimes operate best when there are clear standards are establishing FIOLs followed by flexible and responsive enforcement. This responsive enforcement is usually categorized by allowing for the subsequent blocking of the same sites under changed domain names, URLs, IP addresses, and more. As such this administrative approach is likely to look like the blacklist of illicit sites that the French were considering, 160 with the procedure for filings mirroring satisfaction of the factors articulated in the Indian and Singaporean experiences in defining FIOLs. It should also allow for blocking orders from the ministries to function like dynamic injunctions by allowing subsequent affidavits to be submitted to allow changes in domain names, URLs, and IP address, to be added to the blacklist. While seemingly effortless, the establishment of a site blocking regime via the ministries may require the ministries to amend Circular 07 to address what happens in the event ISPs do not comply with an administrative order. This is so site blocking orders from the ministry are obeyed and have teeth. 160 Nigel Cory, supra note 6; Ernesto Van der Sar, supra note 118. 161 There has been some commentary that such unilateral power over copyright law can have effects on the nature of free speech, particularly in countries with more communistic style governments, See Stephen McIntyre, The Yang Obeys, but the Yin Ignores: Copyright Law and Speech Suppression in the People's Republic of China., 29 UCLA PAC. BASIN L.J. 75, 77-81(2011). 2. Administrative Approach Furthermore, an administrative driven approach to site blocking would not be difficult for the Vietnamese government to implement but may require an increase in the number and effectiveness of its administrative personnel to adequately address the various petitions that will inevitably be filed. It should also be noted that such an approach does place a disproportionate amount of power to censor the internet in the hands of the Vietnamese government.161 Despite this, an administrative approach to site blocking in Vietnam seems like the most efficient and painless approach possible. 162 UTV Software Communication Ltd. V 1337X, (2019) Civil Suit No. 768 of 2018, ¶ 59 (Del. H.C.) (Apr. 10, 2019) (India). 163 Singapore, Copyright Act (Chapter 63 of Singapore Laws), Article 193DDA (revised 31st January 2006). CONCLUSION The growth and expansion of Vietnam’s online marketplace poses serious challenges to effective copyright enforcement, especially in light of its intellectual property laws. Vietnam’s increasing economic development and accession to various economic treaties makes it an increasingly attractive location for investment in the global economy. This includes many domestic and foreign authors and copyright holders who may want to sell and distribute their works to a captive audience. However, the prevalence of online piracy in Vietnam poses a major obstacle to the development of Vietnam’s creative economy. Vietnam’s recent ratification of the CPTPP and accession to the WCT late last year, make how Vietnam eventually addresses online piracy all the more pertinent. At first glance, Vietnam’s online piracy appears to be a challenging problem. Fortunately, many nations have effectively dealt with the threat of online piracy to Effective Anti-Piracy in Vietnam: A Journey Through Site Blocking 72 copyright through the implementation of site blocking. Vietnam’s Asian neighbors are no strangers to site blocking and can serve as guides for what might be possible in Vietnam. Ideally, good site blocking regimes possess clear rules which allow for the identification of FIOLs and responsive enforcement mechanisms to address guileful bad actors. In common law jurisdictions, like India162 and Singapore163, this has resulted in clear factor tests followed by dynamic injunctions. In contrast, civil law jurisdictions have also found success in well-constructed broad statutes that allow their courts to effectively address the idiosyncrasies of the case in front of them. The bitter pill is that Vietnam has a myriad of options and jurisdictions from which it can take lessons to implement an efficient copyright enforcement regime that includes site blocking as one of its tools. However, none of those options that currently exist can be implemented without some changes to Vietnam’s existing framework. This is further exacerbated by the reality that Vietnam’s obligations to the CPTPP and the WCT have come due which requires Vietnam to act now.

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English

Also

Neophilologus

public-domain

ALSO. Giinther, in his English Synonyms explained and illustrated, states that "Neither too nor also are found at the head of a sentence". This is evidently a slip, for Smith, in Synonyms Discriminated, concludes his article on Also, too, likewise, besides with the words: 'Grammatically, Too cannot begin a sentence, while Also can'. g Smith gives no examples, however. M/itzner, Engl. Orammalik III, p. 371, has a quotation from the A. I7. (1611): 'I can no more go out and come in: also the Lord hath said unto me, Thou shalt not go over this Jordan (Deuter. 31. 2). Poutsma, on p. 298 Part. I of Orammar of Late Modern English, states the following rule: "Also precedes the clause it belongs to." There is however but one quotation, in which, moreover, also may be reckoned to belong to a clause understood: 'These are the more patent facts, which are to be deduced from this hat. Also, by the way, that it is ex- tremely improbable that he has gas laid on in his house (Sh. Holmes, Blue Carbuncle). Kriiger, Schmidt, and Storm are silent on the subject. ) g j That also really does occur at the head of a sentence, is proved by the following quotations: 1. This Ronald has faced death and danger and learnt the meaning of responsibility. Also he is the head of the house. (Rita, The Pointing" Finger. Edition Van Hasselt. StoKes and Sketches, p. 24). 1. This Ronald has faced death and danger and learnt the meaning of responsibility. Also he is the head of the house. (Rita, The Pointing" Finger. Edition Van Hasselt. StoKes and Sketches, p. 24). g p ) 2'. Ernest Saunders follows her lead. Also he regards me with pity because I don't play golf (Barry Pain, The One Before, Nelson's Ed., p. 147). 3. He says he'll never give you up. I'm to tell you that. Also, only he didn't say I was to tell you this-he is scared pea-green by grandfather, and' doesn't dare say so much as "boo" to him. (Bar. v. Hutten, Pare, p. 154). y p 4. Traquair had a good opportunity of studying her face, which pleased him well. Also he observed that she wore the deepest mourning (A. S. Swan, A Mask of Oold, p. 103). f p ) 5. ALSO. And we have grown up[ Our hair is braided around our head, our shirts are long, and we have a figure! To say nothing of a lover." . .... "Also, we laugh at the unfortunate who ventures to love, and not to please us." (Bar. v. Hutten, Pare, p. 110). f p 5. And we have grown up[ Our hair is braided around our head, our shirts are long, and we have a figure! To say nothing of a lover." . .... g g y g "Also, we laugh at the unfortunate who ventures to love, and not to please us." (Bar. v. Hutten, Pare, p. 110). 6. For if the oak is to become a stately tree we must provide against the crowding of timber. Also the tree beset with parasites prospers not. G. Meredith, The Egoist, Ch. I, 6. For if the oak is to become a stately tree we must provide against the crowding of timber. Also the tree beset with parasites prospers not. G. Meredith, The Egoist, Ch. I, 6. For if the oak is to become a stately tree we must provide against the crowding of timber. Also the tree beset with parasites prospers not. G. Meredith, The Egoist, Ch. I, W.A. VAN DONGEN. Rotterdam. van Dongen. van Dongen. van Dongen. van Dongen. 158 Also. Also. BOEKAANKONDIOING. Le tome XXXIX, 2e pattie, des Notices et Extraits des Manuscrits de la Biblioth~que nationale et autres biblioth~ques contient une Notice du Ma- nuscrit franeals 12483, par M. Arthur l_~ng'fors. Ce manuscrit est un recueiI compos6 en l'honneur de la Vierge eta depuis long-temps attir~ l'attention des savants, mais M. Ungfors est le premier qui l'ait 6tudi~ m6thodiqt:ement

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https://revistas.ucm.es/index.php/RCED/article/download/RCED0505220623A/15998

Spanish; Castilian

Cómo aprenden los profesores. Un estudio empírico basado en entrevistas

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ABSTRACT The main aim of this study is to describe the different conceptions that teachers hold about their own lear- ning, and also their relationship to their teaching practice as well as their epistemological beliefs, cognitive and metacognitive strategies and values. The implications of these results for teachers' knowledge acquisi- tion and effective training will be commented, in order to highlight some scientific criteria for these inter- ventions. The socioconstructive character of intelligence and learning is emphasised. Key words: teachers' conceptions of learning, cognitive restructuring, multiperspectivism, socioconstructi- vism, epistemological beliefs and strategies of learning. SUMARIO: 1. Introducción. 2. Cómo aprenden los profesores. De las competencias a las capacidades y a las concepciones. 2.1. Concepción empirista-conductual. 2.2 Concepción cognitivo-constructivista. 2.3. Concepción situado-sociohistórica. 3. Relatividad de las concepciones del aprendizaje de los profesores. 4. Agrupación de las concepciones del aprendizaje de los profesores. 5. Las concepciones específicas se rela- cionan con otras dimensiones. 6. ¿Pueden identificarse tipologías de profesores? 7. Relación de las concep- ciones del aprendizaje de los profesores con su práctica docente. 8. Diferencias, según el nivel educativo, en el aprendizaje de los profesores. 9. Conclusiones y consecuencias para el desarrollo profesional de los pro- fesores. 10. Referencias bibliográficas. Manuel PINTOR GARCÍA y Carmen VIZCARRO GUARCH Manuel PINTOR GARCÍA y Carmen VIZCARRO GUARCH Universidad Autónoma de Madrid Email: [email protected] RESUMEN La principal pretensión del estudio es reflejar cómo los profesores construyen su aprendizaje así como la rela- ción existente entre estas diferentes formas de concebir el aprendizaje y sus prácticas docentes, sus creencias epistemológicas, sus estrategias cognitivas y metacognitivas, y su referencia a los valores. Con ello preten- demos aportar elementos de reflexión para revisar y reorientar cómo conocemos los profesores y los criterios en los que basamos nuestros proyectos de formación. A lo largo del trabajo se insiste en el carácter social - socioconstructivismo- de la inteligencia, el aprendizaje y el conocimiento. Palabras clave: concepciones del aprendizaje, reestructuración cognitiva, multiperspectivismo, sociocons- tructivismo, creencias epistemológicas, estrategias de aprendizaje. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 623 ISSN 1130-2496 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 1. INTRODUCCIÓN La importancia concedida de forma continuada durante más de cinco décadas al aprendizaje -aun dentro de diversos enfoques y tradiciones psicopedagógicos- no ISSN 1130-2496 623 ISSN 1130-2496 ISSN 1130-2496 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... precisa demasiada justificación. Es más, hoy día, en nuestra sociedad del conoci- miento, el aprendizaje ha invadido todos los ámbitos de la vida. Se ha diversificado y globalizado -con ayuda del conocimiento a través de la red, todavía por compro- bar su alcance y posibilidades de gestión-, de tal manera que en todas las etapas del ciclo vital podemos construir el sentido de nuestra experiencia, según nuestros inte- reses profesionales, lúdicos y socioculturales. Pues bien, nos vamos a centrar en el aprendizaje de los profesores, para descen- trarnos un poco de su preocupación máxima: cómo aprenden los alumnos. En con- creto, pensamos que la forma como los profesores organizan su enseñanza estaría relacionada con el modo como entienden el aprendizaje, de modo que entender cómo conciben su propio aprendizaje resulta básico si se pretende conseguir una docencia y un aprendizaje de calidad. Ciertamente, suponemos que como seres humanos compartimos básicamente las mismas cualidades; pero también es impor- tante observar posibles diferencias, quizá relacionadas con distintas experiencias o posición. En todo caso, nos acercaremos directamente a las concepciones de su pro- pio aprendizaje, relacionándolas con su metodología o práctica docente. Conocer cómo aprendemos puede ser básico para mejorar nuestro desarrollo personal y profesional, además de tomar conciencia de posibles contradicciones que desvirtúan las prácticas docentes, llenas, en tantas ocasiones, de cómodas rutinas que nos defienden de la presión de los estudiantes e incluso de nosotros mismos. Además, las instituciones que gestionan la formación inicial y permanente de los docentes de todos los niveles deben hacer un esfuerzo para que su servicio esté regi- do por criterios de calidad, por encima del seguidismo, el oportunismo, la moda o lo políticamente correcto. Entendemos que una revisión de nuestra práctica docente -además de las obvias cuestiones propias de la disciplina, las tecnologías y la sociología de la educación y de nuestras aulas- debe fundamentarse en el conocimiento de los procesos de apren- dizaje y enseñanza, de los principios de la psicología del aprendizaje y la educa- ción1. No basta la práctica ni la experiencia, tantas veces ideologizada y parcial, que puede ser objeto de deformación. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 624 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 1. INTRODUCCIÓN Incluso la teoría es pluriparadigmática y debe ser objeto de debate y control de calidad, para una posterior toma de decisiones en diversos ámbitos de competencia y actuación. Por eso, el acercamiento al conoci- miento y a la formación, que se demanda o se ofrece, debe ser crítico. Sobre estos presupuestos debería plantearse la planificación y la gestión de la formación -que desee obtener la confianza de sus potenciales usuarios o clientes2- en cualquier ámbito de conocimiento. El proceso de aprendizaje de los profesores puede no sólo ser inducido, sino catalizado, por proyectos de formación con base científica y coherencia epistemoló- gica, que son facilitadores de la reestructuración conceptual. Parece existir eviden- cia de la relación entre el cambio cognitivo de los profesores (desarrollo personal, flexibilidad y multiperspectivismo) el compromiso libremente asumido, su partici- 624 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... pación significativa en foros de discusión (Gustafson y Rowell, 1995) y la duración de la experiencia. Pero para ello las instituciones de formación deberían aproximar- se a constituirse ellas mismas como comunidades de aprendizaje, para ser conse- cuentes, y no sólo proporcionar consumibles y pastillas de conocimiento. López Camps y Leal (2002) refieren que la propia OIT concibe las competencias profesio- nales como una construcción social de aprendizajes significativos y útiles para el desempeño productivo en una situación real laboral, partiendo para ello de sus cono- cimientos previos, articulando trabajo y aprendizaje, concordando con el modelo del práctico reflexivo de Schön (1987) y Shavelson (1982). Así mismo, afirman que las instituciones formativas, especialmente las dependientes de las administraciones públicas, deben actuar como una organización cualificadora, inteligente, que apren- de y se dota de nuevas competencias -laborales y educativas- para sí y para sus miembros. Así pues, para la efectiva formación de los profesores, se impone facilitar el cambio a partir de las concepciones ingenuas o implícitas del aprendizaje hacia otras más fundamentadas y significativas desde el punto de vista científico, aunque, por ello mismo, siempre plurales, falibles y criticables. Por eso, nos importa descubrir cuáles son las concepciones que los profesores tienen de su propio aprendizaje, cuál es su distribución. Es más, pretendemos relacionar estas concepciones con la meto- dología o práctica docente, para así apreciar su grado de coherencia. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 2. CÓMO APRENDEN LOS PROFESORES. DE LAS COMPETENCIAS A LAS CAPACIDADES Y A LAS CONCEPCIONES Estimamos que, previamente a la adquisición de determinadas competencias profesionales, poseemos en ciernes unas capacidades específicas para determinados dominios de conocimiento o de acción, o bien generales; pero que están integradas en un sistema de representaciones -más o menos elaborado y consciente-, entre las que descuellan las concepciones del aprendizaje, cuya relevancia tratamos de expre- sar gráficamente en la siguiente Figura 1: 625 625 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Figura 1. Papel de las concepciones del aprendizaje en el desarrollo de las competencias del profesorado El esquema trata de insistir en la importancia de la demanda de formación, ges- tionada si es posible en equipo por sus propios actores -en el entorno de su comuni- dad de aprendizaje- y basada en criterios, cuyo fundamento teórico está relacionado con las concepciones del aprendizaje. Éstas son -desde sus versiones más implícitas, cotidianas e inconscientes, hasta las más elaboradas y científicas- fuente de conoci- miento y clave para la toma de decisiones acerca de las actividades y proyectos de formación que se ofertan. Para indagar dichas concepciones, nos ha parecido crucial esta vez partir de cómo aprendemos los profesores. Para ello, nos basamos en el análisis de 54 entre- vistas en profundidad a profesores de enseñanza secundaria y universitaria (Pintor, 2005) S d di li i d ló i (Pi 2002) l CONCEPCIONES DEL APRENDIZAJE Criterios y objetivos Evaluación y retroalimentación del sistema Capacidades y competencias personales y de la institución Análisis de los problemas, experiencias, intereses, motivaciones y necesidades de los profesores (Descubrir la demanda formativa) Procedimientos, actividades y proyectos de formación (Oferta de la institución para liderar las propuestas formativas - individuales o de equipo- de los profesores) CONCEPCIONES DEL APRENDIZAJE Criterios y objetivos Evaluación y retroalimentación del sistema Capacidades y competencias personales y de la institución Análisis de los problemas, experiencias, intereses, motivaciones y necesidades de los profesores (Descubrir la demanda formativa) Procedimientos, actividades y proyectos de formación (Oferta de la institución para liderar las propuestas formativas - individuales o de equipo- de los profesores) Capacidades y competencias personales y de la institución El esquema trata de insistir en la importancia de la demanda de formación, ges- tionada si es posible en equipo por sus propios actores -en el entorno de su comuni- dad de aprendizaje- y basada en criterios, cuyo fundamento teórico está relacionado con las concepciones del aprendizaje. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 2. CÓMO APRENDEN LOS PROFESORES. DE LAS COMPETENCIAS A LAS CAPACIDADES Y A LAS CONCEPCIONES Éstas son -desde sus versiones más implícitas, cotidianas e inconscientes, hasta las más elaboradas y científicas- fuente de conoci- miento y clave para la toma de decisiones acerca de las actividades y proyectos de formación que se ofertan. Para indagar dichas concepciones, nos ha parecido crucial esta vez partir de cómo aprendemos los profesores. Para ello, nos basamos en el análisis de 54 entre- vistas en profundidad a profesores de enseñanza secundaria y universitaria (Pintor, 2005). Se trata de un estudio cualitativo metodológicamente (Pintor, 2002), en el que se buscan las cualidades componentes de la experiencia de los profesores acerca del aprendizaje y el valor y dimensiones que ellos les atribuyen. Posteriormente -una vez validada la codificación de las variables por jueces externos y aplicado el índi- ce criterial de Hambleton y Rovinelli (Hambleton, 1984)- se han sometido los datos a un estudio estadístico relacional, con objeto de obtener agrupaciones estadística- mente significativas y asociaciones de las características encontradas y de los perfi- les de los conjuntos de profesores. Se ha tratado de contrastar empíricamente las aportaciones de la literatura psi- cológica (Greeno, Collins y Resnick, 1995; Rodrigo, Rodríguez y Marrero, 1993; Porlán, Rivero y Martín, 1997) sobre cómo se produce el aprendizaje, pero también las actitudes -reflejadas en los propios análisis psicológicos- ante el conocimiento, 626 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... que se han decantado a través de la historia del pensamiento (racionalismo versus empirismo, positivismo y neopositivismo, neomarxismo, hermenéutica,...). Los diversos enfoques no son excluyentes, sino, a veces, complementarios y dependientes del propósito y el escenario que los inspira. Pozo insiste en la preven- ción de los exclusivismos y dogmatismos, incluso del constructivista: "El proyecto epistemológico constructivista, en contra de lo que suponen muchos de sus defen- sores, especialmente en el ámbito educativo o instruccional, es compatible no sólo con una psicología racionalista (o innatista) sino con un enfoque empirista, como el defendido por el asociacionismo cognitivo" (2003:27). Dichas orientaciones operan en diversos niveles. Las podemos codificar y emplear de modo plenamente consciente, explícito y científico; o bien poseerlas como ideas previas, implícitas e inconscientes de uso cotidiano, económico y adap- tativo. Por eso son también difíciles de cambiar por otras más oportunas. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 2. CÓMO APRENDEN LOS PROFESORES. DE LAS COMPETENCIAS A LAS CAPACIDADES Y A LAS CONCEPCIONES Pero esto ¿en función de qué criterios?, ¿cómo y hacia dónde es deseable nuestro cambio cog- nitivo?, ¿somos siempre consecuentes con nuestras orientaciones preferidas? Pues bien, tratemos de clarificar cómo es el aprendizaje de nuestros profesores y sus per- files, para posteriormente hacer una valoración de las alternativas. Como resultado de la investigación, hemos hallado tres enfoques o concepcio- nes -representadas en la Tabla 1-, con dos matices específicos en cada una, que se detallarán en su momento. Seguramente estas concepciones están presentes en el repertorio de competen- cias y actuación de un mismo profesor, en buena medida; y una misma persona que aprende puede utilizar alternativamente cualidades de cualquiera de ellas. Las tres han contribuido a la comprensión del conocimiento científico, a entender lo que es la cognición y el aprendizaje, así como a la orientación de la práctica educativa. Sin embargo, posiblemente ciertos tipos de aprendizajes y adquisiciones son explicados con mayor abundancia por alguna de ellas. Greeno, Collins y Resnick (1995) esti- man que la contemplación de los problemas de aprendizaje, dentro y fuera de la escuela, desde esta triple perspectiva, tendría la virtud de hacer un uso pluralista de los recursos personales e institucionales, tanto de orden material como social. Pasamos a comentar a continuación los principales hallazgos de este estudio. En primer lugar, una descripción fenomenológica extraída directamente, mediante aná- lisis de contenido de las entrevistas realizadas a los profesores. A continuación comentaremos el resultado de varios análisis estadísticos que nos permiten vislum- brar ciertas asociaciones y relaciones de interés. En un primer momento, se analizó el contenido de las entrevistas sostenidas con los profesores de forma estrictamente cualitativa. Estos contenidos fueron codifica- dos según ciertas categorías conceptuales que fueron sometidas posteriormente a los correspondientes análisis estadísticos. La primera codificación arrojó las siguientes dimensiones del aprendizaje que comprobamos tenían cierta correspondencia con nuestros modelos de partida. 627 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Tabla 1. Concepciones del aprendizaje (elaborada a partir de Greeno, Collins y Resnick, 1995) CONDUCTUAL EMPIRISTA COGNITIVO CONSTRUCTIVISTA SITUADO SOCIOHISTÓRICA Conocimiento Conjunto acumulado de asociaciones, conexiones y habilidades basadas en la experiencia. Relación instrumental Comprensión de conceptos y teorías en ámbitos específicos. Relación comunicativa Representaciones mentales socialmente distribuidas en comunidades de práctica y su ambiente cultural y físico (herramientas, artefactos, teorías…). Construcción social Aprendizaje Adquisición de asociaciones. 2. CÓMO APRENDEN LOS PROFESORES. DE LAS COMPETENCIAS A LAS CAPACIDADES Y A LAS CONCEPCIONES Centrados en los contenidos declarativos Transformación de la información y organización del conocimiento en estructuras o esquemas. Estratégico Interiorización de las representaciones sociales por interacción entre las personas en entorno físico y tecnológico. Uso de herramientas, tanto físicas como culturales Transferencia a nuevos contextos Por similitud de estímulos en situaciones diversas Reinterpretación y revisión de representaciones. Analogías Praxis en contextos diversos, que posibilita la percepción de regularidades en la variedad. Relativismo. Transdisciplinaridad Motivación Incentivos, éxitos Intrínseca Valoración social de funciones y objetos Tareas Prácticas, ejercicios Reelaboración de la información. Confrontación y cambio conceptual Ecológicamente válidas Tabla 1. Concepciones del aprendizaje (elaborada a partir de Greeno, Collins y Resnick, 1995) Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 2.1. CONCEPCIÓN EMPIRISTA-CONDUCTUAL La perspectiva empirista-conductual se basa en varias corrientes de pensamien- to: el asociacionismo, basado en el empirismo clásico de Locke y Hume y renovado por el positivismo; el conductismo, que se centra en la observación y medición de objetivos comportamentales -niveles de pericia e incluso de éxito- y el análisis de tareas; el conexionismo, que concibe el conocimiento como patrones de conexiones entre redes neuronales; e incluso ciertos enfoques de la psicología de la información que consideran la mente como un sistema de cómputo, predicción y control. Los profesores que mantienen esta concepción han afirmado que se aprende a través de la experiencia. En ella se encuentran y acumulan regularidades entre los acontecimientos, se establecen relaciones y adquieren habilidades y hábitos de todo tipo, que se pueden ir consolidando y legalizando. Desde este enfoque, la práctica se constituye en criterio básico de verificación del aprendizaje. Estos nuevos conteni- dos prácticamente siempre se pueden confirmar o verificar recurriendo a los hechos 628 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 Cómo aprenden los profesores... anuel Pintor García y Carmen Vizcarro Guarch Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... o a la experimentación, asociación de estímulos, resultados de una acción, éxitos, fracasos, etc. Se pueden considerar dentro de este modelo dos subcategorías o concepciones específicas del aprendizaje de los profesores: — a) ejercicio: el aprendizaje se adquiriría a través del ejercicio ("drill") como actividad mecanizada o ejecución sensorial o motriz de dominio y con- trol -según la ley skinneriana del aprendizaje referente al número o repe- tición de las respuestas exitosas-, por contraposición a acción como pra- xis o transformación, más propia de la subcategoría acción/interacción, que es parte integrante de la concepción situado-sociohistórica. La maes- tría, como buena ejecución o capacitación, alude a un conjunto de hechos, prácticas, realizaciones o comportamientos, conscientes o rutinarios, que se dominan. Saber es hacer con eficacia y buenos resultados; y ; — b) verificación de datos, o comprobación de hechos, relaciones y asociacio- nes, o bien predicción y control de fenómenos -al estilo de la concepción habitual del método científico y sus fases-, incluyendo las normas o leyes que los gobiernan o explican por su conexión y cuantificación; el criterio de certeza viene postulado por el neopositivista principio de correspon- dencia entre las proposiciones del lenguaje y lo que las cosas son. 2.1. CONCEPCIÓN EMPIRISTA-CONDUCTUAL Se sos- tiene que el conocimiento es un correlato de lo que ocurre en el mundo. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 2.2. CONCEPCIÓN COGNITIVO-CONSTRUCTIVISTA La orientación cognitivo-constructivista, siguiendo a Greeno, Collins y Resnick, (1995) es heredera de tres líneas de pensamiento en la historia de la psicología: la psicología de la Gestalt, que afirma la posibilidad del conocimiento por la configu- ración estructural de su objeto; el constructivismo, desde Piaget; y, siempre según los mencionados autores, el procesamiento simbólico de la información -desarrolla- do en la ciencia cognitiva americana por Chomsky, Simon, Newell, entre otros-, que enfatiza los procesos de comprensión del lenguaje, el razonamiento y la solución de problemas. Remotamente, estimamos, hunde sus raíces en el yo pensante cartesiano y en la síntesis kantiana entre sujeto y objeto. Bastantes profesoras y profesores entrevistados han comunicado que, en su pro- pia experiencia como aprendices, adquieren conocimiento cambiando y construyen- do nuevas representaciones y significados. El conocimiento es, para estos profeso- res, la comprensión de conceptos y teorías, por incipientes que éstas sean (previas, espontáneas, legas, cotidianas, quizá de "usar y tirar", según el contexto ). El sujeto construye su conocimiento: reelabora y transforma la información, la enriquece, confronta teorías y puede cambiar sus conceptos y concepciones o teorías, por frag- 629 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... mentadas que éstas puedan ser o parecer. Hace, pues, una necesaria referencia a la propia coherencia del desarrollo personal y no a una pretendida correspondencia con una exterioridad presuntamente objetiva. Conocer es construir individualmente y no interiorizar meramente representaciones o normas sociales. Dentro de este modelo se pueden agrupar las contestaciones de los profesores en dos subcategorías o con- cepciones específicas del aprendizaje: — a) enriquecimiento cognitivo , que sería un proceso de desarrollo mental no sustantivo, al menos inicialmente, a partir de lo meramente acumulativo; permanece en los matices, atributos, características no esenciales a los conceptos, pero puede replantearlos en nuevas perspectivas o enfoques; — b) reestructuración conceptual o cambio cognitivo , mediante el cual los pro- fesores relativizan y usan el pensamiento complejo, redefinen fórmulas, cambian de conceptos -o el significado de éstos- sustantivamente, no sólo de matices o de carga afectiva; incluso el objeto y la metodología de su materia puede cambiar. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 2.3. CONCEPCIÓN SITUADO-SOCIOHISTÓRICA El enfoque situado-sociohistórico considera que el conocimiento está distribui- do socialmente en un entorno cultural y material. Es, a la vez, atributo del grupo que lleva a cabo actividades cooperativas, y del individuo, que participa en la actividad comunitaria. Las tradiciones de esta perspectiva situada serían el pragmatismo de Dewey, el perspectivismo orteguiano, la etnografía, la psicología ecológica, la socio- logía del conocimiento, la fenomenología hermenéutica desde Heidegger a Gadamer, la escuela sociohistórica de Vygotsky y la teoría de la práctica comunica- tiva de Habermas y Apel, epígonos de la Escuela de Francfort. Su misma denomi- nación indica su carácter interdisciplinar, en el que se integran la antropología, la psico-socio-lingüística, la filosofía de la mente y del lenguaje -sentido y acción como sistemas interrelacionados-, e incluso la etología. Supone un giro que la psi- cología del aprendizaje está operando actualmente hacia el socioconstructivismo, lo cual, no sin sorpresa, hemos podido comprobar en nuestro trabajo. Se da este tipo de aprendizaje en la interacción humana y en un contexto. El conocimiento se halla distribuido socialmente y cambia dialécticamente por con- trastes entre las ideas y situaciones históricas, en los cambios de los instrumentos materiales o condiciones; entre las poliédricas perspectivas de las disciplinas que estudian los mismos y, a la vez, cambiantes objetos de conocimiento; y, por supues- to, se constituye con la mediación de los iguales como mentores. Es en la comuni- dad concreta de aprendizaje o investigación donde se establece el alcance de los sig- nificados. Incluso los procesos sociales deben ser tratados como conocimiento 630 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... (Salomon, 1993, 2002), que se genera y se gestiona social e institucionalmente. 2.3. CONCEPCIÓN SITUADO-SOCIOHISTÓRICA También dentro de este modelo hemos podido agrupar, de forma relevante, las con- testaciones de los profesores en dos subcategorías o concepciones específicas del aprendizaje: — a) acción-interacción, que pone el acento en la relación humana, en el grupo (de colegas o con alumnos; en clase o en departamentos o fuera) formal o informal en el que se aprende; en estos encuentros los conceptos se mati- zan e incluso cambian; en ocasiones achacan algunos de estos cambios a las innovaciones tecnológicas, que pueden generan nuevas formas de aprender y de comprender; la acción se acerca a la praxis como transfor- mación no sólo de las relaciones cognitivas, sino también de las personas; — b) multiperspectivismo dialéctico y relativista, por sus contrastes, paradojas, rebasamiento de fronteras entre los conceptos entre sí, entre las discipli- nas, llegándose a la interdisciplinaridad y la transversalidad de los temas. Característico es la transferencia de perspectivas a situaciones y entornos diversos, así como su carácter crítico y reflexivo. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 3. RELATIVIDAD DE LAS CONCEPCIONES DELAPRENDIZAJE DE LOS PROFESORES Las concepciones no son permanentes ni monolíticas, sino que parecen activar- se en función de los contextos y de las tareas. Kember (1997) subraya que la fron- tera entre las diversas orientaciones y sus connotaciones es borrosa y los profesores son incoherentes y fluctúan en sus opiniones y estrategias. También es verdad que estos resquicios de ambigüedad lo son también de libertad y de educabilidad; es decir, de capacidad de maniobra formativa de los profesores hacia cotas de mayor coherencia epistemológica y fecundidad conceptual. De este modo, afirmaciones correspondientes a los distintos paradigmas coexisten y, como puede observarse en la Figura 2, el porcentaje de adscripción a las mencionadas concepciones cambia, si se interroga sobre la metodología en clase y otras prácticas docentes. Cuando apa- rece el compromiso y el entorno no es tan favorable, se es mucho más empirista-con- ductual, mucho menos cognitivo-constructivista y menos situado-sociohistórico. Mas en nuestro estudio cualitativo no es tan relevante quedarnos en las cifras, sino más bien descifrar el contenido y significado de las dimensiones o cualidades encontradas. Para ello vamos a fijarnos a continuación en su agrupación, apoyándo- nos en el análisis de conglomerados y su representación gráfica. 631 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... 4. AGRUPACIONES DE LAS CONCEPCIONES DEL APRENDIZAJE DE LOS PROFESORES Figura 2. Valores relativos de las concepciones 24 46 30 45 32 23 0 20 40 60 EMPIRISTA COGNITIVA SOCIOHISTÓRICA CONCEPCIONES CONCEPCIONES +METODOLOGÍA Figura 2. Valores relativos de las concepciones 24 46 30 45 32 23 0 20 40 60 EMPIRISTA COGNITIVA SOCIOHISTÓRICA CONCEPCIONES CONCEPCIONES +METODOLOGÍA Figura 2. Valores relativos de las concepciones 24 46 30 45 32 23 0 20 40 60 EMPIRISTA COGNITIVA SOCIOHISTÓRICA CONCEPCIONES CONCEPCIONES +METODOLOGÍA Figura 2. Valores relativos de las concepciones Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 4. AGRUPACIONES DE LAS CONCEPCIONES DEL APRENDIZAJE DE LOS PROFESORES Una vez llevado a cabo el análisis de contenido del discurso de los profesores, nos interesó someter dichos resultados a un análisis jerárquico de clusters o conglo- merados que nos aportara datos cuantitativos sobre la asociación de los elementos antes mencionados. En la Figura 3 puede verse la estructura de las agrupaciones de las concepciones del aprendizaje de los profesores -las generales en negrita en el gráfico, y flanquea- das por su par de específicas-, obtenidas mediante el análisis de conglomerados. A la vez que se diferencian, han quedado asociadas de forma peculiar: Figura 3. Conglomerados relativos a las concepciones del aprendizaje de los profeso- res (distancias reescaladas de 0 a 25 por la similitud de las respuestas) Organización bipolar de las concepciones: emergencia del socioconstructivismo 0 5 10 15 20 25 +---------+---------+---------+---------+---------+ VERIFICACIÓN Ø8ØØØÞ polo empirista-conductual EMPIRIST-CONDUCTØÝ ßØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØÞ EJERCICIO ØØØØØÝ Ù MULTPERSPECTIV ØØØ8ØØØØØØØÞ Ù SITUADO-SOCIOHISTÓRÝ ßØØØØØØØØØØØØØØØØØØØØØØØØØØØØØÞ Ù ACCIÓN-INTERACCIÓNØØØØØØØØØÝ polo socioconstructivista ßØØØØØØØÝ REESTRUCTURACIÓNØØØØØØØØØØØ8ØØØØØÞ Ù COGNITIVO-CONSTRUCTIVISTAØØÝ ßØØØØØØØØØØØØØØØØØØØØØØØÝ ENRIQUECIMIENTO CONCEPTUALØØØØØØØÝ Figura 3. Conglomerados relativos a las concepciones del aprendizaje de los profeso- res (distancias reescaladas de 0 a 25 por la similitud de las respuestas) Organización bipolar de las concepciones: emergencia del socioconstructivismo 0 5 10 15 20 25 +---------+---------+---------+---------+---------+ VERIFICACIÓN Ø8ØØØÞ polo empirista-conductual EMPIRIST-CONDUCTØÝ ßØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØÞ EJERCICIO ØØØØØÝ Ù MULTPERSPECTIV ØØØ8ØØØØØØØÞ Ù SITUADO-SOCIOHISTÓRÝ ßØØØØØØØØØØØØØØØØØØØØØØØØØØØØØÞ Ù ACCIÓN-INTERACCIÓNØØØØØØØØØÝ polo socioconstructivista ßØØØØØØØÝ REESTRUCTURACIÓNØØØØØØØØØØØ8ØØØØØÞ Ù COGNITIVO-CONSTRUCTIVISTAØØÝ ßØØØØØØØØØØØØØØØØØØØØØØØÝ ENRIQUECIMIENTO CONCEPTUALØØØØØØØÝ Descubrimos así mismo en el anterior gráfico o dendograma que las categorías o concepciones cognitiva y situada se asocian, integrando una díada o polo socio- constructivista -más complejo y denso conceptualmente-, frente al polo empirista- 632 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... conductual. Se evidencia también una cercanía más primigenia -menor distancia reescalada-, y que se repetirá, en el constructo empirista-conductual. Es decir, estas dimensiones empiristas suelen presentar una simultaneidad en las verbalizaciones de los profesores. Realmente, la validación de las concepciones específicas, como integrantes de los modelos de las concepciones del aprendizaje, tal como los profesores lo practi- can y conciben, es el núcleo del presente trabajo. Ello ha servido para profundizar cualitativamente a través de ellas en la manera de ser de las concepciones, facilitan- do su estudio y sus relaciones, a la vez que desvelan una parte de su complejidad. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 5. LAS CONCEPCIONES ESPECÍFICAS SE RELACIONAN CON OTRAS DIMENSIONES Parece también conveniente analizar las relaciones de las anteriores concepcio- nes con las dimensiones más importantes sobre las que se interrogó a los profesores en las entrevistas: estrategias cognitivas y metacognitivas, valores, epistemología y motivación. Éstas ofrecen, mediante la observación de las relaciones obtenidas en el dendograma, la posibilidad de una caracterización enriquecedora: 0 5 10 15 20 25 +---------+---------+---------+---------+---------+ EJERCICICIO Ø8ØØØØØÞObjetivismo VERIFICACIÓN ØÝ ßØØØØØØØØØØØØØØØØØØØØØÞ CIENCIA CONOCE REALIDADÝ ßØØØØØØØØØØØØØØØØØØØÞ Cª NO CONOCE REALIDAD8ØØØØØÞ Ù Ù APRENDO VALORES ØØØØØÝ ßØØØØØØØØØØØØØØØØØÝ Ù ACCIÓN-INTERACCIÓNØØØ8ØÞ Ù Relativismo epistemológ.y criticismoÙ MULTPERSPECTIVISMOØØØÝ ßØØØÝ Ù LOS CONCEPTOS CAMBIANØØÝ Ù REESTRUCTURACIÓNØØØ8ØØØÞ Ù ESTRATEGIAS DE APRØÝ ßØÞ Ù APR PARA CLASES Ø8ØØØÞ Ù Ù Estrategias y motivaciones Ù LOGRO ØÝ ßØÝ ßØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØÝ METACOGNICIÓN ØØØØØØØÝ Ù ENRIQUECIMIENTO ØØØØØØØØØÝ 0 5 10 15 20 25 +---------+---------+---------+---------+---------+ EJERCICICIO Ø8ØØØØØÞObjetivismo VERIFICACIÓN ØÝ ßØØØØØØØØØØØØØØØØØØØØØÞ CIENCIA CONOCE REALIDADÝ ßØØØØØØØØØØØØØØØØØØØÞ Cª NO CONOCE REALIDAD8ØØØØØÞ Ù Ù APRENDO VALORES ØØØØØÝ ßØØØØØØØØØØØØØØØØØÝ Ù ACCIÓN-INTERACCIÓNØØØ8ØÞ Ù Relativismo epistemológ.y criticismoÙ MULTPERSPECTIVISMOØØØÝ ßØØØÝ Ù LOS CONCEPTOS CAMBIANØØÝ Ù REESTRUCTURACIÓNØØØ8ØØØÞ Ù ESTRATEGIAS DE APRØÝ ßØÞ Ù APR PARA CLASES Ø8ØØØÞ Ù Ù Estrategias y motivaciones Ù LOGRO ØÝ ßØÝ ßØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØÝ METACOGNICIÓN ØØØØØØØÝ Ù ENRIQUECIMIENTO ØØØØØØØØØÝ En efecto, los tres grupos o clusters obtenidos en torno a las concepciones espe- cíficas parecen aportar algún rasgo digno de consideración a cada una de nuestras tres categorías o concepciones del aprendizaje: El objetivismo subrayaría la importancia que la concepción empirista-conduc- tual da al método experimental y a la propia concordancia de los datos de cualquier aprendizaje con la experiencia como fuente principal y fundamento del conocimien- to. Queda asociada con una variable que implica una epistemología realista: "Con la ciencia podemos conocer la realidad tal como es", lo cual puede constituir un refle- jo del ya mencionado principio neopositivista de correspondencia entre las repre- 633 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... sentaciones, las proposiciones del discurso de la ciencia y los sucesos del mundo de la experiencia o de la realidad. El relativismo epistemológico iría más unido a la concepción situado-sociohis- tórica, cuyo componente dialéctico y crítico va en paralelo con el cambio concep- tual en las personas y en la ciencia. Las estrategias de más alto nivel y las motivaciones intrínsecas estarían más bien contenidas en el grupo dimensional de la concepción cognitivo-constructivista. Y todo ello podemos esquematizarlo en la siguiente tabla: Tabla 2. 5. LAS CONCEPCIONES ESPECÍFICAS SE RELACIONAN CON OTRAS DIMENSIONES Perfil de las concepciones del aprendizaje de los profesores y dimensiones que se les asocian CONCEPCIONES Carácter Dimensiones asociadas Empirista-conductual Objetivista Epistemología realista Cognitivo- constructivista Estratégico y motivador Estrategias cognitivas profundas, metacognición y motivación Situada-sociohistórica Relativista y crítico Epistemología relativista, criticismo hacia los valores Tabla 2. Perfil de las concepciones del aprendizaje de los profesores y dimensiones que se les asocian Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 6. ¿PUEDEN IDENTIFICARSE TIPOLOGÍAS DE PROFESORES? Si agrupamos a los profesores por la similitud de sus respuestas, comprobamos la consistencia del modelo de las concepciones. De nuevo el análisis de conglome- rados genera automáticamente agrupaciones de profesores que tienen sentido con- ceptual. Podremos caracterizar éstas al relacionarlas, mediante las correspondientes tablas de contingencia, con otras dimensiones que los profesores han revelado sobre su experiencia de aprendizaje. En conclusión, resultan significativos -con un potente coeficiente de contingen- cia - cinco de los seis grupos o clusters obtenidos -el 65 % del total de la muestra- , que recogemos en la siguiente tabla: 634 Revista Complutense de Educación Vol 16 Núm 2 (2005) 623 644 Tabla 3. Grupos de profesores que -dentro de sus conglomerados- se asocian significa- tivamente a concepciones del aprendizaje Tipologías de profesores % n 1. Situado-sociohistóricos: grupos I y II 17 9 2. Cognitivo-constructivistas: grupos III y IV 33 18 3. Empiristas: grupo V 15 8 Total 65 35 Tabla 3. Grupos de profesores que -dentro de sus conglomerados- se asocian significa- tivamente a concepciones del aprendizaje 634 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Así pues, al haberse efectuado una asociación significativa, quedaría explicado el 65 % del espacio del problema. En efecto, dentro de los conglomerados el I se acerca más a la concepción socioconstructivista canónica, mientras en el II, aún situándose próximo a ésta, puede considerarse una categoría más de transición. En conjunto, el 17 % de la muestra queda relacionado significativamente con la orien- tación situado-sociohistórica; por eso lo hemos etiquetado con ese apelativo. Entre los conglomerados III (con connotaciones empiristas) y IV de sujetos (con un perfil más estrictamente constructivista), el 33 % de los profesores queda clasificado den- tro de la categoría cognitivo-constructivista. Finalmente, dentro del conglomerado V, hemos podido encontrar un 15 % de profesores que sostienen una concepción empirista-conductual. En el VI no se ha podido establecer una relación significativa. En total, se pronostica una clasificación correcta del 65 % de los profesores dentro de los modelos que guiaron nuestra investigación. Parece constituirse un continuo, cuya culminación es el socioconstructivismo, que compagina la capacidad de reestructuración cognitiva con la interacción social, el relativismo epistemológico y el multiperspectivismo. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 6. ¿PUEDEN IDENTIFICARSE TIPOLOGÍAS DE PROFESORES? Sin embargo, hay una cons- tante en el presente estudio: los profesores, aun de orientación general sociocons- tructivista se tornan en su práctica docente empiristas y se centran en la práctica del ejercicio como ejecución y dominio de tareas concretas (drills) o tratan, ante todo, de controlar y verificar conocimientos, probablemente en busca de seguridad, efica- cia y valoración social por parte de alumnos y compañeros. O de la institución edu- cativa. Queda también reflejada la ya mencionada incoherencia en la práctica con res- pecto a las propias concepciones (Bolhius y Voeten, 2004); y también la gradualidad en la composición de las agrupaciones, lo cual puede ser una huella que nos mues- tra la transición de unos estilos a otros y, de alguna manera, la posibilidad de cam- bio. Esto puede ser un motivo de reflexión a la hora de realizar una oferta de base científica y crítica de formación/acción inicial y permanente del profesorado. Si aceptamos que la construcción de nuevos aprendizajes se fundamenta en la base de conocimientos ya consolidados, sería interesante que esta formación procediera a partir de los elementos correspondientes a los distintos paradigmas en los que se sitúan los profesores. De este modo, los profesores podrían disponer de una gama de recursos docentes más amplia en base a la cual organizar para sus estudiantes expe- riencias de aprendizaje distintas y complementarias (como exige la diversidad de los aprendizajes: declarativo, procedimental, de actitudes y valores...). Así mismo, hemos de volver a subrayar que la práctica de una concepción del aprendizaje suele ser situacional; y, esto, dicho no sólo como toma de decisión indi- vidual, sino como inmersión en un entorno social de aprendizaje, que tiende a faci- litar determinados tipos de decisiones y estilos de aprendizaje. Moore (2002: 19), refiriéndose a Perry, pionero en el estudio de las creencias epistemológicas, habla, en este sentido, de la progresiva caída desde un mundo de absolutos a otro de con- 635 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... textos, compromisos, búsqueda de sentido y elecciones idiosincrásicas. Y termina citándole (ibid.: 26): "Las personas son demasiado complejas como para ser encla- sadas en teorías y categorías. Teorías y categorías sólo iluminan ciertos aspectos de las personas ... nosotros queremos desplegar nuestra eficacia como educadores. Pero eficacia implica poder, y ahí está el conflicto. 6. ¿PUEDEN IDENTIFICARSE TIPOLOGÍAS DE PROFESORES? Si el poder sirve para etiquetar a los estudiantes [nosotros nos podríamos referir también a los profesores] y así pretender contribuir a su desarrollo, entonces les deshumanizamos a ellos y a nosotros mis- mos". Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 7. RELACIÓN DE LAS CONCEPCIONES DEL APRENDIZAJE DE LOS PROFESORES CON SU PRÁCTICA DOCENTE Con el fin de describir la relación entre las concepciones del aprendizaje y las prácticas docentes, recurrimos de nuevo al análisis de conglomerados que nos per- mitiera describir posibles asociaciones consistentes. A diferencia de lo ya expuesto en el epígrafe 5, aquí la información proporcionada por los profesores se refiere a su propia práctica docente. Veamos en el dendograma de la Figura 4 la forma característica en que se agru- pan las dimensiones de la metodología docente en torno a las concepciones especí- ficas del aprendizaje de los profesores. *METODOLOGÍA De nuevo nos encontramos con la diferenciación de las tres concepciones del aprendizaje, advirtiendo la ya repetida y comentada supraordinación con dos polos: el socioconstructivista y el objetivo. Así pues, cada concepción lleva aparejada el polo metodológico que nocionalmente le corresponde y aglutina diversas cualidades de las prácticas docentes como se describe en la Tabla 4 . Tabla 4. Polaridades de la práctica docente y su relación con las concepciones del aprendizaje CONCEPCIONES POLOS METODOLÓGICOS Empirista- conductual Objetivo (centrados en los contenidos, necesidad de proteger el yo de la crítica negativa, TIC) y asociativo (ejercicios) Cognitivo- constructivista Intersubjetivo-constructivo (centrados en el alumno, las estrategias cognitivas y metacognitivas de aprendizaje y la acción) Situado- sociohistórica Crítico (referencia necesaria del conocimiento a los valores éticos y cívicos, interacción, mediación de las TIC, relativismo y falibilidad del conocimiento, pluralismo multiperspectivista) Tabla 4. Polaridades de la práctica docente y su relación con las concepciones del aprendizaje CONCEPCIONES POLOS METODOLÓGICOS Empirista- conductual Objetivo (centrados en los contenidos, necesidad de proteger el yo de la crítica negativa, TIC) y asociativo (ejercicios) Cognitivo- constructivista Intersubjetivo-constructivo (centrados en el alumno, las estrategias cognitivas y metacognitivas de aprendizaje y la acción) Situado- sociohistórica Crítico (referencia necesaria del conocimiento a los valores éticos y cívicos, interacción, mediación de las TIC, relativismo y falibilidad del conocimiento, pluralismo multiperspectivista) Tabla 4. Polaridades de la práctica docente y su relación con las concepciones del aprendizaje Estas conclusiones refuerzan nuestra suposición de que aprendizaje y enseñanza son dos caras de la misma moneda, aun con todas las salvedades e incongruencias, debidas a condiciones personales y a los entornos de aprendizaje. Una vez más que- remos expresar, a la vista de la contundencia, pero al mismo tiempo de la elasticidad de los resultados, que las concepciones del aprendizaje, y de la enseñanza, no son dimensiones monolíticas y persistentes, sino teorías en acción con distinto grado de formalización. Éstas responden a la solución de problemas y a la necesaria toma de decisiones al hilo de cada día; y, por todo ello, son susceptibles de cambio, hacia mayores cotas de excelencia en perspectivas teóricas y epistemológicas. Figura 4. Asociación de concepciones del aprendizaje con prácticas docentes Figura 4. Asociación de concepciones del aprendizaje con prácticas docentes Ù 0 5 10 15 20 25 +---------+---------+---------+---------+---------+ EJERCICIO Ø8ØØØÞ VERIFICACIÓN ØÝ ßØØØØØÞ Concepción empirista-conductual MET*EJERCICIO ØØØØØÝ ßØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØØÞ MET CONTENIDOS Ø8ØÞ Ù polo Ù MET TIC(Tecnolog)Ý ßØØØØØØØÝ objetivo Ù MET PROTEJO MI YOØØÝ Ù REESTRUCTURACIÓNØØØÞ Ù MET METACOGNICIÓNØØÝ Ù MET ESTRAT DE APRØØ9ØÞ Ù MET REESTRUCTURAC ØÝ ßØØØÞ Conc. cognit-constructivista MET CENTRADO EN ALUMNÝ ßØØØØØØØØØØØØØØØÞ polo Ù ENRIQUECIMIENTO ØØØØØØØØØÝ ßØØØØØØØØØØØØØØØØØØØØØØØÝ ACCINTERACCIÓN Ø8ØØØØØØØØØØØÞ Ù socioconstructivista MET ACCIÓN ØÝ ßØØØØØØØØØØØÝ MULTIPERSPECTIV Ø8ØÞ ÙConc. situada-sociohistórica MET MULTIPERSP ØÝ ßØØØØØØØØØÝ MET VALORES ØØØÝ 636 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Cómo aprenden los profesores... Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 8. DIFERENCIAS, SEGÚN EL NIVEL EDUCATIVO, EN ELAPRENDIZAJE DE LOS PROFESORES Nos preguntamos también si estas distintas concepciones se correspondían con algunas características de los profesores como el nivel educativo, el género, la expe- riencia docente o el ámbito del conocimiento. Aquí mencionaremos solamente, para finalizar, los hallazgos correspondientes al nivel educativo. Sólo encontramos diferencias en cuanto a la frecuencia, mayor de la esperada, del posicionamiento empirista-conductual por parte de los profesores de secundaria. 637 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Ya hemos visto la relación de las concepciones del aprendizaje con la práctica docente. Por otra parte, esta orientación empiricista puede tener serios riesgos para la formación del profesorado, ya que se pudiera anclar en un nivel de sobrevalora- ción de la facticidad. Cabría señalar, por último, sus consecuencias respecto al esti- lo de aprendizaje de los estudiantes (Kember, 1997). Por otra parte, en el nivel universitario se utilizan significativamente más las estrategias profundas de elaboración y organización en el propio aprendizaje. Pero los profesores pueden ser poco consecuentes, al no hacerlas revertir a los alumnos en su práctica docente. Otras diferencias con respecto a algunas dimensiones relevantes (p.e., metacog- nición o valores) son de alcance más limitado y, suponemos que podrían ser, con- tingentes con los entornos de aprendizaje. Por lo demás, el paralelismo entre los niveles educativos es casi total. En cuanto a su relación con la metodología docente, los profesores universita- rios tienden a centrarse más en los contenidos y menos en la acción-interacción en clase, y a usar más las tecnologías; por el contrario, en secundaria se centrarían más en los alumnos y su aprendizaje. Pero estas conclusiones requerirían un estudio más centrado en esta cuestión. Además, dentro de la concepción cognitivo-constructivista, los profesores de secundaria se relacionan significativamente con la práctica de ciertas estrategias de aprendizaje, especialmente entre los reestructuradores, que son más metacognitivos y críticos en su referencia curricular a los valores que los universitarios. Dentro de la concepción histórico-situada, existen ciertas diferencias significati- vas estadísticamente en su práctica docente: en secundaria se mantiene una episte- mología relativista -lo cual supone tener una concepción pluralista y falibilista del conocimiento y de la ciencia-, se centra más el profesorado en la acción-interacción y en el aprendizaje de los alumnos, así como en el uso de las tecnologías. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 8. DIFERENCIAS, SEGÚN EL NIVEL EDUCATIVO, EN ELAPRENDIZAJE DE LOS PROFESORES Sin embar- go, los universitarios que mantienen esta concepción situada se comprometen más aún en el conocimiento crítico de los valores éticos y cívicos. A pesar de las anteriores sugerencias, podríamos concluir que con nuestros datos, necesariamente limitados, no tiene mucho sentido globalizar las diferencias - apenas si se han encontrado en dimensiones generales-, sino buscarlas en ámbitos, entornos y enfoques del aprendizaje, para, precisamente en esas relaciones, poder intervenir personal o socialmente para optimizar el propio estilo, enfoque o concep- ción del aprendizaje. O para transformarlo, en el sentido de una posible progresión socioconstructivista que los conglomerados que hemos descrito podrían sugerir, como indicador de riqueza, densidad conceptual y coherencia epistemológica. Quizá en este punto final haya de adoptarse una postura relativista: efectivamente, las dife- rencias son relativas a las fuentes -no sólo de orden cognitivo- de las variaciones. Boulton-Lewis y otros (2001) las hacen depender de la preparación profesional y del entorno. 638 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Revisemos los aspectos diferenciales. La aparición de un conjunto de profesores de secundaria de orientación empirista-conductual, más allá de lo esperable estadís- ticamente, nos advierte sobre la importancia de la intervención en formación del pro- fesorado, pues suele coincidir la adhesión a esta concepción empiricista con un tipo de aprendizaje y enseñanza centrado en los contenidos y en la persona del profesor, como figura de autoridad y depositario de lo que ha de saberse con certeza. Pero todo ello puede dar lugar a un estilo de aprendizaje superficial (Gow y Kember, 1993, en Kember y Gow, 1994; Entwistle, 1981). Sin embargo, y en el mismo nivel de secundaria, la variabilidad es grande, for- mándose una potente bolsa de posibilidades de cambio. En él encontramos un grupo de profesores cuya concepción específica es la reestructuración cognitiva, y que se involucra en el aprendizaje desde la perspectiva de los alumnos (cfr. Fensham y Marton, 1991, en Boulton-Lewis et al., o. c.), llevando a cabo una práctica docente estratégica, metacognitiva y motivacional, que facilita un estilo de aprendizaje pro- fundo entre los alumnos. Esta característica la hemos hallado con carácter general en secundaria -como tendencia-, junto a la facilitación de la interacción entre los alum- nos. Por otra parte, Mellado (1998; en Boulton-Lewis et al., o. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 8. DIFERENCIAS, SEGÚN EL NIVEL EDUCATIVO, EN ELAPRENDIZAJE DE LOS PROFESORES c.) habla de una mayor imprevisibilidad en secundaria en la relación entre concepciones de la ense- ñanza y la práctica docente; sin embargo, de acuerdo con otros autores, la incohe- rencia de dichas relaciones -debido al fuerte carácter episódico que poseen- parece tener carácter general (Larsson, 1987, en Rodrigo et al., 1993). No obstante, la cri- sis que aún sufre especialmente la educación secundaria y el cansancio profesional, pueden polarizar aún más la variabilidad y los contrastes. En el nivel universitario observamos, como cabía esperar, una mayor valoración de los contenidos. A la vez, se ignora como conjunto, la multidisciplinaridad. Todo lo cual puede hacernos pensar que se están sobredimensionando los aspectos de la propia matriz disciplinar. Ya Kember (1997) señaló que hay profesores universita- rios que no se consideran profesores, sino expertos. Parece que se centran en sus pro- pias estrategias y habilidades cognitivas, pero la cultura institucional y otros facto- res externos pudieran dificultar una transmisión intencional de las mismas. Por otra parte, en el sector de profesores universitarios con concepción situado- sociohistórica se observa cómo dan una sobresaliente importancia a la referencia a los valores ético-cívicos en el desarrollo curricular de sus disciplinas. Esto puede ser expresivo de las tendencias universitarias actuales a favor de la transversalidad y la comprensión y respuesta a los problemas sociales que globalmente nos acucian (Michavila y Martínez, 2002). Otros temas conexos (a propósito de Seddoh, 2002) son: la profesionalización de la educación superior -Declaración mundial de 1998-, la carrera docente en los nive- les no universitarios, la productividad y la evaluación de procesos, resultados e ins- tituciones. También urge encontrar sistemas estables de comunicación -redes- y colaboración entre los niveles educativos, particularmente en el terreno de la inves- 639 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... tigación/acción -con ofertas de formación en común o al menos referencias mutuas- , dentro del espacio común europeo -Comisión de Educación-, e incluso global - directrices de la UNESCO-. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 9. CONCLUSIONES Y CONSECUENCIAS PARA EL DESARROLLO PRO- FESIONAL DE LOS PROFESORES Si aceptamos que el aprendizaje se construye sobre los conoci- mientos ya consolidados, sería aconsejable partir de estas concepciones para llevar- las a su ulterior desarrollo. De hecho, podríamos pensar que a pesar de las regulari- dades que hemos observado, las concepciones no se presentan como algo monolíti- co y rígido, sino que existe amplias posibilidades de cambio, desarrollo y enriqueci- miento e incluso cambio conceptual hacia posiciones que epistemológicamente podemos considerar más complejas y ricas en su proyección docente, en una línea más flexible y multiperspectivista. En congruencia con esta perspectiva socioconstructivista, la formación y el des- arrollo debe prestar suficiente atención a las comunidades de aprendizaje -departa- mentos, centros de enseñanza y centros de profesores, e incluso servicios docentes y no docentes de las administraciones- , ya que ellos son el caldo de cultivo origi- nario, donde también aparecen las concepciones del aprendizaje como teorías acti- vas y situadas acerca de cómo acceder al conocimiento y cambiarlo. Y esto, como el tránsito al conocimiento científico, no es un proceso natural (Rodrigo y Correa, 1999), sino más bien cultural. Constituye un debate social sin fin, sobre el que se puede intervenir para que prevalezcan el rigor intelectual y los logros científicos por encima de los intereses, que, por legítimos que sean, merecen que se les ponga en su sitio. Por todo ello, parece tan importante como difícil la intervención institucional en los procesos de formación de los profesores, con base en criterios científicos. Es obvia la necesidad de la formación inicial, siempre en equipo y en entornos signifi- cativos de aprendizaje; pero consideramos igualmente relevante, a la vista de las ten- dencias que hemos podido detectar en nuestro trabajo, volcarse igualmente en la for- mación continua para lograr una transformación de la cultura institucional (Vizcarro, 2002). El constituyente principal -estratégico- de los programas de acción/investiga- ción/formación se referiría a los aspectos científicos y epistemológicos de las con- cepciones del aprendizaje de los profesores y de los alumnos, así como de la prácti- ca docente (Gow y Kember, 1993, en Kember y Gow, 1994) -dentro y fuera de la clase- e investigadora. 9. CONCLUSIONES Y CONSECUENCIAS PARA EL DESARROLLO PRO- FESIONAL DE LOS PROFESORES Ha sido una compensación el diálogo compartido durante largas horas con tan- tos profesionales de la enseñanza y el aprendizaje a quienes agradecemos su gene- rosa colaboración, sin la que estos estudios no hubiesen sido posibles. En un traba- jo cualitativo como éste, no importan las cifras sino la tematización lograda de sus imprevisibles experiencias de aprendizaje, cuyas dimensiones hemos podido rela- cionar y perfilar en torno a las concepciones que tienen de su propio aprendizaje y su influencia en la metodología docente. Con las limitaciones inherentes a nuestra opción metodológica de corte cuanti- tativo y que cubre sólo una pequeña muestra de profesores, podemos afirmar que hemos constatado la existencia de distintas concepciones del aprendizaje entre pro- fesores de secundaria y de universidad, que se corresponden con los modelos histó- ricos propuestos por la Psicología del aprendizaje. Estas concepciones no se refieren en exclusiva a cómo se produce el aprendizaje, sino que son conjuntos complejos de dimensiones que engloban aspectos como motivaciones, estrategias cognitivas y metacognitivas o la referencia a valores. Si quisiésemos esbozar con una imagen global lo esencial del artículo, afirmarí- amos que las concepciones empirista-conductual, la cognitivo-constructivista -espe- cialmente en su versión reestructuración cognitiva y cambio conceptual- y la situa- do-sociohistórica, constituyen una síntesis de su experiencia de aprendizaje, que está relacionada con su metodología docente, sus estrategias de aprendizaje cognitivas y metacognitivas, con sus motivaciones y sus posicionamientos epistemológicos. Así mismo, hemos podido comprobar que reestructuración cognitiva, acción-interacción y multiperspectivismo -componentes estas dos de la concepción situada- conforman la perspectiva socioconstructivista, que aúna la necesaria individualidad del conoci- miento con su origen también necesariamente social. Así pues, estas concepciones parecen tener relación con las prácticas docentes de los profesores y también con las estrategias que desarrollan los estudiantes, según han puesto de relieve diferentes estudios (Entwistle, 1981; Gow y Kember, 1994; Kember, 1997), de manera que a una estrategia docente profunda corresponden estrategias de elaboración y organización por parte de los estudiantes. Esta relación señala la necesidad e interés de la formación y el desarrollo profesional de los docen- tes, que abordaremos a continuación. 640 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Como es natural, estas concepciones de los profesores deberían ser la base en la que se sustentan los programas y actividades de formación y desarrollo profesional de los docentes. Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 9. CONCLUSIONES Y CONSECUENCIAS PARA EL DESARROLLO PRO- FESIONAL DE LOS PROFESORES Las concepciones específicas del aprendizaje socioconstruc- tivista que se ofrecerían para debatir, entre otras posibilidades, girarían en torno a propuestas similares a las representadas por las subcategorías más fecundas halladas en este trabajo: la reestructuración cognitiva y el cambio conceptual, la acción e interacción transformadora del objeto de conocimiento y el multiperspectivismo crí- tico y dialéctico. Estimamos que se halla en línea con el modelo del práctico refle- xivo (Schön, 1987), la investigación-acción (Elliot, 1993) y la racionalidad comuni- cativa y emancipatoria (Habermas, 1992; Rodrigo, Rodríguez y Marrero, 1993). Obviamente, habría otros contenidos tácticos referentes a habilidades -teorías en 641 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... uso, cfr. Coffey y Gibbs, 2001), contenidos disciplinares, empleo significativo de las herramientas tecnológicas y cuestiones siempre candentes de sociología de la edu- cación (Esteve, Franco y Vera, 1995; Seddoh, 2002). A la vista del impresionante despliegue de estrategias cognitivas y metacogniti- vas de aprendizaje, motivaciones, criterios epistemológicos, referencias a valores y riqueza de motivaciones, se puede estimar que este colectivo profesional es, en su conjunto, profundamente reflexivo y creativo. Su rasgo dominante común -que no discrimina grupos, es el enriquecimiento cognitivo para el desarrollo personal, por más que se ejerza muchas veces de forma errática y no planificada, dentro de un con- tinuo que admite ciertas gradaciones: desde la mera acumulación de contenidos hasta la adquisición de nuevas cualidades, como tránsito -no sin crisis- a la rees- tructuración conceptual. La densidad de los contenidos de su aprendizaje y la ampli- tud de sus recursos le confieren un alto rango de calidad no sólo técnica, sino huma- na, que abarca todos los aspectos de su vida laboral, social y personal. Dada esta riqueza en la experiencia de aprendizaje de los profesores, sería una irreparable pérdida no superar la situación actual de marginación de los profesiona- les de la enseñanza y la educación respecto de la investigación educativa (Gore y Gitlin, 2004), parte integrante de la función docente. Aunque es verdad también que la experiencia puede ser un elemento retardatario y conservador. Quizá sea preciso que en todos los niveles educativos se profundice en la cultura del aprendizaje y se promuevan equipos y comunidades de aprendizaje, investigación y acción (Elliot, 1993; Imbernón, 2002), y se posibilite que el centro educativo sea una verdadera unidad de cambio (Marcelo, 1995). Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 642 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 10. REFERENCIAS BIBLIOGRÁFICAS BOLHUIS, S.; VOETEN, M. J. M. (2004) Teachers conceptions of student learning and own learning. Teachers and teaching: theory and practice, 10, 1, 77-98. BOULTON LEWIS, G. M., SMITH, D.J.H., MCCRINDLE, A. R., BURNETT, P.C., CAMP- BELL, K.J. (2001) Secondary teachers' conceptions of teaching and learning. Learning and Instruction, 11, 35-51. COFFEY, M.; GIBBS, G. (2001) The strategic goals of training of university teachers. En RUST, Ch. (Ed.) Improving student learning. Oxford: Oxford Brookes University. ELLIOT, J. (1993) El cambio educativo desde la investigación-acción. Madrid: Morata. ENTWISTLE, N. (1981) Styles of Learning. An integrated outline of educational psycho- logy. Devon: J. Wiley & Son. ESTEVE, J. M.; FRANCO, S.; VERA, J. (1995) Los profesores ante el cambio social. Barcelona: Anthropos. GORE, J. M.; GITLIN, A. D. (2004) [Re]Visioning the academic-teacher divide: power and knowledge in the educational community. Teachers and Teaching, 10 (1) 36-58. GREENO, J. G.; COLLINS, A.; RESNICK, L. B. (1995) Cognition and learning. En D. BERLINER; R. CALFEE (Eds.) Handbook of educational psychology. GUSTAFSON, B. J.; ROWELL, P. M. (1995) Elementary preservice teachers: constructing conceptions about learning science and the nature of science. International Journal of Science Education, 17 (5), 589-605. HABERMAS, J. (1992) Ciencia y técnica como "ideología". Madrid: Tecnos. HABERMAS, J. (1992) Ciencia y técnica como "ideología". Madrid: Tecnos. HAMBLETON, K. H. (1984) Validating the test scores. En R. A. BERK. A guide to criterion- referenced test construction. Baltimore: The John Hopkins University Press. , ( ) g g eferenced test construction. Baltimore: The John Hopkins University Press. Ó IMBERNÓN, F. (Coord.) (2002) La investigación educativa como herramienta de forma- ción del profesorado. Barcelona: Graó. KEMBER, D. (1997) A reconceptualisation of the research into university academics' con- ceptions of teaching. Learning and Instruction, 7 (3) 255-275. KEMBER, D; GOW, L. (1994) Orientations to teaching and their effects on the quality of student learning, Journal of higher education, 65 (1) 58-74. g f g LÓPEZ CAMPS, J.; LEAL, I. (2002) Cómo aprender en la sociedad del conocimiento. Madrid: EPISE. MARCELO, C. (1995) Formación del profesorado para el cambio educativo. Barcelona: EUB. MARTON, F.; BOOTH, S. (1997) Learning and awareness. New Jersey: LEA. Í MARTON, F.; BOOTH, S. (1997) Learning and awareness. New Jersey: LEA. Í MICHAVILA, F.; MARTÍNEZ, J. (Eds.) (2002) El carácter transversal en la educación uni- versitaria. Madrid: Cátedra UNESCO de la UPM y Dir. Gral. de U. de la Comunidad de Madrid. 9. CONCLUSIONES Y CONSECUENCIAS PARA EL DESARROLLO PRO- FESIONAL DE LOS PROFESORES Los profesores tienen una forma peculiar de aprender y de concebir el aprendi- zaje que probablemente les distingue de la sustentada por otros profesionales. Pecharromán (2004) ha encontrado que los profesores se hallan en estadios episte- mológicos más evolucionados que otros adultos universitarios, siendo menor su objetivismo y mayor su constructivismo, aunque con variabilidad. Probablemente la función educativa nos sitúa permanentemente, al menos en potencia, en un clima de aprendizaje estimulante intelectualmente. No es de extrañar que su satisfacción pro- fesional y su clima de aprendizaje sean aceptables, aunque dentro de un amplio mar- gen de diferencias. Finalmente, podemos mencionar de pasada que hemos detectado algunas carac- terísticas que, sin embargo, se refieren a grupos de profesores excesivamene limita- dos (por ejemplo, profesores de secundaria con una posición constrructivista). Dadas las limitaciones numéricas de estos subgrupos, no nos detendremos en estas pecu- liaridades. Probablemente la educación esté necesitada de profesionales reflexivos, libera- dos del cúmulo de variables extrañas que la contaminan, para -al decir de Novac y Gowin (1988)- poder liberar sentimientos y significados compartidos que confor- man los acontecimientos educativos y la vida de los profesores y de los estudiantes. 642 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 Manuel Pintor García y Carmen Vizcarro Guarch Cómo aprenden los profesores... Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 10. REFERENCIAS BIBLIOGRÁFICAS MOORE, W. S. (2002) Understanding learning in posmodern world: reconsidering the Perry scheme of intelectual and ethical development. En B. K. Hofer y P. R. Pintrich. Personal epistemology. NJ: LEA. NOVAC, J. D.; GOWIN, D. B. (1988) Aprendiendo a aprender. Barcelona: Martínez Roca. PECHARROMÁN, I. (2004) Epistemologías cotidianas en el contexto académico y en adul- tos. Tesis doctoral, Universidad Autónoma de Madrid. 643 Revista Complutense de Educación Vol. 16 Núm. 2 (2005) 623 - 644 Cómo aprenden los profesores... Manuel Pintor García y Carmen Vizcarro Guarch PINTOR, M. (2002) Fenomenología y fenomenografía. Punto de encuentro entre la filosofía y la ciencia en el mundo del aprendizaje. Paideia, 59, 19-41. (2005) Las concepciones del aprendizaje de los profesores. Tesis doctoral: Universidad Autónoma de Madrid. Á Í PINTOR, M. (2002) Fenomenología y fenomenografía. Punto de encuentro entre la filosofía y la ciencia en el mundo del aprendizaje. Paideia, 59, 19-41. (2005) L i d l di j d l f T i d t l U i id d PORLÁN, R.; RIVERO, A.; MARTÍN, R. (1997) Conocimiento profesional y epistemología de los profesores, I: Teoría, métodos e instrumentos. Enseñanza de las ciencias, 15 (2) 155- 171. POZO, J. I. (2003) Adquisición de conocimiento. Madrid: Morata. Í RODRIGO, Mª. J.; RODRÍGUEZ, A. y MARRERO, J. (1993) Las teorías implícitas. Una aproximación al conocimiento cotidiano. Madrid: Visor. RODRIGO, Mª.J.; CORREA, N. (1999) Teorías implícitas, modelos mentales y cambio edu- cativo. En J.I. POZO Y C. MONEREO (Comps.) El aprendizaje estratégico. Madrid: Santillana. SALOMON, G. (2002) La educación superior frente a los desafíos de la era de la información. Boletín de la Red Estatal de Docencia Universitaria, 2 (2) 5-12. (1993) Distributed cognition. Psychological and educational considerations. NY: Cambridge University Press. RODRIGO, Mª.J.; CORREA, N. (1999) Teorías implícitas, modelos mentales y cambio edu- cativo. En J.I. POZO Y C. MONEREO (Comps.) El aprendizaje estratégico. Madrid: Santillana. SALOMON, G. (2002) La educación superior frente a los desafíos de la era de la información. Boletín de la Red Estatal de Docencia Universitaria, 2 (2) 5-12. (1993) Distributed cognition. Psychological and educational considerations. NY: Cambridge University Press. SEDDOH, K. F. (2002) Future international tendencies in the contents of higher education for lifelog training. En F. MICHAVILA Y J. MARTÍNEZ (Eds.) El carácter transversal en la edu- cación universitaria. Madrid: U. Politécnica de M. y C. de M. SHAVELSON, R. J. 10. REFERENCIAS BIBLIOGRÁFICAS (1982) Review of research on teachers pedagogical judgements, plans and decisions. The Rand Paper Series. Sta. Monica SHAVELSON, R. J. (1982) Review of research on teachers pedagogical judgements, plans and decisions. The Rand Paper Series. Sta. Monica SHÖN, D. A. (1987) Educating the reflective practitioner. Toward a new design for teaching and learning in the professions. San Francisco. Jossey-Bass Publishers. VIZCARRO, C. (2002) Innovación y métodos de enseñanza. En F. MICHAVILA Y J. MARTÍNEZ (Eds.) El carácter transversal en la educación universitaria. Madrid: U. Politécnica de M. y U. C. de M. VIZCARRO, C. (2002) Innovación y métodos de enseñanza. En F. MICHAVILA Y J. MARTÍNEZ (Eds.) El carácter transversal en la educación universitaria. Madrid: U. Politécnica de M. y U. C. de M. 644 644

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https://journals.hioa.no/index.php/yrke/article/download/2008/1821

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Fra fagarbeider til yrkesfaglærer: Førsteårsstudenters opplevelser av undervisning og veiledning i akademiske skriveferdigheter

Scandinavian Journal of Vocations in Development

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Marit Lindset & Britt Karin Støen Utvær (2017). Fra fagarbeider til yrkesfaglærer: Førsteårsstudenters opple- velser av undervisning og veiledning i akademiske skriveferdigheter. Scandinavian Journal of Vocations in Development. http://dx.doi.org/10.7577/sjvd.2007 Marit Lindset & Britt Karin Støen Utvær (2017). Fra fagarbeider til yrkesfaglærer: Førsteårsstudenters opple- velser av undervisning og veiledning i akademiske skriveferdigheter. Scandinavian Journal of Vocations in Development. http://dx.doi.org/10.7577/sjvd.2007 Marit Lindset & Britt Karin Støen Utvær (2017). Fra fagarbeider til yrkesfaglærer: Førsteårsstudenters opple- velser av undervisning og veiledning i akademiske skriveferdigheter. Scandinavian Journal of Vocations in Development. Peer reviewed article Fagfellevurdert artikkel Forfattere: Marit Lindset & Britt Karin Støen Utvær Nøkkelord: akademisk skriving, yrkesfaglærerutdanning, skrivekurs, tilbakemeldinger på oppgaver 1 Sammendrag Introduksjon: Yrkesfaglærerstudentene begynner på en utdanning der gode skriveferdigheter er en forutsetning for å klare seg gjennom utdanningen. Som lærerutdannere opplever vi at mange studenter strever med å beherske de kravene som stilles til akademisk skriving i høyere utdanning. Dette var bakgrunnen for at to lærere ved 3-årig yrkesfaglærerutdanningen i Trondheim startet en studie hvor formålet var å utforske hvordan førsteårstudentene opplevde et innføringskurs i skriving av akademiske oppgaver og ulike former for tilbakemelding på oppgaver i studiet (skriftlige, muntlige og digitale). Teori: Studien støtter seg i hovedsak til Illeris sine perspektiver på læring. Metode: Dataene utgjør 24 refleksjonslogger av 12 førsteårsstudenter i perioden 2014 – 2015. Analysene baserer seg på Lindseth og Norbergs fenomenologiske- hermeneutiske fremgangsmåte. Resultat: Studien viser at studentene ser et innføringskurs i akademisk skriving som helt nødvendig og av stor betydning for utvikling av deres skriveferdigheter og for videreutvikling av oppgaver. Konkrete, skriftlige tilbakemeldinger direkte i teksten sammen med en oppsummerende tekst til slutt er en form for tilbakemelding studentene opplever som viktig og nyttig. Muntlige tilbakemeldinger virker oppklarende og motiverende. Sammen med muntlige tilbakemeldinger fremheves digitale tilbakemeldinger som svært viktig for å skape trygghet og motivasjon for å jobbe videre. Konklusjon: Førsteårsstudenter forteller om stor usikkerhet og sårbarhet i skrivearbeidet. De opplever et skrivekurs som vesentlig for å mestre kravene som stilles til akademisk skriving i høyere utdanning og ser kvaliteter ved både skriftlige, muntlige og digitale tilbakemeldinger i arbeidet med å ferdigstille oppgaver i studiet. Skriftlige tilbakemeldinger oppleves som helt nødvendige. Nøkkelord: akademisk skriving, yrkesfaglærerutdanning, skrivekurs, tilbakemeldinger på oppgaver kelord: akademisk skriving, yrkesfaglærerutdanning, skrivekurs, tilbakemeldinger på 2 Abstract Introduction: Students pursuing a bachelor’s degree in the Vocational Teacher Education program find that good writing skills are a prerequisite to complete their education. As educators in this program, we experience that many students struggle to master the requirements for academic writing in higher education. For this reason, two teachers from the Vocational Teacher Education bachelor’s degree program in Trondheim undertook a study with the purpose of exploring how first-year students experienced an introductory course in academic writing and different forms of feedback on assignments (written, oral, and digital). Theory: The study relies mainly on Illeris’s Learning Theory. Method: Data collection is based on 24 reflection logs of 12 first-year students during 2014– 2015. The analysis is based on Lindseth and Norberg’s phenomenological–hermeneutical method. Result: The findings of the study show that students experience the introductory course in academic writing as absolutely necessary and of great importance for the development of their writing competence. Specifically, responses written directly in the text in addition to a summary are forms of feedback students perceive as important and helpful. Along with oral responses, digital responses are central for developing confidence and motivation to continue working. Conclusion: First-year students express insecurity and vulnerability in regard to the writing process. They experience the writing course as essential and see aspects of written, oral, and digital guidance as important to the process of completing academic assignments in Vocational Teacher Education. Written feedback is perceived as absolutely necessary. eywords: academic writing, Vocational Teacher Education, course in academic writing, Keywords: academic writing, Vocational Teacher Education, course in academic writing, tutor response tor response 3 Introduksjon Mange studenter opplever at det er vanskelig å beherske de kravene som stilles til akademisk skriving i høyere utdanning, spesielt det første studieåret. Studier viser at dette gjelder spesielt studenter i høyere utdanning som er tatt opp på grunnlag av realkompetanse (Grepperud, Rønning & Støkken, 2006; Hoel & Rokkones, 2012). Dette er også vår erfaring som mangeårige lærere ved 3-årig yrkesfaglærerutdanning i Trondheim. Med utgangspunkt i fagarbeidere som ønsker å kvalifisere seg til et læreryrke, har vi gjennom flere år arbeidet med å utvikle undervisning og veiledning. Hensikten er å kvalifisere, veilede og støtte studenter i prosessen med å skrive akademiske tekster som utgjør mange oppgaver i studiet. Gjennom å utvikle vår undervisning og anvende ulike strategier for veiledning ønsker vi å bidra til at studentene utvikler sine skriveferdigheter og opplever mestring i studiet. Å utvikle skriveferdigheter i første studieår vil danne et godt grunnlag for skriveprosesser i de følgende studieårene. Mye av vurderingene i studiet tar utgangspunkt i skriftlig arbeid. Å mestre studiet er derfor avhengig av hvorvidt studentene lykkes i det skriftlige arbeidet (Hoel & Rokkones, 2012). Som en følge av Kunnskapsløftet har grunnleggende ferdigheter i alle fag fått en sentral plass både i grunnopplæringen og i lærerutdanningen (Kunnskapsdepartementet, 2013; Som en følge av Kunnskapsløftet har grunnleggende ferdigheter i alle fag fått en sentral plass både i grunnopplæringen og i lærerutdanningen (Kunnskapsdepartementet, 2013; Utdanningsdirektoratet, 2006). Forskrift til rammeplan for treårig yrkesfaglærerutdanning fra 2013 understreker at yrkesfaglærere gjennom studiet skal arbeide systematisk med disse ferdighetene (Kunnskapsdepartementet, 2013). Ettersom grunnleggende ferdigheter i skriving nå er lagt til alle fag i skolen må både lærere og lærerutdannere sette seg bedre inn i mange sider ved skriving enn tidligere. Mens skriving og skriveopplæring tidligere var norsklæreres sitt domene, angår dette nå alle lærere uavhengig av fag (Hoel, 2008). Med dette som utgangspunkt startet to lærere ved yrkesfaglærerutdanning ved NTNU høsten 2014 et prosjekt med målsetting om å utvikle studentenes kompetanse i å skrive oppgaver innenfor universitet og høyskoler der akademiske tekster er rådende. Med akademiske skriving menes at man uttrykker seg i et eksakt og objektivt språk, videre at teksten har en klar struktur og logisk oppbygning. Kritisk refleksjon, aktivt bruk av referanser og korrekt kildehenvisninger er en 4 4 selvfølge (Busch, 2013). Den vanligste strukturen på akademiske tekster er IMRoD1 (Hoel, 2008). selvfølge (Busch, 2013). Den vanligste strukturen på akademiske tekster er IMRoD1 (Hoel, 2008). Prosjektet tar utgangspunkt i førsteårsstudentene. 1IMRoD er en norsk betegnelse for IMRAD som står for Introdution, Material/Method, Results og Discussion. Introduksjon Studenter opplever tidlig i utdanningsløpet at kravene som stilles til akademisk skriving kan være vanskelig å fylle, samtidig som første studieåret danner grunnlaget for de to neste årene i lærerutdanningen. Å komme godt i gang med skriving tidlig i studiet anser vi som viktig, ikke minst fordi vurderingsordningen i de fleste emner det første studieåret er mappeeksamen som inneholder oppgaver med IMRoD- struktur. Prosjektet tar utgangspunkt i førsteårsstudentene. Studenter opplever tidlig i utdanningsløpet at kravene som stilles til akademisk skriving kan være vanskelig å fylle, samtidig som første studieåret danner grunnlaget for de to neste årene i lærerutdanningen. Å komme godt i gang med skriving tidlig i studiet anser vi som viktig, ikke minst fordi vurderingsordningen i de fleste emner det første studieåret er mappeeksamen som inneholder oppgaver med IMRoD- struktur. For å styrke studentenes kompetanse og mestringsopplevelse i arbeidet med disse oppgavene har vi iverksatt to tiltak. Vi har utviklet et todagers kurs med innføring i akademisk skriving. Dette kurset blir tilbudt på den første samlingen i studiet. Det andre tiltaket er iverksetting av flere former for tilbakemeldinger på de ulike oppgavene. Helt siden studiet ble etablert i 2007 har studentene fått skriftlige tilbakemeldinger på oppgaver. I dette prosjektet har vi i tillegg innført muntlige tilbakemeldinger på oppgaver en gang per semester samt digitale tilbakemeldinger til en del studenter. Digital tilbakemelding innebærer muntlig formidling via lyd- og bilde. Studier viser at veiledning og tilbakemelding har betydning for studenters opplevelser av mestring, gjennomføring og karakterer (Firing, Klomsten & Moen, 2013). Det er derimot relativt lite forskning på hvordan lærerinstitusjonene driver opplæring i akademisk skriving, og hvordan dette forstås og oppleves av studentene (Handal, Lycke & Lauvås, 2013). Hensikten med denne artikkelen er å gjøre rede for hvordan innføringskurset og de ulike formene for tilbakemeldinger oppleves av studentene. Med dette som utgangspunkt belyses følgende problemsstilling: Hvordan opplever førsteårsstudenter undervisning og veiledning i prosessen med å skrive akademiske oppgaver? Våre forskningsspørsmål er: a) Hvordan opplever studentene innføringskurset i oppgaveskriving? b) Hvordan opplever studentene skriftlige, muntlige og digitale tilbakemeldinger på oppgaver i studiet? I artikkelen bruker vi både begrepene veiledning og tilbakemelding. Veiledning er ifølge Handal og Lauvås (2013) en læringsprosess som foregår mellom student og veileder og er, 1IMRoD er en norsk betegnelse for IMRAD som står for Introdution, Material/Method, Results og Discussion. 5 slik vi ser det, et bredere begrep enn tilbakemelding. Introduksjon I artikkelen bruker vi tilbakemelding når vi omtaler lærernes kommentarer gitt på studentenes oppgaver, veiledning bruker vi når dialog mellom lærer og student også inngår. slik vi ser det, et bredere begrep enn tilbakemelding. I artikkelen bruker vi tilbakemelding når vi omtaler lærernes kommentarer gitt på studentenes oppgaver, veiledning bruker vi når dialog mellom lærer og student også inngår. Teori Å mestre de kravene som stilles til å skrive akademiske oppgaver i lærerstudiet forutsetter læring og utvikling. Studentenes lærings- og utviklingsarbeid blir i denne studien knyttet til Illeris sine perspektiver på læring. Som hjelp og støtte i prosessen med å skrive oppgaver er lærernes veiledning sentral. I vårt arbeid med veiledning og tilbakemelding på studentenes oppgaver har vi valgt å støtte oss til Tveiten, Hoel, Handal, Lycke og Lauvås og Gamlem. Læring Illeris ser på læring som et komplekst og mangesidig begrep og beskriver læring som «enhver prosess som hos levende organismer fører til en varig kapasitetsendring som ikke bare skyldes glemsel, biologisk modning eller aldring» (2012, s. 16). Gjennom mange års arbeid har han utviklet to modeller hvor han søker å favne denne kompleksiteten. I modellene synliggjøres det han benevner som læringens to prosesser og tre dimensjoner. De to prosessene omtaler han som tilegnelsesprosessen og samspillsprosessen (Figur 1). Illeris hevder at begge må være aktive for at vi skal lære. Tilegnelsesprosessen er en indre mental tilegnelse og bearbeidelse av de nye impulsene, og samspillsprosessen en prosess basert på samspillet mellom individet og dets omgivelser Tilegnelses- og samspillsprosessen skjer innenfor rammene av en sosial og samfunnsmessig kontekst (Figur 2). De tre dimensjonene i læring benevner Illeris (2012) som innhold, drivkraft og samspill (Figur 2). Innholdsdimensjonen dreier seg om kunnskap, forståelse og ferdigheter. Gjennom den prøver vi generelt å skape mening og mestring og derigjennom styrker vi vår funksjonalitet. Drivkraftdimensjonen omfatter motivasjon, følelser og vilje. Gjennom den prøver vi generelt å opprettholde en mental og kroppslig balanse, og samtidig utvikler vi vår sensitivitet. Samspillsdimensjonen omfatter handling, kommunikasjon og samarbeid. Gjennom den prøver vi generelt å oppnå en sosial og samfunnsmessig integrasjon som vi finner akseptabel, og samtidig utvikler vi vår sosialitet. Illeris sin grunnleggende tese er at all 6 læring involverer disse to prosessene og tre dimensjonene, og at alle disse alltid må tas med i betraktningen hvis en forståelse eller analyse av en læringssituasjon eller et læringsforløp skal være utførlig. læring involverer disse to prosessene og tre dimensjonene, og at alle disse alltid må tas med i betraktningen hvis en forståelse eller analyse av en læringssituasjon eller et læringsforløp skal være utførlig. Figur 2: Læringens dimensjoner (Illeris, 2012, s. 43). Figur 1: Læringens fundamentale prosesser (Illeris, 2012, s. 41). Figur 2: Læringens dimensjoner (Illeris, 2012, s. 43). Figur 1: Læringens fundamentale prosesser (Illeris, 2012, s. 41). Figur 1: Læringens fundamentale prosesser (Illeris, 2012, s. 41). Figur 1: Læringens fundamentale prosesser (Illeris, 2012, s. 41). Figur 2: Læringens dimensjoner (Illeris, 2012, s. 43). Veiledning Definisjoner av veiledningsbegrepet er mange. Sentralt i forståelsen av begrepet står ofte både læringsaspektet og det relasjonelle aspektet. Fokuset er rettet mot den lærende og at veiledning inkluderer en form for samspill eller samarbeid (Tveiten, 2013). Ifølge Tveiten (2013, s. 21) defineres veiledningsbegrepet som «… en formell, relasjonell og pedagogisk istandsettingsprosess som har til hensikt at fokuspersonens mestringskompetanse styrkes gjennom dialog basert på kunnskap og humanistiske verdier». Istandsettingsprosessen har en start og en avslutning, og man skal selv være i stand til å gjøre det man har forutsetning til. Veiledningsansvaret er delt, der veilederen ansvarliggjøres med å legge til rette for istandsetting. Fokuspersonens ansvar blir å videreføre det som han eller hun er i stand til, for eksempel endringer. Dette er en formell virksomhet som går inn i et arbeidsforhold. Den rasjonelle delen henspeiler relasjonen mellom veileder og fokuspersonen, mens den pedagogiske istandsettingsprosessen inkluderer læring, vekst, utvikling og mestring. Videre mener Tveiten (2013) at dialog er hovedformen i veiledningen som baseres på humanistiske verdier og kunnskaper. 7 Lærernes tilbakemeldinger som en del av veiledningen Tilbakemeldinger er, ifølge Gamlem (2014), informasjon som blir gitt, mottatt eller søkt som handler om kvalitetsaspekt ved egen prestasjon eller forståelse. Formålet med en tilbakemelding er at informasjonen skal gi retning for videre arbeid og læring. De bør være av en karakter som gir studenten en opplevelse av meningsfull informasjon som kan brukes og blir brukt fordi den kan styrke deres forståelse, kunnskap og kompetanse gjennom lærling (Gamlem, 2015). Ifølge Hoel (2008) vil skriftlige tilbakemeldinger fra veileder til student inngå i en læringsprosess, der formålet er å føre studenten inn i en fagkultur og utvikling som fagperson. Dette innebærer at studenten får øving i kritisk sans og refleksjon. Lauvås og Handal (1998) har gjennom mange års arbeid med veiledning i høyere utdanning utarbeidet strategier for veiledning. Disse innbefatter å synligjøre det som er bra, å peke på det som er verneverdig i teksten, å klargjøre det som er uklart, gi retning til det uferdige arbeidet og gi energi og lyst til å arbeide med det som gjenstår. I en studie fra 2013 er disse strategiene videreført og videreutviklet. Handal, Lycke og Lauvås (2013) oppsummerer veiledningsstrategier innenfor høyere utdanning i følgende kategorier: vurderende kommentarer (anerkjennende og kritiske), råd, metakommunikasjon, invitasjon til studenten om å ta del i dialogen og prosess- og produktveiledning. Metodologisk tilnærming Med bakgrunn i problemstillingen, som er knyttet til studenters opplevelse av undervisning og veiledning, har studien en fenomenologisk- hermeneutisk tilnærming. Fenomenologien har som formål å fokusere på beskrivelser av menneskers erfaringer, nærmere bestemt deres livsverden (Alvesson & Sköldberg, 2008). Den tyske filosofen Edmund Husserl (1859-1938) regnes som den moderne fenomenologiens grunnlegger. Livsverdensbegrepet som Husserl utviklet er en betegnelse på den kunnskap som danner et nødvendig utgangspunkt for all forståelse, og som vi stort sett tar for gitt. Husserls ide var at forskeren skulle legge bort mest mulig av faktuelle teorier om verden, og gå Zu den Sachen selbst (Alvesson & Sköldberg, 2008, s. 165). Dette kan forstås som å utforske vesenets egenart ved å skildre og forstå det som utforskes så nøytralt som mulig, uten annen påvirkning. 8 Målet med hermeneutikken er å oppnå en gyldig forståelse av meningen i teksten. Utgangspunktet er å forstå teksten, og at meningen hos en del kan forstås hvis den settes i sammenheng med helheten (Alvesson & Sköldberg, 2008, s. 193). Sentralt begrep er den hermeneutiske sirkelen, der delen kan oppfattes ut i fra helheten og omvendt. Den hermeneutiske sirkelen har følgende grunnsteg: 1)Tolkningsmønsteret vokser frem fra særegenheter i teksten. Et mønster som bringer en dypere forståelse- refleksjon og forforståelse. 2) Tekst – plassere teksten i sin kontekst. Tolkningsmønsteret justeres via større innsikt- og hva som oppfattes som fakta. 3) Dialog- stiller spørsmål til teksten. Forforståelsen endres underveis. Veksler mellom gammel og ny forståelse. Er i dialog med leseren. 4) Deltolkninger skjer under hele prosessen sett ut fra visse bakgrunnsforestillinger (Alvesson & Sköldberg, 2008, s. 194). Målet med hermeneutikken er å oppnå en gyldig forståelse av meningen i teksten. Målet med hermeneutikken er å oppnå en gyldig forståelse av meningen i teksten. Ut i fra den nevnte metodologiske tilnærming har vi i analysearbeidet valgt Lindseth og Norbergs (2004) fenomenologiske-hermeneutiske fremgangsmåte basert på Giorgis (2009) trinnvise fortetning. Denne metoden bevarer meningen til den enkelte student, samtidig som den gir rom til tolking av tekstene (Lindseth & Norberg, 2004). Forskningsetiske prinsipper ble fulgt gjennom studien (Kvale, Brinkmann, Anderssen & Rygge, 2015). Studentene ble informert før oppstart av studien, med informasjon om at det var frivillig å skrive logg og at de kunne trekke seg underveis. Forfatterne var begge involvert i både undervisning av skriveverkstedet og i tilbakemeldingene på oppgavene, noe som har metodisk utfordringer som vi har prøvd å være bevisst. Utvalg Deltagerne i denne studien er 12 førsteårsstudenter ved yrkesfaglærerutdanningen i Trondheim. Yrkesfaglærerutdanningen er samlingsbasert studium med tre samlinger hvert semester. Studentene har fagbakgrunn innenfor helse- og oppvekstfag. Eksempler på yrker er helsefagarbeider, barne- og ungdomsarbeider og hudpleier. Alle er kvinner med en aldersspredning fra 27 - 46 år. Gjennomsnittsalderen er 39 år. Studentene har mange års yrkeserfaring innenfor sin yrkeskompetanse/fagbrevområde. De ble rekruttert til studien gjennom å takke ja til studieplass høsten 2014. Deltagerne i denne studien er 12 førsteårsstudenter ved yrkesfaglærerutdanningen i Trondheim. Yrkesfaglærerutdanningen er samlingsbasert studium med tre samlinger hvert semester. Studentene har fagbakgrunn innenfor helse- og oppvekstfag. Eksempler på yrker er helsefagarbeider, barne- og ungdomsarbeider og hudpleier. Alle er kvinner med en aldersspredning fra 27 - 46 år. Gjennomsnittsalderen er 39 år. Studentene har mange års yrkeserfaring innenfor sin yrkeskompetanse/fagbrevområde. De ble rekruttert til studien gjennom å takke ja til studieplass høsten 2014. 9 Tiltakene Det første tiltaket, et todagers kurs med innføring i å skrive en akademisk oppgave, ble organisert på den første samlingen. Undervisningen var lagt opp som en veksling mellom å presentere teori om å skrive en slik oppgave og praktiske øvelser. Vi startet med en gjennomgang av kjennetegn ved akademiske skriving som beskrevet innledningsvis (Busch, 2013). Deretter hadde vi en gjennomgang av IMRoD- struktur (Hoel, 2008). Mellom presentasjonene hadde vi lagt inn tid til praktiske øvelser. På dag to ble det presentert en klargjøring av hva som ligger i ulike begrep som ofte brukes i oppgavetekster (eksempelvis beskriv, nevn, drøft) samt en gjennomgang av krav til litteraturlister i akademiske tekster og hvordan kildene refereres til i flytende tekst (VIKO, 2014). Ved kursets slutt hadde studentene utarbeidet en skisse til en akademisk oppgave med forside, innholdsfortegnelse, innledning med en egenformulert problemstilling, hoveddel, avslutning og litteraturliste. Det andre tiltaket var ulike og varierte former for tilbakemeldinger på oppgaver. Alle studenter fikk skriftlige tilbakemeldinger på fem oppgave det første semesteret (to i gruppe, tre individuelle). I tillegg fikk de en muntlig tilbakemelding (30 minutter per student) hvor de selv kunne bestemme hva som skulle være i fokus. Den muntlige tilbakemeldingen ble gitt mens studentene var på sin tredje og siste samling i første semester. Noen studenter fikk også digitale tilbakemeldinger. De digitale tilbakemeldingene ble gitt ved hjelp av en skjermvideo produsert ved hjelp av programvarer Screencast-O-Matic hvor lærerne ga muntlige tilbakemeldinger til studentene, samtidig med at studentenes oppgaver ble synlig i skjermbildet. Videoene hadde en varighet på ca. 15 minutter, og ble sendt tilbake til studentene sammen med den skriftlige tilbakemeldingen på læringsplattformen It’s learning. De digitale tilbakemeldingene ble gitt på en gruppeoppgave og en individuell oppgave. Studentene fikk beskjed om å lese den skriftlige tilbakemeldingen før de så videoen. De tre formene for tilbakemelding er i utgangspunktet gitt på de samme språklige og tekstlige forhold slik at de i hovedsak har samme innhold. De skriftlige tilbakemeldingene danner dermed utgangspunktet for de muntlige og digitale. Vi vil med utgangspunkt i teori om tilbakemelding (Lauvås & Handal, 1998; Handal, Lycke & Lauvås, 2013) gi eksempler på konkrete tilbakemeldinger som ble gitt i de skriftlige oppgavene. Eksempel på en tilbakemelding på det som er bra er kommentarer som «Ryddig innholdsfortegnelse». Eksempel på en verneverdig tilbakemelding i teksten er «Casen du har laget gir en god beskrivelse av situasjonen og er et godt utgangspunkt for drøfting». Tiltakene Eksempel på klargjørende 10 tilbakemelding er «Mye bra! Hvis du skal få til mer drøfting kan du prøve å se saken fra et annet synspunkt. Hva hadde skjedd hvis hjelpepleieren ikke var så imøtekommende?» Eksempel på tilbakemeldinger som gir retning til det uferdige arbeidet er «Se nærmere på strukturen- alle oppgaver skal ha en innledning, hoveddel og avslutning». Eksempel på tilbakemeldinger som gir lyst og energi til å arbeide med det som gjenstår er «Du er på rett vei, stor forbedring fra sist!» Til sist kommer et eksempel på en tilbakemelding på invitasjon til studenten om å ta del i dialogen og prosessen: «Fint at du peker på læreren som rollemodell. Hvordan kan læreren som rollemodell bidra til relasjonsbygging mellom lærer-elev?» Etter at vi har gitt kommentarer direkte i teksten, avslutter vi med en kort oppsummerende tekst der vi trekker ut noen essenser. Datainnsamling Dataene i studien er samlet inn gjennom to refleksjonslogger. Studentene skulle svare på spørsmål om deres opplevelser av innføringskurset og de ulike formene for tilbakemeldinger som ble gitt. I den første loggen ble blant annet følgende spørsmål stilt: Hvilke opplevelser har du av skrivekurset? I den andre refleksjonsloggen ble følgende spørsmål stilt: 1) Kan du beskrive dine opplevelser med å få skriftlig tilbakemelding på ulike oppgave? 2) Kan du beskrive dine opplevelser med å få muntlig tilbakemelding? 3) Kan du beskrive dine opplevelser med å få digital tilbakemelding? Studentene leverte refleksjonsloggene på It’s learning etter første og tredje samling. Alle 12 bevarte samtlige spørsmål på begge loggene (til sammen 24 logger). Analyse Det er også fint å få tilbakemelding på det som er bra. Det gjør noe med selvfølelsen og gjør ar man har lyst til å fortsette med skriveprosessen. Føler at noe skriftlig arbeid blir bra, men blir også usikker Tilbakemelding på hva som kan forandres for å bli bedre og for å komme videre er bra Tilbakemelding på det som er bra påvirker selvfølelsen og lysten til å fortsette med skrivingen Å føle på usikkerhet Å få tilbakemelding på hva som kan forandres Å få ros påvirker selvfølelsen og lysten til videre arbeid Å få bekreftende tilbakemelding Å få bekreftende tilbakemelding Å få motiverende tilbakemelding Tabell 1: Eksempel på koding av data. Tabell 1: Eksempel på koding av data. Tabell 1: Eksempel på koding av data. Analyse Analysearbeidet er som tidligere nevnt basert på Lindseth og Norbergs fenomenologiske- hermeneutiske fremgangsmåte (2004). De anvender formuleringer i et fenomenologisk språk ved utforming av temaene. Bruk av verb blir benyttet i stedet for substantiver, for eksempel: Å få bekreftende tilbakemelding. Fremgangsmåten beskrives i 3 faser: 1) Naiv lesning. Vi leste gjennom loggene flere ganger for å kunne forstå meningen som en helhet. Dette er den første antagelsen om meningen i teksten, derfor må den valideres i den påfølgende analysen. 2) Strukturell analyse er i dette tilfelle en tematisk strukturell analyse som innebærer at en leser hele teksten, deretter deles teksten i flere meningsenheter, det vil si utdrag fra teksten. Analysearbeidet er som tidligere nevnt basert på Lindseth og Norbergs fenomenologiske- hermeneutiske fremgangsmåte (2004). De anvender formuleringer i et fenomenologisk språk ved utforming av temaene. Bruk av verb blir benyttet i stedet for substantiver, for eksempel: Å få bekreftende tilbakemelding. Fremgangsmåten beskrives i 3 faser: 1) Naiv lesning. Vi leste gjennom loggene flere ganger for å kunne forstå meningen som en helhet. Dette er den første antagelsen om meningen i teksten, derfor må den valideres i den påfølgende analysen. 2 Strukturell analyse er i dette tilfelle en tematisk strukturell analyse som innebærer at en leser hele teksten, deretter deles teksten i flere meningsenheter, det vil si utdrag fra teksten. Deretter ble teksten delte opp i meningsenheter – deler av en setning. Meningsenhetene ble fortettet og skrevet om til dagligspråk med korte formuleringer (sammentrekning). Deretter blir teksten delt opp i undertema og tema. 3) Helhetlig forståelse. I den siste fasen blir Deretter ble teksten delte opp i meningsenheter – deler av en setning. Meningsenhetene ble fortettet og skrevet om til dagligspråk med korte formuleringer (sammentrekning). Deretter blir teksten delt opp i undertema og tema. 3) Helhetlig forståelse. I den siste fasen blir 11 undertema og tema reflektert over og satt i sammenheng med problemstillingen, samtidig som teksten ble lest gjennom som en helhet med en naiv forståelse. Et eksempel på denne kodingen presenteres i tabellen nedenfor. Meningsenhet Sammentrekning Undertema Tema Noe av det jeg skriver føler jeg blir bra … mens andre ting jeg skriver kan jeg bli litt usikker på om jeg er inne på noe eller helt på villspor. Derfor syns jeg det er kjempeviktig med skriftlig tilbakemelding. Godt å få høre hva jeg kan forandre på for å bli bedre (…) man har noe å jobbe videre på. Innføringskurs i akademisk skriving Et innføringskurs i akademisk skriving med presentasjon av teori, forklaringer og praktiske øvinger var det første tiltaket vi iverksatte. To av temaene som stiger fram av analysen er at skrivekurset ble opplevd som nyttig og at samarbeid med medstudenter var av stor betydning for læringsutbytte. Alle studentene gir på ulike vis uttrykk for at de har hatt stort utbytte av å delta i undervisningen, og mange gir uttrykk for at de ikke har noe kjennskap til akademisk skriving fra før. En student skriver: «….jeg visste ikke noe om hvordan en oppgave skal skrives». Andre sier: «Nå har jeg en mye større forståelse av hvordan en oppgave skal bygges opp og hva den skal inneholde». Noen konkretiserer hva som var nyttig. Eksempler på dette er: «Jeg fikk en del informasjon om hvordan innhold og oppsett skal være» og «Kjempebra at vi studenter fikk øve på å problemformulere, samt å komme litt i gang med en oppgave som øving». «Skrivekurset var et «must» for min del!» er det en student som oppsummerer. Illeris (2012) hevder at all læring involverer både tilegnelse- og samspillsprosesser. Det faglige innholdet i presentasjonene fra skrivekurset favner innholdsdimensjonen mens de praktiske øvelsene favner samspillsdimensjonen. For oss ble det veldig tydelig at samspillet mellom studentene i de praktiske øvelsene og samspillet med oss som lærerne i undervisningen hadde stor betydning for læringsutbytte og opplevelse av kursets nytteverdi i sin helhet. Opplevelse av nyttig innhold og godt samspill er en viktig drivkraft i læreprosessen og bidrar til vilje og motivasjon til å fortsette skrivearbeidet ifølge Illeris (2012). Vi opplever at studentenes logger gjenspeiler dette samtidig som vi ser tydelig at akademisk skriving kan virke vanskelig og skremmende i starten av et universitetsstudium. Vi fikk også erfare at dårlig samspill kan ha motsatt effekt. En student skriver: «For min del ble samarbeidet med medstudent lite vellykket, som førte til at jeg ble sittende igjen med lite utbytte av dagen». Opplevelsen av mislykket samarbeid kan svekke motivasjonen og viljen til videre innsats. Rienecker og Jørgensen (2006) peker på at å lære akademisk skriving og å lære fag støtter hverandre gjensidig. Dette innebærer at arbeid med skriving i fagene i lærerutdanningen ikke nødvendigvis går på bekostning av opplæring i andre fag. Den samme forståelsen finner vi i Kunnskapssløftet der ideen om skriving i alle fag skal styrke både skrivekompetanse og læring i de ulike fagene. Resultater og diskusjon Akademisk skriving er et nytt landskap å bevege seg inn i for mange yrkesfaglærerstudenter. De skal ikke bare uttrykke seg på en relevant måte, de skal også forholde seg til klare retningslinjer for innhold, struktur og referansesystemer som er sentrale spilleregler når de skal besvare oppgaver i studiet. For å veilede og støtte studentene i læringsprosessen av disse spillereglene har vi i tillegg til å undervise i akademisk skriving tatt i bruk ulike former for tilbakemelding på oppgaver. Ifølge Boge, Markhus, Moe og Ødegaard (2002) innebærer veiledning i profesjonsutdanningene både undervisning og studieveiledning. Med utgangspunkt i skrivekurset og de ulike formene for tilbakemelding vil vi i det følgende diskutere temaene som kom til syne gjennom analysen av studentenes refleksjonslogger. I tillegg har vi valgt å avslutte med noen betraktninger om skriving av refleksjonslogger i lærerutdanning. 12 Ulike former for tilbakemelding Det andre tiltaket vi iverksatte var skriftlige, muntlige og digitale tilbakemeldinger på oppgaver. Følgende temaer kom til syne basert på spørsmål om hvordan studentene opplevde å få ulike former for tilbakemelding: Å få bekreftende tilbakemelding, Å få oppklarende tilbakemelding, Å få motiverende tilbakemelding og Å ha relasjon i veiledning. Resultatene knyttet til de skriftlige tilbakemeldingene presenteres først, deretter resultatene knyttet til de muntlige og digitale tilbakemeldingene. Innføringskurs i akademisk skriving Hoel (2008) understreker at det er viktig at studentene får en innføring i akademisk skriving på et tidlig tidspunkt i studiet, slik at de får en fast ramme å forholde seg til. Hun gir to konkrete råd til nye studenter. Det ene handler om å skape en god struktur med overordnede og underordnede nivå i teksten. Det andre er å være pinlig nøyaktig med referanser, sitat og litteraturliste. Å mestre dette håndverket gir trygghet, skriver hun. 13 Tiltak som fremmer trygghet er noe førsteårsstudenter i yrkesfag sårt trenger, noe vi vil komme tilbake til. Tiltak som fremmer trygghet er noe førsteårsstudenter i yrkesfag sårt trenger, noe vi vil komme tilbake til. Skriftlige tilbakemeldinger Helt siden studiets oppstart har studentene fått skriftlige tilbakemeldinger. Studentene opplever at skriftlige tilbakemeldinger viser vei i det videre skrivearbeidet. En student skriver at det er godt å få høre hva som kan forandres i oppgaven for å kunne gjøre den bedre, og at tilbakemeldinger gjør at man vet hva man kan arbeide videre med. Å få kommentarer direkte i teksten oppleves som konkret og nyttig. Konkrete kommentarer gjør det lettere når oppgaven skal videreutvikles. Likeså peker studentene på at «Fine og oppløftende avslutninger gir motivasjon og mening». Studentene beskriver her det som skjer i møtet mellom læringsinnhold, drivkraft og omverden illustrert i Figur 1 og 2 (Illeris, 2012). Skriftlige tilbakemeldinger i teksten gir mening og sammen med motivasjon for læring tilegner studenten seg nye kunnskaper og ferdigheter i skrivearbeidet. Læringen skjer i individet, men foregår i et samspill med læreren som har gitt disse kommentarene. Selve innholdet i kommentarene er av stor betydning. Flere studenter peker på hvor viktig det er å få ros. En student sier: «Det er fint å få tilbakemelding på det som er bra. Det gjør noe med selvfølelsen og gjør at man har lyst til å fortsette med skriveprosessen». En annen student skriver at det er viktig med kommentarer som viser hva som er bra, men også kommentarer på hva som det ikke trengs å gjøres noe med. Slike kommentarer virker også som bekreftelse på at en er på riktig vei. En student skriver: «Noe av det jeg skriver føler jeg blir bra, mens andre ting jeg skriver kan jeg bli litt usikker på om jeg er inne på noe, eller helt på villspor». Hun 14 skriver videre at skriftlige tilbakemeldinger derfor er svært viktig. Hun opplever at lærerens kommentarer bekrefter eller avkrefter om hun er på riktig vei. Det virker som at kommentarbobler underveis i teksten som roser, oppklarer og bekrefter, med klare instrukser om hva som er bra og hva som kan videreutvikles oppleves som nyttig. Dette er i samsvar med andre veiledningsstudier (Gamlem, 2015; Hoel, 2008; Lauvås & Handal, 1998). Studentene peker også på ulike utfordringer knyttet til skriftlige tilbakemeldinger. Flere studenter skriver at det er ikke alltid de forstår hva tilbakemeldingene betyr eller hvordan de skal tolke dem. Etter hvert som studentene får flere tilbakemeldinger blir dette lettere. Skriftlige tilbakemeldinger En students skriver: «Etter å ha sammenlignet den skriftlige tilbakemeldingen på begge individuelle oppgave skjønner jeg bedre budskapet i dem, samt at jeg begynner å forstå ordlyden i lærerens språk». Som veiledere er det viktig å være å være klar over at kommentarer kan oppfattes og tolkes ulikt av studentene. Hvor godt student og lærer kjenner hverandre og hvor studenten er i sin læringskurve har betydning både for hvordan tilbakemeldinger blir tatt imot og hvordan innholdet i dem blir forstått. Det er derfor viktig å bli kjent med studentene slik at læreren kan tilpasse antallet kommentarer, balansen mellom utviklingskommentarer og ros og ikke minst innholdet i responsen til den enkelte student. I følge både Illeris (2012) og Tveiten (2013) er relasjonen mellom veileder og student helt sentral for læring, vekst, utvikling og mestring. Videre er det viktig at studentene arbeider aktivt med tilbakemeldingene slik at de trener seg opp til å tolke veiledningskommentarer. En student viser dette når hun skriver at etter flere tilbakemeldinger skjønner hun bedre lærerens «språk» og «budskap». Ifølge Tveiten (2013) er ansvaret i veiledningen delt. Veilederen har blant annet ansvar for å legge til rette for at studenten skal ha mulighet til å videreføre det som hun eller han er i stand til. Studenten har ansvar for å gjøre endringer knyttet til veilederes kommentarer som oppfattes som meningsfulle. Andre utfordringer knyttet til skriftlige tilbakemeldinger er omfanget av kommentarer. En student forteller at det oppleves som vanskelig å utvikle seg hvis det er få tilbakemeldinger. Vedkommende foretrekker heller mange og negative tilbakemeldinger enn få og bare positive. Noen studenter er dermed mer opptatt av utviklingskommentarer enn mengde ros, mens andre mister motivasjonen om tilbakemeldingen ikke inneholder tilstrekkelig med ros. En student skriver: «Uansett hvor dårlig oppgaven var følte jeg det burde kommet fram noe som motiverte meg for videre arbeid (…) men motivasjonen forsvant med tilbakemeldingen». 15 Med få og uklare kommentarer i tilbakemeldingen er det vanskelig å vite hva en skal jobbe videre med. Er det derimot mange kommentarer, kan det også virke demotiverende. Antallet kommentarer må derfor sees i sammenheng med hva som står i dem. Noen studenter utrykker klart at de fortrekker mange kommentarer som kan gjøre oppgaven bedre fremfor påpekning om hva som er bra, andre sier de mister motivasjonen dersom det er mange kommentarer som peker på hva som kan utvikle teksten videre og få kommentarer med ros. Skriftlige tilbakemeldinger En del studenter gir uttrykk for stor usikkerhet og sårbarhet. De bruker uttrykk som «redd for å være på villspor». Noen ber også om skriftlige kommentarer som bekrefter hvilke deler av teksten som er bra og hvilke deler som ikke krever endringer. For noen er det ikke tilstrekkelig at deler av teksten står urørt, det må en konkret bekreftelse til for at de skal være sikre på at innholdet holder mål. Andre studenter etterspør tilbakemeldinger på hvor de ligger i nivå slik at de vet at oppgaven holder til en ståkarakter. I starten av et nytt studium er det derfor svært viktig med mye bekreftelse og ros. Studenter skriver at selv om den innleverte teksten er mangelfull, så oppleves ros som motivasjon til å videreutvikle oppgavene. Ros fremmer studentenes motivasjon, mestring og selvfølelse. Dette samsvarer med Lauvås og Handal (1998) som understreker viktigheten av å løfte opp det som er bra i en tekst. Å utvikle skriveferdigheter tar tid. Når en blir bedre kjent med den enkelte, kan en som veileder utfordre mer. Å trygge studentene i starten av et studium er derfor en viktig del av veiledningsarbeidet (Lauvås, Hofgaard & Handal, 2013). Gamlem (2014) peker på viktigheten av emosjonell støtte. I sitt doktorgradsarbeid skriver hun at emosjonell støtte er sentralt for å skape trygghet og et positivt læringsmiljø. Hun skriver videre at det er behov for å styrke både lærere og elever sin kompetanse på tilbakemelding og samhandling som støtte for læring. Muntlige tilbakemeldinger Muntlige tilbakemeldinger på oppgavene ble gitt mens studentene var på sin siste samling i første semester. De skriftlige tilbakemeldingene tidligere i semesteret dannet utgangspunktet for de muntlige. På spørsmål om hvilke opplevelser studentene har av muntlige tilbakemeldinger, kommer det tydelig fram at også denne formen for veiledning er viktig for den videre skriveprosessen. Studentene løfter spesielt frem at misforståelser i de skriftlige tilbakemeldingene ofte ble oppklart gjennom dialog med læreren. En av studentene meddeler: «Muntlig tilbakemelding syns jeg var bra å få fordi noe av den skriftlige tilbakemeldingen kan jeg bli usikker på. Litt usikker på om jeg forstod helt hva lærerne mente». Videre kommenterer en annen student: «Det er enklere å spørre om råd og forstå hva som menes med 16 de ulike kommentarene ved å snakke sammen». Studenten som forteller om motivasjonen som forsvant med den skriftlige tilbakemeldingen, svarer slik på spørsmålet om opplevelser med muntlige tilbakemeldinger: «I etterkant av denne samtalen følte jeg meg motivert for å jobbe mer med oppgaven og ser absolutt ikke mørkt på å sette i gang med framtidig oppgaver». Utsagnene over illustrerer at studentene opplever at dialog og personlig møte med læreren er viktig. En student skriver: «… fint å snakke med læreren innimellom». En annen student uttrykker: «Det å snakke i telefonen for å få oppklaring på punkter i oppgave er ikke det samme som å sitte i samme rom». Muntlige tilbakemeldinger bidrar til å bygge relasjoner mellom lærer og student i tillegg til at denne formen for tilbakemelding oppleves både oppklarende, bekreftende og motiverende. Studien tyder på at studentene har behov for muntlige tilbakemeldinger i tillegg til de skriftlige. Å ha mulighet for å snakke med læreren innimellom beskrives av flere studentene som en fordel. De opplever da å ha mulighet å stille spørsmål og oppklare det som de ikke forstod i de skriftlige tilbakemeldingene. Muntlig tilbakemelding etter en skriftlig ser derfor ut til å være hensiktsmessig. Illeris (2012) fremhever også betydningen av samspillsdimensjonen i sine modeller for læring. Lærer og student inngår i en felles handling om oppgaven der kommunikasjon og samarbeid er viktige prosesser for videre utvikling i skrivearbeidet. Digitale tilbakemeldinger Digitale tilbakemeldinger ble gitt ved at studentene fikk se oppgaven med lærerens kommentarer i skjermbildet samtidig som læreren snakket om innholdet i de skriftlige kommentarene. En student skriver dette: Jeg skjønte responsen bedre når jeg leser selv og får det som film. Føler bedre at jeg «fikk kontakt» og var mer konsentrert enn når jeg bare får skriftlig respons på ark. Jeg satt igjen med et mer positivt bilde på oppgaven enn hvis det bare var skrevet. Det er tydelig at å lytte til hva som blir sagt og se kroppsspråket til læreren påvirker hvordan innholdet i kommentarene oppleves og blir mottatt. Tilbakemeldinger gitt på denne måten virker ifølge studentene positivt inn på hvordan kommentarene forstås. En annen student skriver: «Det er nesten som å ha veilederen hos meg gjennom veiledningen, og det er en stor fordel for meg». Flere utsagn tyder også på at digitale tilbakemeldinger gir studentene en 17 følelse av at læreren bryr seg om dem. En student uttrykker dette slik: «Følte at læreren bryr seg mer og får kontakt med oss studenter med å «gidde» å sette seg fremfor et kamera å fortelle». En annen side ved digitale tilbakemeldinger som ble synlig i studentenes logger, er muligheten til å se og høre tilbakemeldingen flere ganger. En student forteller at hun i arbeidet med oppgaven videre så videoen flere ganger. Hun skriver: «Det er lettere å gå tilbake og gjøre endringer i oppgaven når en får slike tilbakemeldinger (…)». Studentene som fikk digitale tilbakemeldinger var entydig positive og svært begeistret for denne formen for veiledning. De opplever at lærerne bryr seg mer om deres faglige utvikling når det settes av tid til muntlig og digital tilbakemelding. Det er liten tvil om at de muntlige og digitale tilbakemeldingene supplerer de skriftlige på måter som oppleves som viktige og positive. Dessuten kan studentene gjennom det digitale mediet danne seg et annet bilde av oppgaven. Flere studenter forteller om at de sitter igjen med et mer positivt bilde av oppgaven etter muntlig og digital respons. Studentene har ikke pekt på utfordringer knyttet til muntlige og digitale tilbakemeldinger, men som lærerutdannere på et samlingsbasert studium bør disse nevnes. En av utfordringene knyttet til å gi flere former for tilbakemelding på de samme oppgavene er bruken av tid. Det må settes av tid til muntlig tilbakemelding til hver enkelt student på tidspunkt som ikke hindrer fellesundervisningen. Dette krever god timeplanlegging. Digitale tilbakemeldinger Å produsere digitale responser på 15 minutter, slik vi gjorde i denne studien, tar gjerne en halv time ekstra når skriftlige kommentarer er utarbeidet i forkant av skjermopptakene. Denne formen for tilbakemelding forutsetter at læreren er villig til å lage digitale tilbakemeldinger samt å dele videoen med andre. Vår erfaring er at dette er vel anvendt tid på denne type studium. Studentenes opplevelser av skrivekurset og ulike former for tilbakemelding viser at yrkesfaglærerstudenter har et stort behov for tett oppfølging og mye støtte i læringsprosessen med å videreutvikle sine skriveferdigheter. Skriving av refleksjonslogger i lærerutdanningen Dataene i denne studien ble samlet inn gjennom refleksjonslogger. I loggskriving fanger man opp studentenes egne erfaringer, forståelse og oppfatninger ut fra gitte spørsmål. Illeris (2012) peker på at refleksjon er tett knyttet til læring. Sammen med skrivekurset og ulike former for 18 tilbakemelding kan regelmessig skriving av refleksjonslogger i lærerutdanningen være et godt hjelpemiddel for å styrke studentenes skriveferdigheter. Illeris (2012) peker på at begrepet refleksjon dreier seg både om ettertanke og om speiling. Ved ettertanke reflekterer man nærmere over noe, mens man ved speiling speiler sin opplevelse eller forståelse av noe i sitt eget selv eller i en annens reaksjon. I forhold til opplæring i akademiske skriving vil vi si at studentene konsentrerer seg om betydningen som undervisning og lærernes tilbakemeldinger har hatt for dem, vurderer dem med sin egen identitet som målestokk. Loggene i denne studien har gitt studentene viktig skrivetrening, og oss lærere viktig informasjon om studentenes behov og tanker om egen skrivekompetanse. Ifølge Eik-Nes (2008) kan loggskriving være et unikt verktøy for å bearbeide usikkerhet når de skal utforme egen rolle som student. Studenter har ulike læreforutsetninger og dermed ulike behov for støtte i skriveprosessen. Logger kan være et multiverktøy som kan fylle ulike behov for de forskjellige lærerstudenter (Klemp, 2012). Hoel (2002) peker på loggskriving som en viktig mediator for læring knyttet til respons som et nødvendig aktiviserende prinsipp. Gjennom loggskriving og responsarbeid kan lærerutdannere støtte og utfordre lærerstudenter i refleksjonsarbeidet. Gjennom regelmessige refleksjonslogger om ulike temaer i studiet kan opplæring i akademisk skriving og fag støtte hverandre gjensidig, slik Rienecker og Jørgensen (2006) peker på. Oppsummering Hensikten med denne studien er å utforske hvordan et innføringskurs i skriving av akademiske oppgaver og ulike former for tilbakemelding oppleves av førsteårsstudentene i yrkesfaglærerutdanningen. Resultat i studien tyder på at studentene opplever at et innføringskurs i akademisk skriving er nødvendig for den videre skriveprosessen. De fleste studentene har liten eller ingen erfaring med oppgaveskriving i høyere utdanning. I tillegg er det relasjonelle aspektet i veiledning sentralt. Ros, støtte og personlig kontakten med lærer fremheves som avgjørende for motivasjon til videre arbeid. Dette er aspekter som på ulike måter er knyttet til både skriftlige, muntlige og digitale tilbakemeldinger. De ulike formene for tilbakemeldinger har sine styrker og sine utfordringer. Skriftlig tilbakemeldinger oppleves som et «must» i lærerutdanningen, kommentarbobler underveis i teksten sammen med en oppsummerende tekst til slutt er en form for skriftlig tilbakemelding studentene opplever som 19 konkret, oppklarende, bekreftende, meningsfull og motiverende. Muntlige tilbakemeldinger har sin styrke i at de oppleves som oppklarende og at dialog med lærer styrker betydningen av de skriftlige tilbakemeldingene. Muligheten til å kunne se og lytte til tilbakemeldingene flere ganger trekkes frem som spesielt gunstig i forhold til de digitale. Resultatene underbygger viktigheten av variert veiledning i arbeidet med å kvalifisere yrkesfaglærerstudenter i akademiske skriveferdigheter. Funnene i denne studien samsvarer godt med Illeris (2012) sin læringsteori der både innhold-, drivkraft- og samspillsdimensjonen gjennom tilegnelses- og samspillsprosesser er involvert i studentenes læringsprosesser. Studentene gir uttrykk for at tilbakemeldinger gir liten læringseffekt hvis veilederen ikke tar hensyn til disse aspektene og kompleksiteten i læringsprosessen. Det er lærerinstitusjonene sitt ansvar å legge til rette for at studentene får tilstrekkelig kunnskap, øving og oppfølging i skrivearbeidet. I Forskrift om rammeplan for den treårige yrkesfaglærerutdanningen står det at studiet skal kvalifisere studenter til å kunne gi læringsfokuserte tilbakemeldinger, og de skal arbeide systematisk med grunnleggende ferdigheter (Kunnskapsdepartementet, 2013). Hellne-Halvorsen (2014) sin doktoravhandling om skrivepraksiser på yrkesfaglige utdanningsprogrammer viser at yrkesfaglærere trenger sammensatte skrivekompetanser. Det er derfor svært viktig at fremtidige yrkesfaglærere får opplæring og trening med ulike skrivekompetanser i løpet av lærerstudiet slik at de kan arbeide systematisk med grunnleggende ferdigheter og tilbakemeldinger i egen undervisning. Vi ser det som nødvendig at skriving får mer plass i yrkesfaglærerutdanningen, både for å holde tritt med de øvrige lærerutdanningene og for at studentene skal stå godt rustet til videre studier og en fremtidig lærerjobb. 20 Referanser Alvesson, M. & Sköldberg, K. (2008). Tolkning och reflektion: Vetenskapsfilosofi och kvalitativ metod. Lund: Studentlitteratur. Boge, M., Markus, G., Moe, R. & Ødegaard, E. E. (2002). Læring gjennom veiledning: Meningsskaping i grupper (2.utg.). Bergen: Fagbokforlaget. Busch, T. (2013). Akademisk skriving for bachelor- og masterstudenter. Bergen: Fagbokforlaget. Eik-Nes, N. L. (2008). Dialogging: Negotiating Disciplinarty Identity through the Medium of Logs. (Doktorgradsavhandling), NTNU, Trondheim. Firing, K., Klomsten, A. T. & Moen, F. (2013). Masterstudenters opplevelser av møter med veileder: Det er veiledning som gjør at en føler en mestrer. Uniped, 36(2), 81-92. Gamlem, S. M. (2014). Tilbakemeldinger som støtte for læring på ungdomssteget. (Doktorgradsavhandling), Universitetet i Stavanger, Stavanger. Gamlem, S. M. (2015). Tilbakemelding for læring og utvikling. Oslo: Gyldendal akademisk. Giorgi, A. (2009). The descriptive phenomenological method in psychology: A modified husserlian approach. Pittsburg: Duquesne University Press. Grepperud, G., Rønning, W. M. & Støkken, A. M. (2006). Studier og hverdagsliv: Voksne studenter i fleksibel læring. Trondheim/ Oslo: VOX. Handal. G., Lycke, K. H., & Lauvås, P. (2013). Strategier i forskningsveiledning?: En analyse av veilederes tilbakemelding på tekst. Uniped, 36(4), 32-44. Handal, G. & Lauvås, P. (2013). Forskningsveilederen (3. utg.). Oslo: Cappelen akademisk. Hellne-Halvorsen, E. B. (2014). Skrivepraksiser i yrkesfaglige utdanningsprogrammer. (Doktorgradsavhandling), Universitetet i Oslo, Oslo. Hoel, T. L. (2002). Interaction and learning potential in e-mail messages. I E. Maagerø & B. Simonsen (Red.), Learning through genres (s. 15–38). Kristiansand: Høyskoleforlaget. Hoel, T. L. (2008). Skriving ved universitet og høgskolar- for lærerar og studentar. Oslo: Universitetsforlaget. Hoel, T. L. & Rokkones, K. E. (2012). Frå tommestokk til pc: Yrkesfaglærarstudentar og skriving. I S. Matre, S. Kibsgaard Sjøhelle & R. Solheim (Red.), Teorier om tekst i møte med skolens lese- og skrivepraksiser, s. 239-249. Oslo: Universitetsforlaget. Illeris, K. (2012). Læring. Oslo: Gyldendal akademisk. Klemp, T. (2012). Praksisloggen som multiverktøy: Hva bruker lærerstudentene den til? I S. Matre & G. Melby (Red.), Å skrive seg inn i læreryrket, s. 89-112. Trondheim: Akademika. Kunnskapsdepartementet (2013). Forskrift om rammeplan for yrkesfaglærerutdanning for trinn 8-13. Hentet den 03.06.2015 fra 21 https://www.regjeringen.no/globalassets/upload/kd/vedlegg/rammeplanen/yrkesfaglaer erutdanning.pdf Kvale, S, Brinkmann, S., Anderssen, T. M. & Rygge, J. (2015). Det kvalitative forskningsintervju (3. utg., 2. opplag). Oslo: Gyldendal akademisk. Lauvås, P. & Handal, G. (1998). Hovedfagveiledning ved Universitet i Oslo: Rapport frå prosjektet «Vitenskapelig veiledning» (rapport 1/1988). Oslo: Universitetet i Oslo. Lindseth, A. & Norberg, A. (2004). A phenomenological method for researching lived experience. Scandinavian Journal of Caring Science, 18(2), 145-153. Rienecker, L. Referanser & Jørgensen, P. S. (2006). Den gode oppgaven: Håndbok i oppgaveskriving på universitet og høyskole. Bergen: Fagbokforlaget. Tveiten, S. (2013). Veiledning: Mer enn ord (4. utg.). Bergen: Fagbokforlaget. Utdanningsdirektoratet (2006). Kunnskapsløftet. Hentet den 25.09.2014 fra http://www.udir.no/Lareplaner/Kunnskapsloftet/ VIKO (2014). VIKO gir hjelp til litteratursøk og oppgaveskriving. Hentet den 25.09.2014 fra http://www.ntnu.no/viko/ 22

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ESTADO DE ESPANTO

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Estado de espanto Astonished state Laurita Ricardo de Salles1 Equipes Laboratório 10 Dimensões/UFRN e Media Lab/UFG2 Resumo O artigo parte da fala de artistas e/ou artistas teóricos (Pierre Reverdy, Octávio Paz) que invocam o que seria um estado de poesia para alcançar o estatuto poético nas obras de arte. Propõe dar voz ao que os artistas dizem sobre o fenômeno poético, estabelecendo laços de parentesco e analogias entre obras e discursos para a emergência do que alguns chamam de encantamento (Gadamer). Certos artistas situam o poético como algo fugidio e raro, oriundo da e além da estratégia de sua produção: os verdadeiros poetas provam da poesia poetisando, criando um momento de ilusão de um fragmento de vida irreal intensa (Reverdy); o poético vai além da linguagem (Paz). O que seria, afinal, esse estado de espanto? O artigo também trata de obras que articulam suas poéticas através da conjugação do imaginário, deslocamentos de sentido e tecnologia, dentre elas, a obra Flauta Mágica da autora do artigo e equipe 10 Dimensões/Media Lab UFG. Palavras-chave: Poética, Artistas, Arte e tecnologia Abstract The article starts from the speech of artists and/or theoretical artists (Pierre Reverdy, Octávio Paz) who invoke what would be a state of poetry to achieve poetic status in works of art. It proposes to give voice to what artists say about the poetic phenomenon, establishing ties of kinship and analogies between works and discourses for the emergence of what some call enchantment (Gadamer). Certain artists place the poetic as something elusive and rare, arising from and beyond the strategy of their production: true poets taste poetry by creating a moment of illusion of a fragment of intense unreal life (Reverdy); the poetic goes beyond language (Peace). What, after all, would be this state of astonishment? The article also deals with works that articulate their poetics through the combination of the imaginary, shifts of meaning and technology, among them, the work Magic Flute by the author of the article. Keywords/Palabras clave/Mots clefs: Poetics, Artists, Art and technology 1 Autor, Artista e pesquisadora na área de Arte e Tecnologia. Pós Doutora em Mídias Interativas pelo Media Lab//UFG. Professora Associada II no CLAV/Curso de Licenciatura em Artes Visuais,DEART/Departamento de Artes/CCHLA/Centro de Ciências Humanas, Letras e Artes/ UFRN/Universidade Federal do Rio Grande do Norte. Ganhou o Premio Vitae de Artes em 1998. 2 Coautor, demais membros das Equipe Laboratório 10 Dimensões: Aquiles Medeiros Filgueira Burlamaqui, Gabriel Gagliano Pinto Alberto, Rogério Júnior Correia Tavares, Rodrigo Montandon Born, Girleno Atos Alves Martins ,Andiara de Freitas Emidio, Nero Rocha de Araujo, Lucas Rodrigues Marques, Leonardo Moniz Sodre Lopes Teixeira, Leonardo Meneses Pereira, Matheus Pereira Batista de Macedo, Vinicius dos Santos Tavares, Mateus Vieira Garcia. Equipe Media Lab/UFG: Cleomar de Sousa Rocha, Hugo Alexandre Dantas do Nascimento, Wilder Fioramonte. 107 ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. Antes do nome [...]A palavra é disfarce de uma coisa mais grave, surda-muda, foi inventada para ser calada. Em momentos de graça, infrequentíssimos, se poderá apanhá-la: um peixe vivo com a mão.[...] (PRADO: 1991, p. 22) Introdução As reflexões sobre poética nos fizeram nos defrontar com a fala de artistas, teóricos-artistas, e ou teóricos que relatam ou narram a experiência da criação e fruição poética como algo que de alguma forma organiza e apresenta um projeto e, simultaneamente, algo indefinível ou uma descoberta. Ou seja, a criação artística e o fenômeno artístico manifestam-se por um conjunto de forças que em alguns aspectos constituem um enigma: a obra de arte apresenta-se. Este texto, então realiza o trabalho de compilar de forma livre e ensaística momentos do discurso ou fala de artistas a teóricos onde este mistério da obra vem à tona, com o intuito de expor em uma primeira abordagem, a existência e persistência dessa espécie de consciência do intangível pelo qual o fenômeno poético é produto. Pierre Reverdy no texto Esta emoção chamada poesia3 qualquer artista cuja ambição e objetivo seja criar por meio de uma obra estética feita por uma emoção que só a arte pode lhe dar.(REVERDY; 2003, pág.1). Reverdy também diz que escrever é também um meio de comunicação mas que o poeta, por outro lado, apresenta o que é indizível e que, no entanto, deve ser dito. [...]Mas, ao final, nada do que foi finalmente dito foi realmente indizível. Este indizível é, provocada pelo que e pelo dizer mas, sobretudo pela forma como se d conclui o autor, os verdadeiros poetas só podem provar a poesia poetizando (REVERDY; 2003, pág.3)4. Otávio Paz também levanta algo nesse sentido afirmando que o poético vai além da linguagem, apontando que Aristóteles caminhava nesta direção: ] nada é comum, exceto a métrica, entre Homero e Empedocles; e por isso com justiça se chama poeta o ou, para sermos exatos, nem toda obra construída sob as leis da métrica (PAZ: 1982, p.16). E acrescenta: Sem deixar de ser linguagem - sentido e transmissão do sentido - o poema é algo que está mais além da linguagem. 3 Versão da autora: Cette émotion appelée poésie, acessível em: -re... Pierre Reverdy, Sable mouvant, Au soleil du plafond, La Liberté des mers, -Alain Hubert, Poésie / Gallimard, 2003 4 108 Todas as citações são do mesmo texto e página. ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. Mas isso que está mais além da linguagem só pode ser conseguido através da linguagem. Um quadro só será poema se for algo mais além da linguagem pictórica.(PAZ: 1982, p.16) Assim, observamos a recorrência no poético desse algo obtido através da linguagem, que se configura via um programa poético e que, paradoxalmente, oferece a quem entra em contato com a obra (Eugen Fink, apud MERLEAU-PONTY:1999,p.10). A metáfora do espanto, a qual eu mesma coloquei no título deste artigo antes de conhecer os antecedentes que reivindicam esse estado provocado pela obra de arte foi salientada por Paul Valéry, no ensaio "Poesia e pensamento abstrato". No caso, ele fala das motivações do poeta, em vida que se espanta: poética, nascem dessas sensações intelectuais súbitas. É a minha própria vida que se espanta, é ela que deve me fornecer, se puder, minhas respostas, pois é somente nas reações de nossa vida que pode residir toda força e como que a necessidade de nossa vontade.O poeta chama esses estados de poéticos, alguns terminam por se concretizar em poemas.(VALÉRY: 1999, p. 204) Valéry também situa os motivos do poeta como algo inesperado: O poeta desperta no homem através de um acontecimento inesperado, um incidente externo ou interno: uma árvore, um rosto, um "motivo", uma emoção, Observem bem esta dualidade possível de entrada em jogo: às vezes, alguma coisa quer se exprimir, às vezes, algum meio de expressão quer alguma coisa para servir. (VALÉRY: 1999, p. 218) O poeta Ferreira Gullar também fala claramente em espanto como instância de motivação para a poesia: [...]a minha poesia nasce do espanto. Qualquer coisa pode espantar um poeta, até um galo cantando no quintal. Arte é uma coisa imprevisível, é descoberta, é uma invenção da vida. E quem diz que fazer poesia é um sofrimento está mentindo: é bom, mesmo quando se escreve sobre uma coisa sofrida. A poesia transfigura as coisas, mesmo quando você está no abismo. A arte existe porque a vida não basta. […] Eu escrevo muito pouco, sem espanto eu não escrevo. (GULLAR: 2010, sem pág.) Porém, a questão do espanto não é nova. A questão do sublime como uma experiência do inefável, indizível ou indescritível foi pensada desde a antiguidade. Kant tratou da questão, assim como o sublime romântico reivindica de alguma maneira esse estado proporcionado pela Arte, em outra chave. Na Crítica da faculdade do juízo, ao final da Analítica do sublime, Kant refere-se ao sublime como um objeto (da natureza) 109 ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. cuja representação determina o ânimo a imaginar a inacessibilidade da natureza como apresentação de ideias que somente pelo fato de poder também pensá-lo prova uma faculdade do ânimo que ). Já o sublime no romantismo envolve o reencontro do homem e a natureza. secretas da natureza, as quais sem essa aparição teriam permanecido eternamente TI: 2011, pág. 118ainda, que: Uma obra de arte autêntica, assim como uma obra da natureza, permanece sempre infinita para o nosso entendimento; ela é contemplada[angeschaut],sentida, faz efeito, mas não pode ser propriamente conhecida, muito menos podem ser expressos em palavras sua essência, seu mérito (GOETHE: 2008, pág.117). Não à toa vamos encontrar em Schiller a categoria de forma viva que segundo Sussekind compreende a forma como instância de um impulso vital: Seu objeto é ao mesmo tempo forma (idéia) e vida (natureza), por isso o conceito de "forma viva" serve para designar a possibilidade de harmonia entre os dois "mundos", da natureza e da cultura, separados na modernidade. Assim como o homem não é exclusivamente matéria nem exclusivamente espírito, a consumação de sua humanidade não pode ser mera vida nem mera forma, ela deve ser "forma viva", criada pelo impulso lúdico.(SUSSEKIND:2005, sem pág.) Paul Ricoeur fala em metáfora viva5, como uma original e específica de apreender o (Ricoeur, 2000, p. 348) onde querela que está em é direito de passar da estrutura, que é para a obra complexa o que o sentido é para o enunciado, ao mundo da obra, que é para esta o que a denotação é para o (Ricoeur, 2000, p. 337). Segundo Braga (BRAGA: 2010, p.14), Ricoeur, pensa que as emoções e sentimentos tem função cognitiva na poesia , onde a tristeza poética seria uma de consciência das (Ricoeur, 2000, p. 348). Paul Ricoeur (RICOEUR: 2000, p. 328,329) defende ao final uma fenomenologia da imaginação onde lembra que o sentido metafórico enquanto tal se forma na espessura do imaginário liberado pelo e cita Gaston Bachelard afirmando a imagem não é um resíduo da impressão, mas uma aurora da assim, 6 palavras Não à toa os últimos teóricos se utilizam de uma construção poética para tentar captar o fenômeno. Afinal, as afirmações da palavra e palavras poderiam estar em um poema. 5 Titulo de um livro do autor: A metáfora viva. 6 Citações de Ricouer a partir do livro de Gaston Bachelard , La Poétique de espace PARIS: PUF, 1957 p 1-2, p.7 tradução a partir da edição brasileira da Editora Martins Fontes 1998, p.7(Nota do tradutor de A metáfora viva) 110 ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. Voz dos artistas Podemos pensar que tais estratagemas apesar de estarem relacionados a períodos e propostas diversas guardam um fundo em comum, vinculado à constatação sobre uma região da criação artística ou a poética capaz de produzir um estado de admiração do mundo alavancada pelo imaginário. É comum na fala dos artistas modernos o relato de um estado de poesia como um momento fugidio e de um encontro não planejado mas surpreendente, como até é denominada uma das últimas obras do cantor e compositor Chico César: o cd Estado de poesia7. A poetisa Cecilia Meirelles escreveu: voam e MEIRELES:1986, p. 627), da mesma maneira o poeta Manoel de As palavras me escondem sem cuidado.Aonde eu não estou as palavras me que são inventadas (BARROS: 2010, pág. 347 ao grau de brinquedo para ser séria (BARROS: 2010, pág. 348 350). Verificamos novamente a reivindicação da imaginação para a realização da poética. É de interesse a reivindicação da imaginação, pois a poesia, por trabalhar com a multiplicidade do sentido, de fato, oferece um campo de latência e de possibilidades ampliadas para o sentido. Podemos dizer que o poético ativa o imaginário que, por sua vez, da corpo às condições para a poética se concretizar, seja no programa da obra, seja na recepção do expectador. Oswald de Andrade reconhece o estado da apreensão inesperada pelo artista Podemos dizer que Valéry concorda com Oswald e considerava que o poeta se afasta de seu estado normal para criar um discurso extraordinário , como uma segunda potência da linguagem. Também fala em estado de poesia e que o poeta transforma o (VALÉRY: 1999, p. 206). Detalhando o pensamento do autor: O poeta é, a meu ver, um homem que, a partir de um incidente, sofre uma transformação oculta. Ele se afasta de seu estado normal de disponibilidade geral e vejo construirse nele um agente, um sistema vivo, construtor de versos. A linguagem com que se ocupa o poeta, domada até certo ponto mas não dominada, passa a ser uma utilidade de segunda ordem, pois alça vôos além de uma utilidade prática a que bem servia para sagrar-se como discurso extraordinário, estranho e misterioso. Essa segunda potência de linguagem se distingue da linguagem comum, prosaica.(VALÉRY: 1995, pág. 1017) um modo mais elevado da linguagem cotidiana. Ao contrário. É a fala cotidiana que 2012, p. 24). Heidegger se dá conta da dificuldade da apreensão do fenômeno da linguagem e diz: que escrever sobre o (2012, p. 09). Bachelard não tentou isso fazer ao dizer que as palavras sonham? 7 111 Estado de poesia é o oitavo álbum do cantor e compositor paraibano Chico César, lançado em 2015. ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. Assim vemos que a arte envolve um discurso autônomo que se constitui como a arte, como bela aparência, opor-senão reside a tarefa da estética, justamente, em fundamentar que a experiência da arte é Trânsito dos sentidos Como estas questões se refletem em obras concretas? Esta é uma problemática de per si difícil de tratar. A obra Flauta Mágica a qual estamos realizando junto ao Laboratório 10 Dimensões no DEART/UFRN8 trabalha com uma espécie de trânsito dos sentidos e de deslocamentos de significados. Faz uma flauta tocar inspirados nos sons do sol e da lua e do espaço e não notas musicais e, sons luzirem nos reflexos luminosos de uma caixa espelhada sonora com alto-falantes e dispositivos luminosos. O interator é convidado a tocar uma flauta que possui três canais de sons melódicos, rítmicos e harmônicos. Ele pode tocar um só canal, ou sobrepor sons, a maneira, digamos, de um arranjo. Ele também pode decidir mudar o banco de sons que está tocando, e dar maior ou menor volume ao conjunto sonoro final. Este será reproduzido através da caixa de sons espelhada com grande intensidade sonora. Esta caixa também tem dispositivos luminosos que operam de acordo com a saída sonora além de rebatimentos luminosos em suas faces espelhadas e opera uma caixa de fumaça que tem duas saídas que interpenetram essa caixa; este conjunto provocará uma miríade de rebatimentos luminosos do som. Render da caixa espelhada da obra Flauta mágica, ainda em fase de construção (Render do Laboratório 10 Dimensões- Nero Rocha, bolsista) 8 A obra está em andamento. Participam os co-autores deste artigo, integrantes do Laboratório 10 Dimensões e Media/Lab/UFG. 112 ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. A obra tenta operar um jogo de trânsito dos sentidos através de uma estratégia simples para construir uma sensação de transfiguração, advinda desses deslocamentos, escorregamentos ou desvios. É como se utilizasse de meios expressivos transfigurados e procura instaurar uma plataforma para o encantamento, onde uma flauta, tocada pelo interator, apresenta-o a uma caixa de som e luz em movimento e refrações de luz e espelho. Há uma estratégia de justaposição de instancias- musico e instaurador de performance sonoro luminosa, em um novo conjunto que adquire sentido, justamente, por esta aproximação inaudita. Conseguirá seu intento? Apenas com a obra pronta poderemos saber se esse enigma da obra apresenta-se como tal, instaurando uma obra que sonhe, parafraseando Bachelard. Flauta da obra Flauta mágica. Mostra os canais de interação com o banco de sons da obra. Diagrama e foto Laboratório 10 Dimensões, bolsista Leonardo Sodré) Caixa espelhada da obra Flauta mágica, ainda a receber auto falantes e leds de iluminação. Já dá para ver os dois canais de passagem para a fumaça. (Foto Laboratório 10 Dimensões, bolsista Leonardo Sodré) Comento a seguir duas obras do artista francês Joanie Lemercier. Ele é focado principalmente em projeções de luz no espaço e sua influência em nossa perceção. Mantém um estúdio desde 2015 em Bruxelas, Bélgica. Desde 2006, Lemercier trabalha com luz projetada e projeções arquitetônicas em todo o mundo. Em 2010, o artista voltou-se para instalações e trabalhos de galeria e expôs no China Museum of Digital Art, (Pequim), Art Basel Miami e Sundance Film Festival 2013.9 As obras deste artista tentam estabelecer imagens no espaço de forma tridimensional. Envolve estabelecer estratégias para realizar isto, e tem levado o artista 9 113 Vide em: https://joanielemercier.com/projects/ ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. na direção de instalações e projeções sobre dispositivos que instauram um receptor proje atmosfera e em escalas imponentes, são o foco do trabalho que articula-se no espaço e apresenta-se como uma féerie10 encantadora e arrebatadora. De fato, o trabalho coloca o expectador em uma condição de maravilhamento. Ressalto em particular a obra Constelações (vide em https://joanielemercier.com/constellations/, partículas de água, luz som, 16 min, 2018) Já a obra Quartzo de 2013 envolve uma instalação com mini peças espelhadas configuradas como uma formação orgânica que sobre projeção de luz apresenta uma miríade de brilhos e dotada de acompanhamento sonoro. Esta formulação da intangibilidade da imagem através da reflexão e refração da luz, confirmou nossa opção para construir a caixa espelhada da obra Flauta mágica, decorrente da obra anterior realizada pelo projeto 10 Dimensões, Mini Paredão eletrônico. Quartzo, instalação audiovisual (Foto do site do artista) Por fim, comentamos a obra Água (2010) da dupla de artistas Rejane Cantoni/Leonardo Crescenti11. Os artistas apresentam sua obra como uma interface tátil-visual onde um espelho flexível e de grande dimensão, sofre deformações em função do peso e da posição dos visitantes. Este recebe uma iluminação potente com projetores de luz que rebatem as refrações de luz no espaço ambiente. Também esta obra se constrói através do uso de um espelho flexível e pelo rebatimento da luz no espaço e constitui uma zona de imersão em ondas de luz para os interatores, os quais passam a estar mergulhados em uma espécie de mar luminoso que se rebate nas paredes e teto onde a obra é apresentada (há várias versões). Esta obra também contribuiu na criação da obra Flauta Mágica, como um vetor confirmatório da direção tomada. 10 Reino das fadas, ou reino do maravilhoso. 11 Vide em : https://www.cantoni-crescenti.com.br/vu4pf4m76z3fepkhn2smhp4se4vl50 114 ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. Obra Água em apresentação na OCA/São Paulo, SP, 2013 (Print screen a partir de foto do site dos artistas) A conclusão provisória é que as obras em pauta se constroem em parte pela luz e reflexão espelhada almejando construir um campo iluminado em diálogo com algo que não é exatamente uma imagem mas que dialoga com o imagético como algo em fluxo no ar, contribuindo para o poético vir a estabelecer um estado de assombro diante do mundo. Referências ANDRADE, O.Pau Brasil, cancioneiro de Oswald de Andrade. Paris: Impresso pelo Sans Pareil, 1925 ASHTON, Dore. Picasso on Art, a selection of views. Nova Iorque: Viking Press, 1972 BRAGA, H.M. A metáfora viva de Paul Ricoeur. In Revista Último Andar, Cadernos de Pesquisa em Ciências da Religião, Programa de Ciências da Religião, PUC/SP, n.18.2010. Acessível em: https://revistas.pucsp.br/ultimoandar/article/view/13291 BARROS, M. de. O livro sobre nada in Manoel de Barros Paulo: Leya, 2010 Poesia Completa , São GADAMER, Hans-Georg . Verdade e método: traços fundamentais de uma hermenêutica filosófica. Petrópolis: Editora Vozes, 1999 GOETHE, J. W. Escritos sobre arte. Introdução, tradução e notas de Marco Aurélio Werle. São Paulo, Associação Editorial Humanitas; São Paulo: Imprensa Oficial do Estado de São Paulo, 2008 GULLAR, F. A Arte existe porque a vida não basta. Entrevista por Luciano Trigo, Jornal O Globo, Rio de Janeiro, 2010 (07/08/2010) Acessível em: http://g1.globo.com/pop-arte/flip/noticia/2010/08/arte-existe-porque-vida-naobasta-diz-ferreira-gullar.html 115 ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. https://repositorio.unesp.br/bitstream/handle/11449/6400/S198288372011000100008.pdf?sequence=1&isAllowed=y HEIDEGGER, Martin. A caminho da linguagem. Petrópolis: Vozes, 2012. KANT, Immnuel. Crítica da faculdade do juízo. Tradução de Valério Rohden e Antônio Marques. Rio de Janeiro: Forense Universitária, 1995. MEIRELES,C. Obra Poética Volume único. Rio de Janeiro: Editora Nova Aguilar, 1986 MERLEAU-PONTY, M. A Fenomenologia da percepção. São Paulo: Martins Fontes, 1999. Acessível em: https://monoskop.org/images/0/07/Merleau_Ponty_Maurice_Fenomenologia_d a_percep%C3%A7%C3%A3o_1999.pdf PAZ, O. O arco e a lira. Rio de Janeiro: Editora Nova Fronteira, 1982 PRADO, Adélia. Poesia Reunida. Rio de Janeiro: Siciliano, 2001 REVERDY, P. Sable mouvant, Au soleil du plafond, La Liberté des mers, seguido -Alain Hubert, Poésie / Gallimard, 2003 Acessível em: -re... RICOEUR, P. A metáfora viva. São Paulo: Edições Loyola, 2000 SUSSEKIND,P.:Schiller e os gregos, in Kriterion vol.46 no.112 Belo Horizonte Dec. 2005 Acessível em: https://doi.org/10.1590/S0100-512X2005000200009 VALÉRY, Paul. Variedades. Org. João Alexandre Barbosa. São Paulo: Iluminuras, 1999.p.193-210. __________. Situação de Baudelaire. In: BAUDELAIRE, Charles. Poesia e prosa. Org. 116 ISSN 2358-0488 Anais do VII Simpósio Internacional de Inovação em Mídias Interativas. HUB Eventos 2020. ROCHA, Cleomar et all (Orgs). São Paulo: Media Lab / BR, PUC-SP, 2020. 

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https://www.cambridge.org/core/services/aop-cambridge-core/content/view/C0F25F64E1E8A249435BEEF7DAFF46C8/S0034412522000531a.pdf/div-class-title-is-the-desire-for-life-rational-div.pdf

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Is the desire for life rational?

Religious studies

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© The Author(s), 2022. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. Christophe de Ray Haberdashers’ Boys’ School, Butterfly Lane, Elstree, Radlett, Borehamwood WD6 3AF, UK Email: [email protected]; [email protected] (Received 20 March 2022; revised 11 August 2022; accepted 12 August 2022; first published online 29 September 2022) (Received 20 March 2022; revised 11 August 2022; accepted 12 August 2022; first published online 29 September 2022) Religious Studies (2023), 59, 681–699 doi:10.1017/S0034412522000531 Religious Studies (2023), 59, 681–699 doi:10.1017/S0034412522000531 Religious Studies (2023), 59, 681–699 doi:10.1017/S0034412522000531 ORIGINAL ARTICLE Abstract The question of the meaning of life has long been thought to be closely intertwined with that of the existence of God. I offer a new theistic, anti-naturalist argument from the meaning of life. It is argued that the desire for life is irrational on naturalism, since there would be no good reason to believe that life is worthwhile on the whole if naturalism were true. As I show, the same cannot be argued of theism. Since it is clear that the desire for life is not irrational, it is concluded that we have strong reason to prefer theism over naturalism. Keywords: Theism; naturalism; meaning in life; desire Introduction: God and meaning in life Is the desire for life rational? Christophe de Ray Haberdashers’ Boys’ School, Butterfly Lane, Elstree, Radlett, Borehamwood WD6 3AF, UK Email: [email protected]; [email protected] Christophe de Ray Haberdashers’ Boys’ School, Butterfly Lane, Elstree, Radlett, Borehamwood WD6 3AF, UK Email: [email protected]; [email protected] Introduction: God and meaning in life So I hated life, because the work that is done under the sun was grievous to me. All of it is meaningless, a chasing after the wind. (Ecc. 2:17) The book of Ecclesiastes is well known for its bleak assessment of the human condition, and for repeatedly equating human life with mere ‘smoke’ (Heb. hevel), usually rendered as ‘vanity’ or ‘meaningless’ by English translations, and with ‘chasing after the wind’. The latter expression indicates that for the Qoheleth (‘Teacher’), the meaninglessness of life lies primarily in its futility. This is an odd claim: activities like chasing after thewind or trying to count allthe grainsof sand on a beach are said to be ‘futile’ because they inevitably end in failure. Specifically, they fail to achieve their desired ends. But virtually all of our goals – having a successful career, getting married, publishing books etc. – require us to be alive in order to achieve them. If so, how can the very act of living be futile? The Qoheleth offers the following answer: ‘What do people get for all the toil and anxious striving with which they labour under the sun? All their days their work is grief and pain; even at night their minds do not rest. This too is meaningless. (Ecc. 2:22–23) In his estimation, all the goods that life has to offer are outweighed by the pain, sorrow, disappointment, and all the other evils that life throws at us (and in any case, our enjoy- ment of them does not last). In short, life is simply not worth the trouble and living is not a worthwhile project. It is in this sense that life is ‘meaningless’. However, the Qoheleth hints at various points that the futility of life may perhaps be only apparent, and that https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press 22000531 Published online by Cambridge University Pres Christophe de Ray 682 we may see why our lives have meaning after all, as long as we take God into account: ‘This too, I see, is from the hand of God, for without him, who can find enjoyment?’ (Ecc. 2:24). The question of the meaning of life has long been thought to be closely intertwined with that of the existence of God. Introduction: God and meaning in life Theistic philosophers often argue that God, and God alone, can ensure that our lives are meaningful.1 How God does this will depend on what a ‘meaningful life’ is taken to be. On one view, human life is meaningful as long there is an objective purpose (or telos) which human beings exist in order to fulfil, inde- pendently of our particular preferences and desires. To use Jean-Paul Sartre’s (1965) well- known example, ‘the purpose of a paper-cutter is to write’ is made true by the fact that paper-cutters were intentionally designed for this reason. In contrast, it would be confused to say that the purpose of mountains is to look beautiful, even if they do in fact look beau- tiful, unless of course one believes that they were designed. Thus, William Lane Craig (2013, 163) argues that if ‘God does not exist, then you are just a miscarriage of nature, thrust into a purposeless universe to live a purposeless life’. Similarly, Thomas Morris (1992, 56) contends that ‘[to] have meaning of any kind, a thing must be brought under the governance of some kind of purposive intention’, and infers from this that human life could only have been endowed with meaning by a transcendent purpose-giver. Combine statements of this sort with the premise that life is meaningful (in the relevant sense), and we have an argument for theism. But it is not obvious that having an objective purpose is either sufficient or necessary to living a meaningful life. Concerning sufficiency, suppose you discovered that you and all other human beings had been created in a lab by highly intelligent aliens, for the sole purposes of harvesting our faeces, which they consider to be a delicacy.2 If this scenario were true, your life would certainly have an objective purpose, in the same way that pens, computers, and aeroplanes do. But intuitively, this does not make your life meaningful – if it is meaningful at all, it is in spite of the purpose for which you were made, not because of it. Concerning necessity, many theists would vigorously deny that God’s life is meaning- less,3 even though it necessarily could not be the product of intentional design.4 We may instead follow the Qoheleth in taking ‘meaningfulness’ to refer to the value or worth of a life. Stewart Goetz (2012, 3) similarly identifies ‘what makes life meaningful’ with ‘what makes life worth living’. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press The argument Once again, I take ‘metaphysical naturalism’ (or simply ‘naturalism’) to be the thesis that the natural order is all that exists, or at least that there are no supernatural beings. The question of the exact boundaries of naturalism is of course a matter of much controversy. For instance, it is unclear whether admitting non-natural (but not supernatural, since causally inert) entities like Moorean-Platonic ‘Goodness’, as done by some atheistic philo- sophers like Erik Wielenberg (2009) and David Enoch (2017), allows one to remain within the naturalist fold. Fortunately, I do not believe that the success of my argument hinges on how naturalism is to be defined. I take a ‘rational’ desire to be a desire that it is rationally permissible to hold (that is, such that it is not irrational to hold it).5 The desire for life is ‘reflective’ as long as we have strong reasons to believe that life is worthwhile (more on this later). (1) The desire for life is rational. (2) The desire for life is irrational, unless it is reflective. (3) If naturalism is true, the desire for life cannot be reflective. (4) Therefore, if naturalism is true, the desire for life is irrational. (5) Therefore, naturalism is false. (4) is entailed by (2) and (3), as is (5) by (1) and (4). Therefore, motivating my argument will only require me to motivate (1), (2) and (3). Introduction: God and meaning in life In that case, theists can avail themselves of the vari- ous moral and axiological arguments on offer. John Cottingham (2005) contends that objective values cannot exist unless they find their source in God, the ultimate ground of value. It follows that life (or anything else) cannot be objectively valuable if atheism is true. Here the atheist may answer that the subjective value of life is all that matters to him: he can desire and enjoy life regardless of whether God exists. It is enough for his life to be meaningful, namely worthwhile, to him, given his goals, beliefs, and values. Whether or not life is also meaningful in some mind-independent sense is irrelevant. In addition, many atheistic philosophers have advanced and defended non-theistic accounts of objective value (e.g. Wielenberg (2009)). I will present and defend a new argument from the meaning of life. The target of my argument will be metaphysical naturalism, the popular view that reality is exhausted by the natural order. I will argue that the desire to live is irrational if naturalism is true. Since the desire to live surely is rational, it follows that we ought to reject metaphysical naturalism. As I will show, traditional theism does not face this problem. And as should become clear, this argument will not at all depend on the claim that life must be meaningful or worthwhile in an objective sense, or that it requires a purpose in order to be meaningful. I will simply take the meaningfulness of a life to be its worthwhileness to the individual who lives it. g I begin by laying out my argument and motivating its premises, before addressing objections. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Religious Studies 683 https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Motivating (1): the desire for life is rational Desires, like beliefs, are intentional mental states, meaning that they represent states of affairs. On one view, a desire just is a kind of belief – specifically, a belief about what is good (cf. Gregory (2021) for a recent discussion). In any case, both beliefs and desires may be justified or unjustified, rational or irrational.6 The rationality (or justification) of both desires and beliefs is at least partly a function of their coherence with our other beliefs and desires. My desire to write this article and to send it for publication is, I take it, a rational one: given my other desires and background beliefs about what is valu- able, it is reasonable for me to desire this. On the other hand, my desire for a cigarette is irrational, given my belief in the harmful effects of smoking and my desire to remain in good health. g The great majority of us desire to live. When asked whether we do, we reply in the affirmative. And we behave in such a way as to continue to live, generally avoiding deci- sions and lifestyles that are likely to result in the cessation of life. We are not indifferent as to whether or not we will continue to live. Some, such as Spinoza, have gone as far as to argue that desiring to ‘persevere in its own being’ (conatus) is part of the essence of any existing thing, including human beings (Della Rocca (2008), 145). We need not go that far, of course. Nor does it obviously follow from the fact that all or most of us desire to live, that we are rational in so desiring. As G. E. Moore (1922/1903, 56) remarked to J. S. Mill regarding the latter’s ‘proof’ of utilitarianism, ‘“desirable” does not mean “able to be desired”’, but rather ‘what ought to be desired or deserves to be desired’. As he goes on to say, the Prayer Book’s talk of ‘good desires’ is surely no tautology (ibid.). Even so, I do not expect many naturalists to take issue with (1). I take this to be a strength of my argument relative the other, related ones mentioned in the introduction: while naturalists can easily deny that human life has an objective purpose or objective value, they are unlikely to disagree that it is rational for them to want to live. Motivating (1): the desire for life is rational Indeed, Christophe de Ray 684 many such naturalists enthusiastically affirm their gratitude for their existence in the world, as documented by Yujin Nagasawa (2018).7 Hence I do not intend to motivate (1) in any depth, though I will offer the following two considerations in its defence. First, it enjoys firm support from common-sense intuition. It just seems intuitively obvious that there is nothing irrational about wanting to live. We don’t typically chastise one another for expressing this want, or for acting in such a way as to satisfy it (it wouldn’t even occur to us to do so). Oppositely, we find expressions of suicidal thoughts deeply disturbing. A plausible explanation of this posits an intuition to the effect that it is rational to desire to live. Second, its denial would severely undermine eminently plausible moral beliefs, such as our belief that we should try to protect people whose lives are endangered. If Bob’s desire to live is irrational, it is hard to see why I ought to save him from drowning if I am able to (compare: I don’t have a moral duty to make sure that Bob successfully counts every blade of grass on his lawn, even if he passionately desires this). One might cite the physical pain of drowning, or the distress that Bob experiences at the thought of losing his life, as grounds for my duty to rescue him. But this would fail to explain why that duty would surely still exist, even if Bob’s imminent demise would somehow be painless, and if he were somehow completely unaware of it.8 The persistence of the moral duty to protect Bob’s life, even in these circumstances, seems best explained by the rational permissibility of Bob’s desire to live: since it is appropriate for Bob to want to live, it is right for me to assist him in satisfying this desire, insofar as I am able to. Note finally that (1) need not be taken to mean that it is never irrational to desire to live, without exception. Naturalists will probably point to cases of extreme pain and mis- ery, in which (they argue) it is no longer rational for the victim to wish to remain alive,9 against an ‘absolutist’ reading of (1). A more moderate reading, according to which it is generally rational to desire to live, barring unusually unfortunate circumstances, will suf- fice for our purposes. Motivating (1): the desire for life is rational Much more could be said in defence of (1), but I must move on to my argument’s other premises. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Reflective desires Could one put forward a cost-benefit analysis, yielding the conclusion that life’s benefits outweigh its costs, and thus is desirable on the whole? I shall return to this question later. For now, let us say that my desire to live is reflective only if I have strong reasons to believe that life is worthwhile. More generally, my desire to engage in some activity A is reflective only if there are strong reasons to believe that A is a worthwhile activity. If a desire for some state of affairs is reflective, having been found to be worthwhile by an individual, it is thereby rational for said individual to desire said state of affairs. The converse of this implication also has some initial plausibility: intuitively, an agent A’s desire for some state of affairs S needs to ‘spring’ from A’s knowledge (or reasonable belief) that S is worthwhile on the whole, in order to be rational.11 For example, consider your desire for some fancy gadget. Suppose that this desire is in fact entirely the product of aggressive, manipulative advertising on the internet, resulting in an intense longing for the gadget. Should you discover, after prolonged soul-searching, that this is indeed how your desire came about, you would be likely to come to the conclusion that you ought to try to withhold the desire, if at all possible. Why would you conclude this? Because you would have become aware that your desire for the gadget is not at all the result of your having any good reasons to believe that the item is indeed worth buying, and that there are in fact no such good reasons. You thus judge the desire to be irrational. Similarly, learning that, while we do desire life, we lack good reasons to believe that living is a worthwhile activity should lead us to conclude that wanting to live is irrational. Note, importantly, that this would be different from acquiring good reasons to believe that life is disvaluable. It may be thought that this is what the above thought experiment really illustrates: since you have a limited amount of money, arbitrarily spending it on a trinket that you know you won’t need would constitute a net cost, and hence it is irrational to desire doing so. Worthwhileness I have said that a life is ‘meaningful’ as long as it is worthwhile for the individual that lives it. Plausibly, the ‘worthwhileness’ of an activity is best thought of as its aggregate value, or the sum of the values of its benefits and costs.10 To say that a life is worthwhile or ‘worth living’, then, is to say that its benefits outweigh its costs – in other words, that being alive is a good thing on the whole. I should clarify that ‘good’ here need not at all be interpreted in an object- ive sense. It may be that value is entirely mind-dependent: for instance, if hedonistic accounts of value are true, my life is only worth living to the extent that it produces pleas- ure, and that the pain that it also inflicts is outweighed by the pleasure. p g y p Some activities are such that it is quite easy to tell whether they are worth engaging in it or not. For example, sticking my hand in a fire ant nest is evidently not a worthwhile activity, given my knowledge of fire ant stings and my desire to avoid pain. Other activ- ities are more difficult to evaluate in this way. If I am offered a new job, accepting the offer would mean acquiring new colleagues, a new workplace, a new schedule, engaging in new kinds of activities, and various other putative costs or benefits which I must care- fully examine, before reaching a conclusion as to the desirability of the job. It should be clear that in this respect, continuing to live is much more like acquiring a new job than like sticking one’s hand in an ant hill. Life involves a wide array of costs and Religious Studies Religious Studies 685 benefits, which are to be weighed up against each other, if one is to present a positive case for the worthwhileness of living. benefits, which are to be weighed up against each other, if one is to present a positive case for the worthwhileness of living. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Can we show that life is worthwhile? Once again, our desire for life is reflective only if we can put forward strong reasons to believe that life is worthwhile. As I indicated earlier, such an argument would need to take the form of a cost-benefit analysis, yielding the conclusion that the benefits of living (happiness, meaningful relationships, virtue, etc.) are worth the costs of living (suffering, to others and oneself), and therefore that living is a worthwhile project. Naturally, not everyone would be in a position to produce the required argument. Take the book of Job, whose eponymous protagonist tragically loses his property and children, and falls dreadfully ill. Though his story does end on a positive note, Job does not know this at this stage of the story. Given his limited evidence, he has no good reason to expect his condition to improve, and is hence in no position to argue that, all things considered, it is a good thing that he is alive. If anything, the opposite seems to be true: ‘May the day I was born be wiped out’ (Job 3:3). Interestingly, the inability to show that life’s benefits are worth its costs appears to be the rule, rather than the exception. Most human beings may not be as unfortunate as Job. Nevertheless, our lives, no matter how happy, are also filled with evils of various kinds. The third chapter of David Benatar’s aptly named Better Never to Have Been begins with the question, ‘How bad is coming to existence?’ and proceeds to offer an extensive survey of human suffering (Benatar (2006), 61–92). ‘Dissatisfaction does and must pervade life’, he tells us, if only because some of our most important desires, such as wanting to stay healthy, young, and not to be separated from our loved ones will inevitably be frustrated by ageing and death. For other desires, like the desire for happiness or friendship, frus- tration is extremely common, and satisfaction is exceedingly brief and gives rise to other desires. Pain, discomfort, stress, boredom, and frustration permeate our lives, rela- tionships, and work. And this is to say nothing of the dreadful tragedies faced by countless individuals, from rape and murder to crippling disease and suicide. Overall, he argues, ‘the quality of even the best lives is very bad’ (ibid., 61). Benatar’s ultimate goal is to show that coming into existence is always a harm. Reflective desires But supposing that you do not know whether you will need the item, and you have no reason at all to believe that buying it will benefit you overall (or that it will cost you overall), desiring to buy the item would still intuitively be irrational. Thus, lacking good reasons to believe that some activity is worthwhile does appear to entail that the desire to engage in said activity is irrational.12 But there may be exceptions to this rule. Intrinsic (or ‘basic’) desires are such that the desired state of affairs is desired for its own sake, not by virtue of some other desired good. David Hume described intrinsic desires as ‘original existences’, holding that they could never be irrational, regardless of our background beliefs (cf. Parfit (1984) for a response to Hume, and Hubin (1991) for a defence) – they are ‘just there’, so to speak. Take the case of our desire for happiness, which is one of the most promising candidates for being an intrinsic desire. If asked why we desire it, we are likely to answer ‘I just do’. Because we do not derive this desire from other desires, we cannot present reasons for desiring it, but it surely does not follow that the desire for happiness is irrational.13 h d b d f l f l k l b This caveat need not worry us, because our desire for life is very unlikely to be an intrinsic desire. For if it was, we would consider being plunged into a dreamless sleep for the rest of our lives (before finally ceasing to exist) to be at least somewhat preferable to being annihilated. But we do not, which strongly suggests that we desire life in virtue of desiring other things, such as happiness or well-being. Thus it appears that if the desire for life were not a reflective desire, it would follow that the desire for life is not a rational desire; hence my argument’s second premise. Christophe de Ray 686 Motivating (3): if naturalism is true, the desire for life cannot be reflectiv Can we show that life is worthwhile? https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Can we show that life is worthwhile? Since none of us choose to be alive, the question whether we are rational to desire our lives should be distinct from the question whether it might have been better had we never been brought into existence. In any case, my aims are markedly more modest: I only wish to argue that, for any given life, it is impossible to show that the benefits of life are worth its costs, and thus that living is a worthwhile project (that is, unless one is a traditional theist, as I will explain shortly). In response to Benatar, Yujin Nagasawa (2008, 676) accuses him of failing to account for the ‘balance of pleasure and pain in life’ (emphasis in original). The pleasures (and other goods) we experience in life can compensate us for the pains (and other evils): ‘Most of us believe – reasonably, I think – that coming into existence is not a serious harm because, roughly speaking, the amount and quality of pleasure one enjoys in one’s life usually com- pensates for the amount and quality of pain one suffers.’ y I am inclined to agree that most of us regard the benefits of being alive as making up for its costs. However, for living to be a worthwhile project, it is not enough for its benefits to ‘compensate’ its costs, it must also outweigh them – in other words, being alive must consti- tute a net gain for the living being. For if the costs and benefits of being alive cancelled each other out, there would ultimately be nothing to gain from being alive, meaning that the state of affairs of being alive would not be desirable (though its opposite, i.e. not being alive, would not be desirable either). Indeed, the appropriate attitude towards a state of affairs whose benefits are equal to its costs is one of indifference or apathy, not desire. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Religious Studies 687 Now, consider the three main theories of well-being, namely hedonism, desire theory, and objective list theory (to use the standard taxonomy, cf. Parfit (1984)). On hedonism, a life goes well to the extent that it contains more pleasure than pain. For any given life, and possibly barring extreme cases like Job’s, it is practically impossible to determine whether it is more pleasurable than painful, largely owing to the immense complexity of our inner lives. Can we show that life is worthwhile? One would need to examine carefully one’s pleasurable and painful experiences, and establish that on the whole, the former last longer, are more frequent, and/or more intense than the latter. Our lives contain simply too many such experiences for this to be feasible. The same applies to desire theory, which states that well-being is entirely dependent on the satisfaction and frustration of desires: we cannot realistically assess whether our lives involve more desire-satisfaction or frustration. Both theories face the additional worry that our memories of past pleasures, pains, and satisfied or fru- strated desires are likely to be distorted, making it difficult to draw a conclusion about the overall desirability of our lives.14 y Objective list theories hold that a life goes well as long as it instantiates a certain number of objective goods (e.g. meaningful relationships, knowledge, virtue) and, presumably, does not instantiate too many objective evils (e.g. having a meaningless job, failed relationships, ignorance, vice, etc.). The main worry here is the is the incommensurability of the different goods and evils: if I have a very successful and meaningful career but all my relationships have ended in dismal failure, how can I argue that the former good outweighs the latter evil? It seems that I could not, because there is no standard method of measuring the value of different goods and evils against each other. I could of course claim to prefer having a successful career over meaningful relationships, but this would bring us back to desire theory and its associated problems. Even if one could devise a system that would ascribe a numerical positive or negative value to any objective good and evil (e.g. +10 for a meaningful career, −7 for a failed relationship), the issue of complexity would arise once more, as the goods and evils to be weighed up against each other would be extremely numerous. Finally, our highly imperfect epistemic access to the future is a concern, regardless of which theory of well-being one opts for. We cannot rule out that we will be struck by sud- den devastating tragedies, like Job. More generally, it is difficult to see how one could pre- dict that one’s life will contain more good than evil. The general worry here is not that we cannot know with absolute certainty that our lives are (or will be) worthwhile. Can we show that life is worthwhile? Rather, the worry is that there are no good reasons to believe that it is: for every instance of good in any given life (happiness, desire- satisfaction, success), there is a corresponding instance of evil (sadness, desire-frustration, failure), and it is practically impossible to show (to oneself or to others) that the former outweigh the latter. Therefore, it appears that the desire for life cannot be a reflective desire, in the sense given above – though this depends on one’s background philosophical commitments, as I will now argue. Theism and naturalism contrasted ‘What people are for’, as G. E. M. Anscombe (1993, 124) put it, ‘is – as guided missiles – to home in on God, God who is the one truth it is infinitely worth knowing, the possession of which you could never get tired of’. The potential for this ‘beatific vision’, entailing full communion with God and perfect joy, is what makes ‘every life, right up to the last, infin- itely precious’ (ibid.). It is difficult to deny that being alive in the world would be desirable, if it really did provide one with an opportunity to achieve this supremely great good (as long as the probability that one actually achieves this is not exceedingly low). Whatever miseries we may suffer while physically alive would pale in comparison to eternal happiness and blessing. Not only this, the biological process of life, however painful at times, would be the process by which we are sanctified, that is, by which our character is formed and pre- pared for union with God. This point is often made in discussions of what Stephen Maitzen (2009, 122) called the ‘Heaven Swamps Everything’ theodicy. But my present aims are more modest than those of a theodicy: I am not arguing that a heavenly afterlife would justify God in causing or allowing suffering, but rather that God, if he exists, could ensure that our lives are worth- while, even in the midst of great suffering.16 Neither am I arguing that there are no other ways in which a perfect being could ensure that our lives are worthwhile (that is, other than by providing an opportunity for supreme happiness after death). Alternative ways may perhaps exist, but motivating (i) only requires me to show that there is at least one way in which he could accomplish such a feat. As to (ii), a world in which human lives are worthwhile is intuitively greater than one in which they are not (all else being equal). Thus, a perfectly good being would prefer to actualize the latter possibility, and should be expected to act accordingly. Since (i) and (ii) jointly entail (iii), it follows that the theist has a valid and sound argument for the con- clusion that life is worthwhile, meaning that the theist’s desire for life can be reflective. g As God does not figure in his ontology, the naturalist obviously lacks access to this argument. Theism and naturalism contrasted Traditional theism (of which classical Christian theism is a subset) holds that the natural order owes its existence to the activity of a transcendent, self-existent, supremely good God. If I believe that such a being exists, successfully arguing that life is worthwhile would not at all require me to produce a cost-benefit analysis of life. It is sufficient to argue that a perfect being would not have brought human beings into existence, unless their lives were worthwhile. This is much easier than weighing up all the many goods and evils of being alive against each other. Consider this simple argument: https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Christophe de Ray 688 (i) God would have the means to ensure that human life is worthwhile. (ii) If (i), God would ensure that human life is worthwhile. (iii) Therefore, God would ensure that human life is worthwhile.1 The plausibility of (i) is manifest if one bears in mind that God’s resources are not exhausted by worldly goods. Indeed, on the classical Christian view, the post-mortem enjoyment of an infinite supernatural good is the very purpose for which we were made. ‘What people are for’, as G. E. M. Anscombe (1993, 124) put it, ‘is – as guided missiles – to home in on God, God who is the one truth it is infinitely worth knowing, the possession of which you could never get tired of’. The potential for this ‘beatific vision’, entailing full communion with God and perfect joy, is what makes ‘every life, right up to the last, infin- itely precious’ (ibid.). It is difficult to deny that being alive in the world would be desirable, if it really did provide one with an opportunity to achieve this supremely great good (as long as the probability that one actually achieves this is not exceedingly low). Whatever miseries we may suffer while physically alive would pale in comparison to eternal happiness and blessing. Not only this, the biological process of life, however painful at times, would be the process by which we are sanctified, that is, by which our character is formed and pre- pared for union with God. The plausibility of (i) is manifest if one bears in mind that God’s resources are not exhausted by worldly goods. Indeed, on the classical Christian view, the post-mortem enjoyment of an infinite supernatural good is the very purpose for which we were made. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Nagasawa’s ‘Problem of evil for atheists’: a comparison The reader may have noticed similarities between the above argument and Yujin Nagasawa’s recent ‘Problem of Evil for Atheists’ (2018). Nagasawa argues that certain kinds of evil raise a difficulty for naturalists18 who hold to ‘existential optimism’ – that is, ‘that the world is overall a good place and that we should be grateful for our existence in it’ (ibid., 157). The cruelty and violence of natural selection threatens to make such opti- mism irrational. If my existence in the world is the end-product of a fundamentally evil system, which required the suffering and death of countless living beings in order to pro- duce me, how could I possibly be thankful for it? And how could I claim that the world is ‘good overall’, if the natural laws that govern it are inherently oppressive? Theists are comparably well equipped to deal with this threat, because theism implies that there is much more to reality than the natural world in its present state. If we think of the actual world as an enormous ‘spacetime worm’, the unsavoury aspects of reality (like natural selection and other ‘systemic evils’) need only constitute a tiny spatio- temporal slither of said worm. In particular, temporary evils, however horrible, may be vastly overshadowed by the existence of a supremely benevolent being and the possibility of a blessed afterlife in perfect communion with him, such that it would be reasonable to be grateful for one’s existence in the world. Naturalists obviously cannot vindicate their optimism in this way, since their ontology includes only the natural world. Existential optimism and the desire for life, while not identical, are closely related: one can hardly rationally desire to live while rejecting the proposition that it is good to be alive. Both my argument and Nagasawa’s aim to show that given the extent of suffering in the world, a positive propositional attitude (i.e. a desire or a belief) towards life is irrational on naturalism. However, since Nagasawa does not put forward any considerations in favour of exist- ential optimism, his argument is not strictly against naturalism, but against the conjunc- tion of naturalism and existential optimism. In contrast, my argument includes the crucial premise that the desire for life is rational (motivated in (3)) which, combined with the premise that the desire for life would not be rational given naturalism, yields the conclu- sion that naturalism is false. Conclusion: (3) is true I have advanced a serious challenge to the claim that the desire for life can be reflective, and presented a solution to this challenge, which is compatible with traditional theism but incompatible with naturalism. The existence of God would give us strong reasons to believe that life is worthwhile. If instead naturalism were true, we would not be entitled to appeal to such reasons, hence (3). Having motivated all key premises, I now wish to compare briefly my argument to another, related one. Theism and naturalism contrasted But could he deploy an analogous argument showing that the naturalistic, evo- lutionary forces that shaped our cognitive faculties would only have given us an innate, distinctive desire for life if life was in fact worthwhile? An evolutionary explanation of the innate desire is easily articulated: being innately disposed to desire life from an early age is conducive to survival and reproductive success. Organisms disposed to desire life (and thus, to fear death) are more likely to survive long enough to reproduce than those that do not, everything else being equal. So, the in-built disposition to desire life was selectively retained in our ancestral populations, and passed on to us through the standard mechanisms of genetic inheritance. Thus, evolutionary psychologist Geoffrey Miller (2007) speaks of the ‘the hardwired fears and reactions that motivate humans to avoid death’: ‘Suffocate me, and I’ll struggle. Shoot me, and I’ll scream. The brain stem and amygdala will always do their job of struggling to preserve one’s life at any cost.’ y Significantly, however, the nature of the evolutionary process is such that the desire for life would have been selected for, regardless of whether life is worthwhile or not. This is so on both subjectivist and objectivist accounts of value. For instance, take the sim- ple hedonistic view that some activity is worthwhile just as long as it is pleasurable on the whole. The sad truth is that wanting to live would still be conducive to greater https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Religious Studies 689 reproductive success, even if life invariably involved far more pain than pleasure. For even in this scenario, human beings who desired to live would fare better in the evolutionary race. The same holds if we take more objectivist positions, such as the view that ‘worth- whileness’ is an irreducible, mind-independent property: whether life instantiates this property or not is irrelevant to natural selection.17 The upshot of this is that holding irrational desires – specifically, desires whose objects are undesirable – may be an evolutionarily beneficial trait. There is hence a crucial asym- metry between theism and naturalism, insofar as theism gives us strong reasons to believe that life is worthwhile, whereas naturalism does not. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press An objection to (2): elitism An objection to (2): elitism My argument’s second premise states that the desire for life cannot be rational unless it is reflective – that is, unless one has good reasons to believe that life is worthwhile. One could object that this is too restrictive, and that it implausibly rules out many patently rational desires. For example, very young children desire food, sleep, affection, play, etc. without giving any thought at all to the value or desirability of such things. Having a reflective desire requires the ability to represent reflectively their own desires to themselves, an ability that such children almost certainly lack. But it seems absurd to call these desires irrational. ‘Rational’ and ‘irrational’ are normative terms, and to call a desire ‘irrational’ is to say that there is a sense in which we ought not to have it. And there is surely no sense in which very young children ought not to desire the goods listed above. g In short, it could be argued that the reflectiveness requirement is unduly elitist, since it denounces the natural desires of very young children as irrational. 19 Since it is indeed implausible to claim that all (non-intrinsic19) desires must be reflect- ive in order to be rational, the objection compels us to specify the conditions under which non-reflectiveness is problematic. Recall the example given above: you learn that your desire for the fancy gadget is the result of manipulative marketing, and this causes you to consider this desire as irrational. Note that in the example, your awareness of the true origins of your desire, and of the absence of reasons to regard it as a worthy object of desire, appears to play an important role. Intuitively, one cannot rationally continue to desire something after learning that there are no good reasons to regard said thing as valuable.20 This seems to motivate the following principle: Loss of Innocence (LI): If you know that you have no good reasons to believe that some state of affairs S is desirable, your (non-intrinsic) desire for S is irrational. LI would explain why the non-intrinsic desires of very young children can still be rational, even if they are not reflective: since they give no thought to the desirability (or lack thereof) of the objects of their desires, they are unaware of any lack of reasons to believe that said objects are desirable. Nagasawa’s ‘Problem of evil for atheists’: a comparison One could say that while both arguments try to show that Christophe de Ray 690 naturalism commits one to a certain kind of pessimism about life in the world, only my argument draws the further conclusion that naturalism must therefore be false. Moreover, it should be noted that the grounds for saddling naturalists with pessimism about life are different in both arguments. In mine, the worry arises from the practical impossibility of determining that life is worthwhile given naturalism, not from the sys- temic nature of natural evil. My argument and Nagasawa’s thus constitute distinct contributions to the field, despite their many similarities. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press An objection to (2): elitism Should they begin to reflect on the value of these objects, they may come to realize that some of them are unworthy of being desired, in which case some of their desires would thereby become irrational. However, LI is insufficient in its present form. Returning to our example, your desire for the gadget did not become irrational upon your discovery of its true origins. Rather, it was irrational all along; you simply became aware of its irrationality. This suggests the following modification to LI: LI-2: If you know or are able to know that you have no good reasons to believe that some state of affairs S is desirable, your (non-intrinsic) desire for S is irrational. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Religious Studies Religious Studies 691 LI by itself sanctions my desire for a cigarette, just as long as I am ignorant of the fact that I have no good reasons to value smoking, even though I have easy epistemic access to this fact. As such, LI is an overly permissive principle, hence the need for LI-2. LI by itself sanctions my desire for a cigarette, just as long as I am ignorant of the fact that I have no good reasons to value smoking, even though I have easy epistemic access to this fact. As such, LI is an overly permissive principle, hence the need for LI-2. LI-2 is also capable of explaining why very young children can have rational, non- reflective (non-intrinsic) desires: they not only lack the knowledge that some of the objects of their desires may not be worthwhile, they are unable to acquire this knowledge, since they lack the relevant metacognitive capacities. In contrast, we adults are able to reflect on our desires and the value of their objects, and are fully aware that some desired states of affairs are not desirable. Let us finally turn to our desire for life, and suppose that we have no reason to believe that living is a worthwhile project, that is, it is an unreflective desire. We are either aware of this, or not. If we are, then by both LI and LI-2, our desire for life is irrational. An objection to (2): elitism If we are not, we are still capable of becoming aware of this, assuming that we are not infants, since we are fully capable of understanding the considerations raised against the claim that we can show life to be a worthwhile project (see above). Thus, by LI-2, our desire for life would still be irrational. Whether we would know it or not, then, our desire for life would be irrational if unreflective, and premise (2) stands unscathed. Another objection to (2): the benefits of desiring to live A second way of responding to (2) appeals to pragmatic considerations. It is sometimes argued that, in cases where it is unclear whether a certain belief B is true, it may be rational to (as it were) elect to hold to hold B, as in Pascal’s wager. Perhaps a similar prin- ciple applies to desire, and the naturalist’s desire for life in particular: since it is unclear, given the evidence, whether or not life is worthwhile on naturalism, it may be rational for the naturalist to elect to desire to live. To show that this choice is in fact rational, the naturalist would need to show that the likely benefits of desiring life would outweigh its likely costs, just as Pascal argued for belief in God. He could, for instance, argue that those who desire to live are thereby more likely to be happy, since they are alive and thus have what they wish. The obvious reply here is that the desire to live carries with it the fear of losing one’s life in a danger- ous world, and the sadness that comes with the knowledge of one’s inevitable demise. Conversely, not desiring to live may deprive one of the happiness and gratitude for being alive, but also of the associated anxiety and sorrow. In each case, it seems impos- sible to show that the benefits outweigh the costs, for the same reasons that it was impos- sible to show that the benefits of being alive outweigh its costs (see above), chiefly, the sheer complexity of our emotional lives. But in that case, the naturalist cannot defend the desire for life on pragmatic grounds. An objection to (3): perspectivism My argument’s third premise states that if naturalism is true, our desire for life cannot be reflective, since there are on naturalism no good reasons to believe that life is worthwhile. ‘Perspectivists’ could object that the truth or falsehood of naturalism is irrelevant, because reasons depend on perspectives, not mind-independent facts. To borrow an example from Bernard Williams (1991, 101), suppose Bill believes – and has every reason to believe – that the contents of his glass is gin and tonic, when it is in fact petrol and ice. Intuitively, he still has a good reason to drink his glass (assuming he enjoys drinking gin and tonic) despite the fact that doing so would make him seriously ill. Similarly, one could argue that the human desire for life can still be rational, despite the truth of naturalism, as long as human beings have compelling reason to believe that theism is true. For then, https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Christophe de Ray 692 they could appeal to the argument above concluding with the proposition that God would ensure that our lives are worthwhile if he existed. It would hence be reasonable for us to believe that living is a worthwhile project. But this would be a case of us forming a reflect- ive desire for life even while naturalism is true, against (3). Needless to say (and notwithstanding the intuitive plausibility of perspectivism), this objection should be deeply unappealing to naturalists, for two reasons. First, it would require the concession that there are compelling reasons to believe that theism is true, and that naturalism is therefore false. Second, and even less attractively, they would need to accept that they themselves cannot have a reflective desire for life, and thus (given (2)) that their desire for life is not rational. This would amount to saying that (1), which claims that the ‘desire for life is rational’, applies to theists, but not to natur- alists. Insofar as most naturalists are probably inclined to agree with (1), I cannot imagine that very many of them would consent to being excluded from its scope. y y g p We may thus leave this objection to the side, and turn to the next one. Another objection to (3): Kahane’s problem Guy Kahane (2018) has argued that the existence of God or any otherworldly good or realm is unnecessary to making our lives worthwhile, since sufferings undergone in this life could in principle be more than compensated for by an eternal, purely physical afterlife involving only worldly goods and pleasures (meaningful friendships, intellec- tually stimulating activities, exciting trips, etc.), whether or not God exists. In that case, it may be thought that the naturalist is no worse off than the theist, since both world-views are compatible with a blissful post-mortem existence, and thus with life being worthwhile. However, recall that the motivation for (3), which states that the desire for life cannot be reflective on naturalism, is that if naturalism were true, we would have no reason to believe that life’s goods would outweigh its evils. This is very different from saying that given naturalism, it is impossible that this would be the case. The latter is far more difficult to substantiate; fortunately only the former is required to motivate (3) (since for my desire for life to be ‘reflective’ just is for me to have strong reasons to believe that living is a worthwhile activity). g y Now, there are indeed conceivable naturalistic scenarios in which human beings enjoy blissful afterlives – say, if immortal aliens somehow upload the consciousness of every human being into an unending ‘Virtual Reality’ world involving all sorts of pleasure.21 But there would be no grounds for believing that this sort of scenario will obtain, given naturalism – only grounds for believing that they could obtain.22 By contrast, I argued that there are strong reasons to expect the God of traditional theism, should he exist, to give us an opportunity for supreme happiness after we die. So, while both theism and naturalism would make possible the worthwhileness of lives, only on the former pos- ition would we be justified in trusting that they are actually worthwhile. I conclude that Kahane’s problem does not apply to my argument. Another objection to (3): life just seems worthwhile Once again, a desire to engage in activity A is ‘reflective’ just as long as there are good reasons to believe that A is a worthwhile activity. I have argued that there are, on natur- alism, no good reasons to believe that life is a worthwhile activity, and thus that the desire for life cannot be reflective on naturalism. The naturalist could object that I have not in fact shown this. Instead, I have shown that there are (on naturalism) no good arguments whose conclusion is that life is worthwhile. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Religious Studies 693 Perhaps, life simply seems worthwhile to us, and this could, in itself, be a good reason for thinking that life is worthwhile, even in the absence of a cost-benefit analysis showing that life is worthwhile. After all, it is widely held that at least some beliefs can be justified simply in virtue of seemings, without the need to be inferred from other beliefs. Phenomenal conservatism states that one can justifiably believe that P just as long as it appears to one that P, unless there are defeaters for the belief that P (for instance, if it seems to me that there is a cat on the mat, my belief that there is a cat on the mat cat can be justified, provided that I have no reasons to doubt this belief). If so, then perhaps it is sufficient that life seems worthwhile to us. The ‘seeming’ in question would have to be an intuitive one (rather than, say, a visual seeming). My defence of premise (1) suggested that the rationality of the desire for life seems intuitively obvious to us, and that this is reason enough to accept the premise. If so, then why shouldn’t the same be said for the worthwhileness of life? Of course, the objection stands only if our intuitive conviction that life is worthwhile, namely, that its benefits outweigh its costs, is faced with no defeaters. It may perhaps be thought that the considerations raised in defence of (3) entail that there can be no such defeaters: due to the extreme complexity of our lives and incommensurability of many goods and evils, we cannot tell whether said evils outweigh the goods, and thus whether the initial optimistic intuition is false. Another objection to (3): life just seems worthwhile An intuitive seeming to the effect that life is worthwhile therefore does not remove the need for an argument showing that it is worthwhile. Without this, the intuition that life is worthwhile will remain faced with an undefeated defeater, in which case there is no good reason to hold this belief. Another objection to (3): life just seems worthwhile But a defeater D for a belief that P need not show that P is false, in order to be effective. All that is needed is for D to constitute significant evidential support for not-P. For example, suppose I believe that it would be worthwhile to take on a new job that I have just been offered. Suppose a former employee assures me that the manager I would be working under is quite an unpleasant and incompetent per- son. This new information would not show that my initial belief was false – for all I know, the generous work benefits and the stimulating nature of the job more than make up for having a sub-par manager. Even so, it could still count as a defeater for my belief that taking the job is worthwhile, insofar as it significantly lowers the justification of said belief, which it may do without compelling me to hold the opposite belief (i.e. ‘taking on the job would not be worthwhile’). This defeater may or may not itself be defeated, depending on whether I can show that the costs of the job are outweighed by its benefits. p g j g y Now, take the fact that to be alive is to risk experiencing horrible tragedies, of the kind only partially enumerated above. This does not entail that life is not worthwhile, and that its evils are not outweighed by its good. But it is certainly evidence in favour of the latter prop- osition. The horrors that so many suffer, and the very real risk that everyone faces of suffer- ing them, would be expected given the hypothesis that life is not worthwhile. This evidence seems sufficiently strong to count as a defeater, that is, to give us a reason to doubt our ini- tial optimistic intuition about the worthwhileness of life, and to suspend judgment on the matter until we acquire reasons to believe that the goods of life are worth its evils. This, I have argued, is precisely what naturalists, unlike theists, cannot access. Contrary to the objection, then, there is a defeater for the widespread belief that life’s evils are outweighed by its costs. In fact, part of the point of my argument is to draw attention to this defeater, before showing that it cannot itself be defeated on naturalism. One last objection to (3) (2*) is far more plausible than (2′), but the change from (3′) to (3*) is not inconsequential. The reformulated argument simply tells us that there are reasons to believe that life is worthwhile, without telling us what those reasons are. If, as I have been arguing, natur- alism entails that there are no such reasons, (3*) entails that naturalism is false. Thus, the naturalist cannot deploy the above argument without presupposing that my argument is unsuccessful (since if it is successful, the above argument would disprove naturalism). To object to my argument in this way would therefore be question-begging. One last objection to (3) Granted that there needs to be an argument showing that life is worthwhile, the naturalist may propose the following one: https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Christophe de Ray 694 (1′) Our desire for life is rational. (1′) Our desire for life is rational. (2′) If our desire for life is rational, then life is worthwhile. (3′) Therefore, life is worthwhile. (1′) is obviously identical to (1), so I cannot reasonably take issue with it. (2′) seems plaus- ible enough, and entails (3′) when conjoined with (1′). Has the naturalist found an argu- ment? If so, it would follow that the desire for life can be reflective on naturalism, against (3). (1′) is obviously identical to (1), so I cannot reasonably take issue with it. (2′) seems plaus- ible enough, and entails (3′) when conjoined with (1′). Has the naturalist found an argu- ment? If so, it would follow that the desire for life can be reflective on naturalism, against (3). g Unfortunately, (2′) is incorrect. For it could be that life is not worthwhile, even though we have strong reason to believe that it is, and are thereby rational in wanting to live. Since it is possible that the antecedent is true while the consequent is false, the implica- tion in (2′) is false. To be sound, the argument should be reformulated as follows: (1*) Our desire to live is rational. ( ) ( ) (2*) If our desire to live is rational, we have strong reason to believe that life is worthwhile. (2*) If our desire to live is rational, we have strong reason to believe that life is worthwhile. ( *) h f h b l h l f h h l (3*) Therefore, we have strong reasons to believe that life is worthwhile. (2*) is far more plausible than (2′), but the change from (3′) to (3*) is not inconsequential. The reformulated argument simply tells us that there are reasons to believe that life is worthwhile, without telling us what those reasons are. If, as I have been arguing, natur- alism entails that there are no such reasons, (3*) entails that naturalism is false. Thus, the naturalist cannot deploy the above argument without presupposing that my argument is unsuccessful (since if it is successful, the above argument would disprove naturalism). To object to my argument in this way would therefore be question-begging. A tu quoque objection: heaven and hell I have argued that given naturalism, there are no good reasons to expect the goods of life to outweigh its evils, and thus that our desire for life cannot be reflective if naturalism is true. By contrast, traditional theism opens up the potential for a state of perfect happi- ness after death, which would vastly outweigh whatever suffering we may have endured during our physical lives. One world-view is thus in a significantly better position to defend the worthwhileness of life than the other. But the naturalist could charge that the asymmetry is illusory, for two reasons. First, consider the candid words of the apocryphal 2 Esdras: Let the human race lament, but let the beasts of the field be glad; let all who have been born lament, but let the four-footed beasts and the flocks rejoice! For it is much better with them than with us; for they do not look for a judgment, nor do they know of any torment or salvation promised to them after death. For what does it profit us that we shall be preserved alive but cruelly tormented? For all who have been born are involved in iniquities, and are full of sins and bur- dened with transgressions. And if we were not to come into judgment after death, perhaps it would have been better for us. (7:65–69) The author laments that the lives of animals are preferable to those of humans, precisely because our souls live on after physical death, and are thus potentially subject to a mis- erable afterlife. The naturalist need not go so far. But he can argue that, just as it is unclear whether worldly goods outweigh worldly evils, it is unclear whether otherworldly https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Religious Studies Religious Studies 695 goods outweigh otherworldly evils. For many traditional theists, being alive gives one an opportunity to progress in virtue and holiness towards the supremely great good of eter- nal salvation. Conversely, it involves the risk of damning one’s soul, through poor life choices and acquired habits. One the face of it, damnation seems at least as bad as salva- tion is good, in which case the cost and the benefit cancel each other out. A tu quoque objection: heaven and hell If so, the trad- itional theist appears to be in no better position than the naturalist to show that life is worthwhile, since expanding one’s ontology beyond the natural fails to increase the net value of being alive. In response, I argued earlier that God, a supremely good being, would ensure that our lives our worthwhile if he existed. Now, if the risk of damnation entailed that life is not in fact clearly worthwhile (as per the above objection), it would not follow that God would not after all ensure that our lives are worthwhile. Rather, it would follow that contrary to traditional Christian teaching, theism is inconsistent with the potential for damnation. Thus, the objection suggests the following argument: (iv) If God exists, life is clearly worthwhile. y v) If there is a real risk of ending up in hell, life is not clearly worthwhile. (v) If there is a real risk of ending up in hell, life is not clearly worthwhile. g p y (vi) Therefore, it cannot be the case that God exists and that there is a real risk of ending up in hell. (vi) Therefore, it cannot be the case that God exists and that there is a real risk of ending up in hell. Various theists have defended (vi). Universalists like Thomas Talbott (2014) and Daniel Howard-Snyder (2003) argue that all human beings will ultimately receive salvation, even if some will need to undergo a lengthy purgatorial experience first. Less radically, annihilationists (e.g. Swinburne (2003), 182)23 contend that damnation amounts to anni- hilation after a certain period of post-mortem suffering, in which case the goodness of salvation would vastly exceed the evil of damnation, since eternal happiness is intuitively far better than the simple cessation of existence is bad. At most, then, the objection shows only that there are compelling reasons to deny that God would allow anyone to suffer everlasting torment in hell. It does not show that theism can give us no positive assurance that our lives are worthwhile. In truth, I am unconvinced by (v) of the above argument. For it seems to me that life could be worthwhile even with the possibility of eternal suffering. Suppose, for instance, that damnation were significantly less likely than salvation, and/or significantly less unpleasant than salvation is pleasant. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press A tu quoque objection: heaven and hell In that case, the potential rewards of being alive would be well worth its risks, as high as these would be. And it would not be difficult for the God of traditional theism to actualize this state of affairs. [here]One possible rejoinder here is that a chance to gain infinite happiness, however great, could never outweigh the risk of infinite pain, however slight, owing to the nature of infinity. But this is simply implausible: suppose I could participate in a contest which I had a reasonable chance of winning, and such that the reward if I won would be an eter- nal, supremely joyful state, while the penalty if I lost would be a barely noticeable but unending itch on my toe. It seems absurd to say that the reward wouldn’t be worth the risk of participating here. p p g One could also object that, just as we are incapable of knowing through introspection whether our earthly lives are more pleasurable than painful (see above), we are likewise in the dark about the moral state of our minds, and thus about whether we will receive sal- vation or damnation. Kantian worries about our inability to know the motives of our actions, and Freudian ones about the opacity of our mental states more generally, spring to mind. But in that case, I am in no epistemic position to argue that God will grant me eternal happiness, and hence that my life is still worthwhile despite its pains. 696 Christophe de Ray But surely I can know that a given project is worthwhile, even if I cannot know what the outcome of engaging in it will be, as when I apply for jobs or write and submit requested revisions to an article for a philosophy journal. If the desired outcome does not obtain, and an undesirable outcome obtains instead – for example, my application is rejected, and I experience sadness and a loss of self-confidence as a result – this would not entail that the project in question was not worthwhile, and that I was wrong to think that it was.24 It is sufficient that, upon deciding whether or not embark on the project, I know that my chances of succeeding are reasonably good. A tu quoque objection: heaven and hell These need not necessarily be higher than my chances of failure, as it is surely sometimes rational to embark on projects with a low chance of success (e.g. applying for a job with a hundred other applicants), but they need to be high enough to warrant the associated risk. But, the objector insists, how can we know that our chances of receiving salvation are ‘reasonably good’ in this sense? I propose the following alternative to the above argument: (iv′) If God exists, life is clearly worthwhile. (iv′) If God exists, life is clearly worthwhile. y (v′) If there is a real risk of ending up in hell, and no reasonable chance of receiving salvation, life is not clearly worthwhile. (v′) If there is a real risk of ending up in hell, and no reasonable chance of receiving salvation, life is not clearly worthwhile. (vi′) Therefore, it cannot be the case that God exists and that there is a real risk of end- ing up in hell, and no reasonable chance of receiving salvation. (vi′) Therefore, it cannot be the case that God exists and that there is a real risk of end- ing up in hell, and no reasonable chance of receiving salvation. (vi′) entails that if theism is true, we can know that we have a reasonable chance of acquiring salvation, and thus that life is worthwhile, even if hell is a real possibility. (vi′) entails that if theism is true, we can know that we have a reasonable chance of acquiring salvation, and thus that life is worthwhile, even if hell is a real possibility. Thus in my view, the possibility of hell would not entail that life is not clearly worth- while. But even if it did, this would not show that theism would fail to give us good rea- sons to believe that life is worthwhile – once again, it would simply show that theism and hell are incompatible. I conclude that the asymmetry between theism and naturalism with respect to the rationality of the desire for life still stands. Can the naturalist turn the tables? In order to show that the desire for life does not pose the same problem to theism, I defended the claim that ‘God would ensure that human life is worthwhile’ (see above). This contention may be thought to backfire severely against theism. Consider the follow- ing argument, suggested (though not defended at length) by Jason Megill and Daniel Linford (2016): (I) If God exists, then all lives have meaning. (II) There is or has been at least one human life that lacked meaning. (III) Therefore, God does not exist. (III) Therefore, God does not exist. Suppose that for a life to ‘have meaning’ here is for it to be worthwhile. If God’s existence would guarantee that all human lives are meaningful in this sense, good evidence of a single meaningless human life would be sufficient to disprove theism. Hence, my argu- ment appears to make theism vulnerable to an easy refutation. Suppose that for a life to ‘have meaning’ here is for it to be worthwhile. If God’s existence would guarantee that all human lives are meaningful in this sense, good evidence of a single meaningless human life would be sufficient to disprove theism. Hence, my argu- ment appears to make theism vulnerable to an easy refutation. However, recall that part of my motivation for the claim that God would ensure the worthwhileness of our lives was the claim that God could do so. As I argued, God’s capacity to provide us with the opportunity to achieve eternal happiness in union with him entails that he is able, if he so wishes, to make it the case that even the worst human lives are worthwhile. Thus, being alive in the world would be desirable just as long as the https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Religious Studies Religious Studies 697 probability that one actually achieves this is not too low, even if the quality of one’s earthly existence is truly wretched. I then argued that if God could accomplish this, he would. If so, and unless the naturalist is able to argue that this is not in fact the case, and that the opportunity for eternal joy would fail to make a miserable life worthwhile, the claim that there is at least one meaningless life (i.e. to claim (II)) would thus simply beg the question against theism. Can the naturalist turn the tables? Furthermore, the naturalist presumably accepts that some lives which contain a sig- nificant amount of misery would nevertheless still be worthwhile given a realistic oppor- tunity for supreme, eternal happiness. In that case, it would seem strange and arbitrary for the naturalist to argue that there is a certain level of earthly misery, beyond which even the chance of attaining a blessed eternity would fail to make life worthwhile. In any event, the onus is on the naturalist to show that such a line exists, and that it is actually crossed by some human lives in the world. Conclusion I have presented and defended an argument against naturalism, from the rationality of the desire for life. The crux of the argument is that there are, on naturalism, no good rea- sons to believe that life is worthwhile, in which case it would not be rational to desire life. But since it surely is rational to desire life, it follows that naturalism must be false. While the argument laid out in this article does not directly support theism (since it concludes with the falsehood of naturalism), it should be clear that it gives us strong rea- son to prefer theism over naturalism, insofar as no parallel argument from the meaning of life can be levelled against theism, as I have argued. I conclude that the meaningfulness of human life constitutes a significant consideration in favour of theism. Notes 1. Metz (2019) helpfully distinguishes between ‘extreme’ and ‘moderate’ supernaturalism, and notes that theistic philosophers have gravitated towards the latter in recent years. Extreme supernaturalists argue that no meaning at all is possible without God, while their moderate counterparts claim that the existence of God would greatly enhance the meaningfulness of our lives. g This scenario is adapted from Thomas Nagel (1971) p g 3. Though some, such as Daniel Hill (2002), ‘bite the bullet’ and accept that God’s life is devoid of meaning. f h d h b d f b f l d b l 3. Though some, such as Daniel Hill (2002), ‘bite the bullet’ and accept that God’s life is devoid of meaning. 4. Some go further and argue that being made for a purpose, even by a perfectly good being, degrades our lives rather than increasing their meaningfulness, since it reduces human to the status of mere artifacts or instru- ments to an end (in Kurt Baier’s words, a ‘gadget, a domestic animal, or perhaps a slave’ (Hill (2000), 120), rather than ends in themselves. 4. Some go further and argue that being made for a purpose, even by a perfectly good being, degrades our lives rather than increasing their meaningfulness, since it reduces human to the status of mere artifacts or instru- ments to an end (in Kurt Baier’s words, a ‘gadget, a domestic animal, or perhaps a slave’ (Hill (2000), 120), rather than ends in themselves. 5. Some philosophers take the stronger view that for a desire to be ‘rational’ is for it to be rationally obligatory. The weaker view outlined above will be assumed for the purpose of my argument. 5. Some philosophers take the stronger view that for a desire to be ‘rational’ is for it to be rationally obligatory. The weaker view outlined above will be assumed for the purpose of my argument I will take ‘justified’ and ‘rational’ to refer to the same property of desires and beliefs. ( ) 6. I will take ‘justified’ and ‘rational’ to refer to the same property of desires and beliefs. 7. For instance, Nagasawa quotes Richard Dawkins (2009) who, despite decrying the violence of the evolutionary process, claims to be ‘grateful to be alive to appreciate [natural] wonders’ like the Grand Canyon and the Milky Way. 7. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Notes Such as the one featured in the ‘San Junipero’ episode of the Black Mirror TV Series. 22. That is, unless Nick Bostrom (2011) is correct in telling us that we ‘almost certainly’ live in a computer simu- lation, and that we have good reason to believe that the creator of said simulation is good enough to be expected to grant us a happy afterlife. Admittedly, this strikes me as more of a form of metaphysical idealism, à la Berkeley, than a bona vide variant of naturalism. Either way, I take it that the overwhelming majority of natur- alists would disagree with Bostrom, and agree with me that there is on naturalism no good reason to expect that we will enjoy a happy afterlife. 23. Evangelical Anglican Theologian John Stott is probably one of the best-known advocates of ‘annihilationism’, although he does not hold to it dogmatically (Edwards and Stott (1989)). Proponents argue that on their view, the damned, while not eternally conscious, still receive ‘everlasting punishment’ (Mt 25:46), since their annihilation is eternal and with no opportunity for forgiveness and repentance. 24. Hence, the fact that someone corrupted his soul to the point of damnation would not entail that living was never a worthwhile project to begin with for this person, even if the person experiences more suffering than happiness as a result. Rather, living was worthwhile for him since it gave him the opportunity for a blessed after- life, which he squandered. Notes As Paul Edwards (1997, 141) puts it, while ‘It makes sense for a person to ask about something “Is it really worthwhile?” or “Is it really worth the trouble?” if he does not regard it as intrinsically valuable’, it ‘does not make sense [however] to ask such a question about something he regards as valuable in its own right’. 14. Benatar (2006, 65) discusses the well-documented Polyanna Principle: we are much more likely to recall posi- tive past experiences than negative ones g j 13. As Paul Edwards (1997, 141) puts it, while ‘It makes sense for a person to ask about something “Is it really worthwhile?” or “Is it really worth the trouble?” if he does not regard it as intrinsically valuable’, it ‘does not make sense [however] to ask such a question about something he regards as valuable in its own right’. q g g g 14. Benatar (2006, 65) discusses the well-documented Polyanna Principle: we are much more likely to recall posi- tive past experiences than negative ones. 15. A closely similar argument is advanced by Jason Megill and Daniel Linford (2016). While their main purpose is to show that God cannot be the source of meaning in life and thus that his existence is not necessary for our lives to be meaningful, it is nevertheless a sufficient condition. In their words, ‘The existence of God would guar- antee that our lives have meaning’ (ibid., 8, emphasis mine). g . My defence of (i) admittedly assumes that continued existence after physical death is metaphysica ssible. 17. This paragraph may remind the reader of so-called ‘evolutionary debunking arguments’ in ethics (e.g. Street (2009)). Unlike such arguments, I am not arguing that evolutionary theory has sceptical implications, but rather that naturalists cannot appeal to evolutionary considerations (in the way that theists can appeal to theological considerations) to show that life must be worthwhile, since the desire for life is a product of evolution. 18. Though Nagasawa does not explicitly refer to metaphysical naturalism, he describes the ontology of atheists as ‘being limited to the material universe’ (Nagasawa (2018),154), indicating that he intends to target naturalists specifically. 19. As we have seen, intrinsic desires present a special case. 20. Again, unless said thing is an intrinsic good. g g g 21. Such as the one featured in the ‘San Junipero’ episode of the Black Mirror TV Series. 21. Notes For instance, Nagasawa quotes Richard Dawkins (2009) who, despite decrying the violence of the evolutionary process, claims to be ‘grateful to be alive to appreciate [natural] wonders’ like the Grand Canyon and the Milky Way. 8. This need not commit us to the claim that being alive is an intrinsic good, desirable in and of itself (indeed, I will argue that it is not). We need only say, for instance, that refraining from saving Bob’s life would deprive him of the goods (e.g. meaningful relationships and projects) in virtue of which he desires to continue to live. This is so, even if Bob’s death is painless, and he is completely unaware of it. 8. This need not commit us to the claim that being alive is an intrinsic good, desirable in and of itself (indeed, I will argue that it is not). We need only say, for instance, that refraining from saving Bob’s life would deprive him of the goods (e.g. meaningful relationships and projects) in virtue of which he desires to continue to live. This is so, even if Bob’s death is painless, and he is completely unaware of it. y Note that I am not saying that the moderate reading of (1) is true and that the absolutist reading is false – on at the former is all that is needed for my argument to go through. 9. Note that I am not saying that the moderate reading of (1) is true and that the absolutist reading is false – only that the former is all that is needed for my argument to go through. 10. This is analogous to aggregationism in ethics, i.e. the view (accepted by utilitarians) that the overall value of the world is the sum of the values of its parts. This construal of the meaningfulness of life in terms of its aggre- gate value would admittedly be resisted by some theistic philosophers, who would argue that life is meaningful 10. This is analogous to aggregationism in ethics, i.e. the view (accepted by utilitarians) that the overall value of the world is the sum of the values of its parts. Notes This construal of the meaningfulness of life in terms of its aggre- gate value would admittedly be resisted by some theistic philosophers, who would argue that life is meaningful https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Pre Christophe de Ray 698 just as long as God has ordained a purpose for it. However, it may simply be that such philosophers and myself are referring to different properties when we speak of life’s ‘meaning’. Indeed, an aggregationist analysis is dif- ficult to resist when one defines meaningfulness as worthwhileness. Hence the aggregationist account of worth- whileness, while not uncontroversial, will be assumed to be true throughout this article. 11 I will qualify this statement later just as long as God has ordained a purpose for it. However, it may simply be that such philosophers and myself are referring to different properties when we speak of life’s ‘meaning’. Indeed, an aggregationist analysis is dif- ficult to resist when one defines meaningfulness as worthwhileness. Hence the aggregationist account of worth- whileness, while not uncontroversial, will be assumed to be true throughout this article. 11 I will qualify this statement later 12. One could object that, if you had an unlimited supply of money, it wouldn’t matter to you that your desire for the gadget resulted from manipulative marketing, or whether or not you have good reasons to believe that the gadget is valuable. Does this show that it is possible to rationally desire something while lacking good reasons to believe that it is worthwhile? Not quite. First, the fact that you wouldn’t care whether the value of the gadget warrants your desire for it doesn’t show that your desire is rational: it could just be that your desire, while irrational, is relatively harmless (much like my irrational desire to stay away from spiders). Second, this would be a case in which acquiring the gadget carries no cost whatsoever (or negligible cost). This is strong rea- son to believe that acquiring the gadget is worthwhile – if it costs nothing, it can only benefit you! Therefore, this would not be a case in which one lacks good reasons to believe that an object of desire is worthwhile. g j 13. https://doi.org/10.1017/S0034412522000531 Published online by Cambridge University Press Benatar D (2006) Better Never to Have Been. Oxford: Clarendon Press. B t N (2011) I fi it thi A l i d M t h i 10 9 59 Baier K (2000) The meaning of life. In Klemke E (ed.), The Meaning of Life. New York: Oxford University Press, pp. 101–132. 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Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces

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Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces ANNA C. TASOLAMPROU 1, ALEXANDROS PITILAKIS 1,2, SERGI ABADAL 3, ODYSSEAS TSILIPAKOS 1, (Senior Member, IEEE), XAVIER TIMONEDA3, HAMIDREZA TAGHVAEE3, MOHAMMAD SAJJAD MIRMOOSA4, FU LIU 4, CHRISTOS LIASKOS 1, (Member, IEEE), AGELIKI TSIOLIARIDOU1, SOTIRIS IOANNIDIS1, NIKOLAOS V. KANTARTZIS 1,2, (Senior Member, IEEE), DIONYSIOS MANESSIS5, JULIUS GEORGIOU 6, (Senior Member, IEEE), ALBERT CABELLOS-APARICIO3, EDUARD ALARCÓN3, ANDREAS PITSILLIDES7, (Senior Member, IEEE), IAN F. AKYILDIZ 7,8, (Fellow, IEEE), SERGEI A. TRETYAKOV4, (Fellow, IEEE), ELEFTHERIOS N. ECONOMOU1,9, MARIA KAFESAKI1,10, AND COSTAS M. SOUKOULIS1,11 1Foundation for Research and Technology Hellas, 71110 Heraklion, Greece 2Department of Electrical and Computer Engineering, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 3NaNoNetworking Center in Catalonia, Universitat Politècnica de Catalunya, 08034 Barcelona, Spain 4Department of Electronics and Nanoengineering, Aalto University, FI-00076 Aalto, Finland 5System Integration and Interconnection Technologies, Fraunhofer IZM, Berlin, Germany 6Department of Electrical and Computer Engineering, University of Cyprus, Nicosia, Cyprus 7Department of Computer Science, University of Cyprus, Nicosia, Cyprus 8School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, GA, USA 9Department of Physics, University of Crete, 70013 Heraklion, Greece 10Department of Materials Science and Technology, University of Crete, 70013 Heraklion, Greece 11Ames Laboratory, Department of Physics and Astronomy, Iowa State University, Ames, IA 50011, USA Corresponding author: Anna C. Tasolamprou ([email protected]) Thi k d b h E U i H i 2020 R h d I i P F E i T i (FETOPEN) responding author: Anna C. Tasolamprou ([email protected] This work was supported by the European Union Horizon 2020 Research and Innovation Programme-Future Emerging Topics (FETOPEN) under Grant 736876. ABSTRACT Software-defined metasurfaces are electromagnetically ultra-thin, artificial components that can provide engineered and externally controllable functionalities. The control over these functionalities is enabled by the metasurface tunability, which is implemented by embedded electronic circuits that modify locally the surface resistance and reactance. Integrating controllers within the metasurface able them to intercommunicate and adaptively reconfigure, thus imparting a desired electromagnetic operation, opens the path towards the creation of an artificially intelligent (AI) fabric where each unit cell can have its own sensing, programmable computing, and actuation facilities. In this work we take a crucial step towards bringing the AI metasurface technology to emerging applications, in particular exploring the wireless mm-wave intercell communication capabilities in a software-defined HyperSurface designed for operation in the microwave regime. Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces We examine three different wireless communication channels within the landscape of the reflective metasurface: Firstly, in the layer where the control electronics of the HyperSurface lie, secondly inside a dedicated layer enclosed between two metallic plates, and, thirdly, inside the metasurface itself. For each case we examine the physical implementation of the mm-wave transceiver nodes, we quantify communication channel metrics, and we identify complexity vs. performance trade-offs. INDEX TERMS Mm-wave communications, millimeter wave wireless communications, intercell wireless networks, mm-wave devices, artificially intelligent materials, software defined metasurfaces, antennas and propagation. INDEX TERMS Mm-wave communications, millimeter wave wireless communications, intercell wireless networks, mm-wave devices, artificially intelligent materials, software defined metasurfaces, antennas and propagation. SPECIAL SECTION ON MILLIMETER-WAVE AND TERAHERTZ PROPAGATION, CHANNEL MODELING AND APPLICATIONS Received June 30, 2019, accepted July 25, 2019, date of publication August 5, 2019, date of current version September 12, 2019. Di i l Obj Id ifi 10 1109/ACCESS 2019 2933355 Received June 30, 2019, accepted July 25, 2019, date of publication August 5, 2019, date of current version September 12, 2019. Digital Object Identifier 10.1109/ACCESS.2019.2933355 The associate editor coordinating the review of this article and approving it for publication was Ning Zhang. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0 I. INTRODUCTION Area as a function of the data rate for state-of-the-art transceivers for Wireless Personal Area Networks (WPAN) and chip-scale applications. Data extracted from [31] and references therein. The wireless approach is enabled by recent advances in on- chip antennas in mm-wave and THz bands [39]–[41], as well as the constant miniaturization of RF transceivers for short- range applications. As shown in Fig. 1, transceivers with multi-Gbps speeds and footprints as small as 0.1 mm2 have been demonstrated. Before assessing the potential applica- bility of existing transceivers, though, a deep understanding of the electromagnetic wave propagation within this new landscape is essential. For instance, it is important to analyze the possible propagation paths and assess the potential inter- ference between the MS operation and the wireless network. Some researchers have studied propagation in environments with metallic enclosures similar to HSFs [42]–[44], whereas others have investigated propagation within computing pack- ages [45]–[47]. However, these works consider structures that differ considerably from HSFs and do not account for their particularities in terms of RF interference. The integration of the computing and intercell communi- cation circuits on a per-cell basis is critical for the realiza- tion of the HSF vision [30]–[32]. A first implementation, as we will see, considers the embedding of multiple chips within the MS structure. Thus, by definition, we need to interconnect (i) the controllers within the same chip and (ii) the multitude of chips within the HSF to provide the highly sought distributed intelligence. In such an integrated environment, wired communication is the default choice as technical know-how from general-purpose Network-on- Chip (NoC) or low-power embedded systems can be effec- tively used [33], [34]. However, for off-chip communication, input/output pin scarcity severely limits the bandwidth and connectivity, whereas the latency and power consumption of transmission links increase significantly with distance. Even within the chip, NoCs may still suffer from power and latency issues in very dense HSFs due to their resemblance to massive manycore processors, where such issues are well known [35]. In such cases, wireless technology has proven to reduce the broadcast latency by an order of magnitude with a similar energy consumption [36]. In this paper, we undertake the thorough analysis of wire- less mm-wave intercell communication in the HSF environ- ment. The wireless intercommunication of the metasurface cells is a major step towards the realization of artificially intelligent fabrics. I. INTRODUCTION periodically aligned subwavelength features, the unit cells, that provide overall control over the metasurface electro- magnetic (EM) properties [1]–[4]. They exhibit a wide vari- ety of exotic electromagnetic functionalities, from perfect and controllable absorption, to beam and wavefront shap- ing, polarization control, broadband pulse delay, or harmonic Metasurfaces (MSs), the two dimensional version of metama- terials, are planar artificial structures with purposely designed 122931 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in t A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the L lamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces generation [5]–[17]. To add reconfigurability, together with the ability to host multiple functionalities within a single MS, recent works have proposed to embed circuits capable of tuning the response of each individual unit cell [18]–[20]. This allows us to drive such reconfigurable MSs via a cen- tralized control unit such as a field-programmable gate array (FPGA) [21], [22]. With the addition of external sensors providing feedback to the control unit, adaptive MSs can be conceived. are analogous to those in on-chip networks [36], [37] and other computing systems [38]. We note that wireless intercell communication refers to information transfer among the unit cells of a single HSF device and that wireless signals are not intended to be leaked outside the HSF. FIGURE 1. Area as a function of the data rate for state-of-the-art transceivers for Wireless Personal Area Networks (WPAN) and chip-scale applications. Data extracted from [31] and references therein. The recent HyperSurFace (HSF) paradigm aims to make a firm step ahead by realizing the vision of an intelligent metasurface fabric where each unit cell not only incorpo- rates its own sensing, programmable computing and actu- ation facilities, but can also exchange information with other unit cells [23], [24]. This allows the MS (i) to be autonomous, adapting to the environment without exter- nal intervention, (ii) to be seamlessly interconnected with other MSs, and subsequently (iii) to implement distributed sensing and intelligence both at the MS or system levels. This approach enables many exciting applications in robotics or within the exploding spectrum of ultra-compact Internet- of-Things (IoT) platforms such as high-capacity wire- less networks, physical-layer security, intelligent wireless environments, distributed beamforming and spatial-index modulations [25]–[29]. FIGURE 1. I. INTRODUCTION (d,e,f) Unit cell side-views (vertical cross-sections) illustrating the three considered communication channels: (d) Communication in the chip layer, (e) communication in a dedicated parallel-plate waveguide, and (f) communication inside the metasurface dielectric. of each unit cell. The controllers adjust the electromag- netic behaviour of the metasurface fabric by attributing addi- tional local resistance and reactance at will [13], [21], [22]. The controller plane is decoupled from the MS thanks to the back plane that separates the patches from the chips. with different advantages and constraints. In Section III, we assess the chip area as a communication channel, a natural choice since there lie all the electronics of the device. Then, in Section IV, we assume a parallel plate waveguide dedicated solely to the intercell mm-wave communication, a choice of high efficiency and security, at the expense of device complexity and volume. Finally, in Section V, we examine the metasurface layer itself as an opportunistic intercell commu- nication channel which minimizes the volume of the device. In all channels, we evaluate the communication performance through frequency- and time-domain numerical calculations providing data for the channel transfer function, mean delay and delay spread. Technical considerations, detailed designs, analysis of obtained simulation results, and discussion are provided for each channel, separately, before drawing the overarching conclusions and outlook of the intercell commu- nication exploration, compared to our preliminary study [31], in Section VI. Our case study MS is designed for perfect tunable absorp- tion and anomalous reflection operation in the microwave regime. For operation in the microwave regime, the lateral size of the metasurface is required to be in the order of tens of centimeters, i.e., at least a few wavelengths. Specifically, the reference MS structure under consideration is designed to operate at f = 5 GHz (λ0 ∼60 mm). It consists of periodically arranged, four-patch unit cells with xy size equal to D × D = 12 mm × 12 mm, as shown in Fig. 2. The size of each patch is w × w = 4.2 mm × 4.2 mm. The thickness of the substrate is h = 1.575 mm and it is made of Rogers RT/Duroid 5880 with permittivity ϵr = 2.2 and loss tangent tan δ = 9 × 10−4. Another high frequency laminate board could be also appropriate for this work. I. INTRODUCTION It provides the HyperSurface the capability of interchanging information through the whole extend of the fabric. This allows the controllers to readapt the meta- surface according to the external conditions even if modified locally, compensate for any malfunctioning cells, select con- figurations that balance power consumption and many others functionalities assessed with the readily assembled wireless solution. In [31], we performed a preliminary assessment of the spectral characteristics of intercell communication in two specific channels and in the frequency domain. Here, we present a rigorous frequency and time domain study towards the exploration of the wireless communication chan- nels within the landscape of a fully functional HSF, consisting of the components effectuating the electromagnetic manipu- lation of microwave radiation, the MS layer and the controller chip. We consider three different communication channels In light of these drawbacks, wireless intercell communi- cation becomes a compelling alternative in large or dense HSFs. The wireless option has the obvious advantage of not requiring wiring between the chips, which facilitates the assembly and improves the modularity of the solution. Moreover, assuming omnidirectionality, the wireless technol- ogy naturally supports broadcasting, thereby facilitating data dissemination and the implementation of distributed intel- ligence. Such enhanced connectivity also allows to imple- ment denser inter-chip network topologies. These advantages VOLUME 7, 2019 122932 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces tion of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasur FIGURE 2. The HyperSurface under study. (a) Top-view of unit cell with geometric parameters and (b) bottom-view of unit cell with the controller chip for the programmable operation. (c) A 5 × 6 unit cell MS operating at 5 GHz under oblique plane wave incidence. (d,e,f) Unit cell side-views (vertical cross-sections) illustrating the three considered communication channels: (d) Communication in the chip layer, (e) communication in a dedicated parallel-plate waveguide, and (f) communication inside the metasurface dielectric. FIGURE 2. The HyperSurface under study. (a) Top-view of unit cell with geometric parameters and (b) bottom-view of unit cell with the controller chip for the programmable operation. (c) A 5 × 6 unit cell MS operating at 5 GHz under oblique plane wave incidence. II. STRUCTURE, ENVIRONMENT DESCRIPTION AND ELECTROMAGNETIC OPERATIONS A. ENVIRONMENT DESCRIPTION We assume at this point that each chip serves four metallic patches. Chips are square with lateral dimensions of 2 mm × 2 mm and are placed 0.15 mm below the back plane. In our case study, we consider a conventional flip chip package which mainly consists of a standard die with attached solder bumps that carry the input/output (I/O) signals. The flip chip package will be assembled with bumps facing down on the backplane. Figure 3 shows a chip schematic layout, which includes, from top to bottom, a stack of materials typ- ically found in conventional chips: low-conductivity silicon die (ϵr = 11.9, ρ = 10 Ω-m, thickness of 0.5 mm), an insu- lator (silicon nitride of thickness 15 µm, ϵr = 7.5, assumed lossless), and a polyimide layer (ϵr = 3.5, 30-µm thick) As a case study we consider the software-defined HSF depicted in Fig. 2. The metasurface (MS) part shown in Fig. 2a consists of a periodic array of electromagnetically thin metallic patches placed over a dielectric substrate back-plated by a metallic layer serving as ground. To enable the software- based MS control, the patches are connected to a group of controller chips that lie below the metallic back plane through vertical vias (isolated from ground by holes) as shown in Fig 2a. The characteristics of the impinging electromag- netic wave that is reflected from the metasurface (see Fig. 2c) can be controlled depending on the electromagnetic features 122933 VOLUME 7, 2019 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in t A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the L lamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces FIGURE 3. Layout of the chip hosting the metasurface controllers. The stackup includes a low-conductivity silicon-die, a silicon nitride insulation layer, and a polyimide passivation layer. The total volume of the chip is approximately 2 mm × 2 mm × 0.8 mm. that propagate omnidirectionally in this environment, the only obstacles being the monopole antennas. This communication channel is completely isolated from the core of the metasurface and therefore does not interfere with the metasurface operation which offers increased security. Section IV evaluates the dedicated layer communication. B. WIRELESS PROPAGATION PATHS The physical landscape of the software-defined HSF offers several opportunities for the propagation of RF signals within the structure for wireless connectivity between the different controllers. The actual implementation depends on the tile lateral dimensions and the targeted wavelength. In this work we consider three distinct communication channels, as seen in Fig. 2(d,e,f): II. STRUCTURE, ENVIRONMENT DESCRIPTION AND ELECTROMAGNETIC OPERATIONS A. ENVIRONMENT DESCRIPTION • Communication in the metasurface layer The last channel under consideration is the space between the metasurface patches and the metallic ground plane, called MS layer. A blind via fed from the chip serves as the antenna, while the metallic patches and the metallic back plane act as a waveguide. The communication and metasurface operation takes place in the same vol- ume which minimizes the overall size of the device. Section V evaluates the communication scenario in the metasurface layer. FIGURE 3. Layout of the chip hosting the metasurface controllers. The stackup includes a low-conductivity silicon-die, a silicon nitride insulation layer, and a polyimide passivation layer. The total volume of the chip is approximately 2 mm × 2 mm × 0.8 mm. We study different channels with respect to their phys- ical constraints and the communication opportunities that they offer. In both chip and dedicated layer communication channels, the wave propagates in a restricted waveguide. The fact that they are completely decoupled from the metasurface layer allows for a wide choice of communication frequen- cies since the corresponding parts of the structure can be adapted accordingly without affecting the operation of the metasurface. On the other hand, the communication channel in the metasurface layer needs to be designed respecting the metasurface operation which requires a specific and invari- able geometry. This posses constraints in the design of the communication channel which will affect the details of the communication. that acts as passivation. The passivation layer allows an extra metallic layer, which in this case is a copper redistribution layer that connects the first metallization layers of the chip with the corresponding solder bumps at the bottom. In our case study, the solder bumps have a diameter of 0.25 mm and a pitch of 0.4 mm. We consider that the space surrounding the solder bumps, between the flip chip and the board, is free of underfilling epoxy material usually added for mechanical robustness. We further assume that the metasurface is not mounted on top of any object and, therefore, that there is nothing preventing signals to propagate below the chips. D. PERFORMANCE METRICS Once the whole matrix of frequency responses H is obtained, a path loss analysis can be performed by fitting the attenuation L over distance d to D. PERFORMANCE METRICS In the structures under study, however, in addition to S21, we also observe the S11 component in order to understand how much of the power is reflected back to the antennas operating as the transmitter/receiver. In the optimum case the antennas need to be impedance matched, that is, we need to achieve a very low value for the magnitude of S11. This translates into negligible losses due to reflection, and high values for the magnitude of S21. However, in the case of noticeable return loss (reflection), we can resolve this issue by employing an external matching circuit. In fact, the important parameter is essentially the transmission coefficient. We note here that the formulation above can be generalized for any transmitter i and receiver j. τij = R τPij(τ)dτ R Pij(τ) dτ . (6) (6) We also evaluate the multipath richness of the channel by obtaining the delay spread τrms of each PDP as We also evaluate the multipath richness of the channel by obtaining the delay spread τrms of each PDP as τ (i,j) rms = sR (τ −τij)2Pij(τ) dτ R Pij(τ) dτ , (7) (7) From a communications channel perspective, the delay spread provides a lower bound of the signal bandwidth that can be decoded correctly. Such a measure is generally referred to as coherence bandwidth and can be calculated for a channel between transmitter i and receiver j as B(i,j) c = 1 τ (i,j) rms . (8) (8) Frequency Domain Analysis. Besides the S-parameters, a metric pertinent to channel characterization is the transfer function, |Hij(f )|, of the channel defined between the trans- mitter i and the receiver j. It is obtained via the following expression In this work we will assume that all wireless channels are broadcast and, therefore, they should be operated at the lowest speed ensuring correct decoding at all nodes. As a result, we will take the worst delay spread as limiting case and use it to evaluate the coherence bandwidth Bc, as follows GiGj|Hij(f )|2 = |Sji(f )|2 (1 −|Sii(f )|2) · (1 −|Sjj(f )|2), (2) (2) τrms = max i,j̸=i τ (i,j) rms ⇒Bc = 1 τrms . (9) (9) (9) where Gi and Gj are the transmitter and receiver antenna gains, while Sji, Sii, and Sjj are the scattering matrix elements defined above. C. OPERATIONAL CONDITIONS Monopoles fed from the chip are inserted in the parallel-plate waveguide formed and excited waves VOLUME 7, 2019 122934 VOLUME 7, 2019 ntercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Met of operation is possible assuming that the properties of the materials involved remain the same. the antennas are matched and their gain is small, Sji alone provides a fair indication of path loss. Time Domain Analysis. In the time domain, the electro- magnetic (EM) solver allows to define an input excitation x(t) at the transmitting antenna. The impulse response hij(t) between transmitter i and receiver j can be derived by the input transmitting signal and the output signal y(t) obtained at the antennas using the classical formulation: D. PERFORMANCE METRICS In the simulations we generally consider a number of antennas evenly distributed across the simulated space. The outcome is the field distribution, the antenna gain, and the coupling between antennas. To see the electromagnetic cou- pling (communication) between any pair of antennas, it is enough to observe the magnitude of the scattering matrix component S21 (a complex number). Generally speaking, for a two-port network the scattering matrix is described as yj(t) = xi(t) ⋆hij(t), (4) (4) where ⋆denotes the convolution operator. Once the impulse response is calculated, it is straightforward to evaluate the Power-Delay Profile (PDP) as S = S11 S12 S21 S22  , (1) (1) Pij(τ) = |hij(t, τ)|2, (5) (5) therefore obtaining a matrix of PDP functions P. To charac- terize the channel in the time domain, we first obtain the mean delay τij defined as where S21, S12 determine the transmitted power from one port to the other, and S11, S22 represent the power reflected out of the network from each port, owing to impedance mis- match. Due to the reciprocity and symmetry of the problem, it holds that S12 = S21 and S22 = S11, respectively. In the structures under study, however, in addition to S21, we also observe the S11 component in order to understand how much of the power is reflected back to the antennas operating as the transmitter/receiver. In the optimum case the antennas need to be impedance matched, that is, we need to achieve a very low value for the magnitude of S11. This translates into negligible losses due to reflection, and high values for the magnitude of S21. However, in the case of noticeable return loss (reflection), we can resolve this issue by employing an external matching circuit. In fact, the important parameter is essentially the transmission coefficient. We note here that the formulation above can be generalized for any transmitter i and receiver j. where S21, S12 determine the transmitted power from one port to the other, and S11, S22 represent the power reflected out of the network from each port, owing to impedance mis- match. Due to the reciprocity and symmetry of the problem, it holds that S12 = S21 and S22 = S11, respectively. C. OPERATIONAL CONDITIONS To ensure that the electromagnetic response of the metasur- face and the wireless communication operation are decoupled for all communication paths, we choose the communication frequency to be greater than 25 GHz. This decoupling is especially important in the case where the metasurface layer hosts both the electromagnetic waves for the metasurface operation as well as the communication signals which is seen in Fig. 2(f) and analysed in Section V. Therefore, in over- all, we investigate the channel communication in the range f = 25 to 200 GHz, although we could investigate lower frequencies for the chip and dedicated channels. The distance between two neighbouring nodes equals D = 12 mm which, in the frequency regime under consideration, is in the order of 5λ0 to 40λ0, respectively. This means that the communi- cation takes place in the near and intermediate field regime. Thus, we cannot resort to simplified farfield calculations and we use full wave electromagnetic analysis for the numerical investigation. For higher frequencies, i.e., for frequencies f > 1 THz (D > 200λ) the full wave analysis becomes cumbersome and we would need to turn to simplified schemes such as ray tracing [48]. It is stressed that even though we perform the analysis for the reference case dimensions, a direct scaling of the structure along with the wavelengths • Communication in the chip layer. The first chan- nel under consideration is the back side of the meta- surface, where the chip and additional circuitry lie, together with any system packaging; it should be noted no structural change to the HSF landscape is assumed. Monopoles can be implemented by means of Through- Silicon Via (TSVs) within the chips or regular vias placed on the chip sides. Information propagates omni- directionally through the system package and part of it penetrates into the chips. Wireless communication in the chip layer is a natural choice since it is the section where all the electronics of the software defined metasur- face are placed. The evaluation of this communication channel is presented in Section III. • Communication in a dedicated layer The second chan- nel under investigation is a dedicated communication layer formed by adding two extra metallic plates below the chip. III. COMMUNICATION IN THE CHIP LAYER In this scenario, antennas are integrated within the chip or at its close vicinities and propagation occurs in two regions: (i) the intra-chip region, in which the waves radiated by the monopole inside the silicon substrate travel through several layers of the chip (mainly the silicon layer); and (ii) the inter- chip region, in which the waves that have left the chip travel through the inter-chip space until they reach the boundaries of another chip. L = 10 n · log10(d/d0) + L0, (3) (3) where L0 is the path loss at the reference distance d0 and n is the path loss exponent [46]. The path loss exponent is around 2 in free space, below 2 in guided or enclosed structures, and above 2 in lossy environments. Since losses in the channel are crucial to determine the power consumption at the transceiver we will report improvements in terms of worst-case Lmax, average Lavg, and path loss exponent n. In the cases where The main advantage of this configuration is that the com- munication channel does not require significant modifica- tions with respect to the original structure. This leads to a very cost-effective implementation that leaves the antenna and 122935 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the L A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfac FIGURE 4. Frequency domain analysis of the chip-layer communication scenario for three different frequencies: 60, 90, and 120 GHz. (a) Total attenuation as a function of distance and (b) directivity patterns. transceiver circuits as the only elements that may incur some area overhead. In fact, antenna and transceiver integration are performed for each chip individually, decoupling this process from the complete system integration and thereby reducing its complexity. Depending on the actual implementation of the system package and on the mounting of the HSF within the propaga- tion environment (e.g., over a wall), this scenario could lead to a totally enclosed volume. This would exclude the possibility of any coupling between ‘external’ electromagnetic radia- tion (e.g., metasurface operation microwaves or other stray incoming signals) and the intercell communication. Still, losses may still arise due to reflections, dissipation in the chip materials, or spreading in undesired areas within the package. A. SIMULATION RESULTS We first simulate a scenario with 5 × 5 chips embedded within the HSF. We model the antenna as a Through-Silicon Via (TSV) at the center of each chip and adjust the length to build antennas with fundamental resonance at f1 = 60 GHz, f2 = 90 GHz, and f3 = 120 GHz. To this end, the lower layers of the chip (the redistribution layer and the array of solder bumps) act as an effective ground plane. Therefore, the TSV can be modeled as a quarter-wave monopole. Since neighboring chips are at a distance of 12 mm, the relative distance among adjacent antennas in terms of the free-space wavelength ranges from 2.4λ0 (60 GHz) to 4.8λ0 (120 GHz). To perform the analysis, we excite the antenna in the bottom-left corner of the system. FIGURE 4. Frequency domain analysis of the chip-layer communication scenario for three different frequencies: 60, 90, and 120 GHz. (a) Total attenuation as a function of distance and (b) directivity patterns. FIGURE 5. Time domain analysis of the chip-layer communication scenario for three different frequencies: 60, 90, and 120 GHz. (a) Mean delay and (b) delay spread. Figure 4(a) shows the path loss as a function of the dis- tance. The first aspect worth noting is the rather large atten- uation in the range of 40–70 dB, which is in part due to the low gain of the antennas placed within the lossy silicon. It is also observed that the losses increase with the distance, as expected. Since the inter-chip medium is air, we can attribute these increasing losses to the reflections caused at the silicon-air interfaces and the spreading of energy in the half-space below the chips. Another remarkable result is the significantly larger attenuation observed at f2 = 90 GHz and f3 = 120 GHz. This is caused by both the larger spreading losses at such frequencies and the low directivity of the antennas at certain directions. The second effect is justified in Fig. 4(b): the use of a monopole within a squared cavity of fixed size leads to a pattern that may be omnidirectional or favor certain directions depending on the communication frequency. We can thus conclude that on-chip antenna design has a great impact on the path loss and should be carefully approached when implementing inter-cell communication within the chip layer. FIGURE 5. III. COMMUNICATION IN THE CHIP LAYER In this work, however, we will not make specific assumptions on system packaging or the mounting of the HSF, leaving the space among and below the chips empty (i.e. filled with air). B. ENHANCING INTER-CHIP PROPAGATION VIA SURFACE WAVES The results shown above confirm that losses, and non- multipath effects, would be the main impairment of commu- nication in the chip layer. One of the reasons is that the system is not completely enclosed and waves may spread out away from the chips. Within this context, it would be interesting to create a wireless channel that propagates around the chips following a path along the surface of the MS back plane. Sur- face waves are bound states that propagate along the interface of two semi-infinite domains and exhibit many interesting features [49]–[52]. A dielectric material close to a metallic plane can support a TM surface wave that travels along the metal-dielectric interface. For this to work, the dielectric layer needs to have a sufficiently high reactance [53]. The reactance Xs is calculated as Xs = 2πf µ0 ϵr −1 ϵr  t + 1 21  (10) FIGURE 6. Effects of adding a dielectric layer to enhance propagation for the case f = 120 GHz. (a) Electric field distribution at the surroundings of the radiating chip without and with dielectric layer (top and bottom, respectively). (b) Path loss comparison. (c) Delay spread comparison. (10) where εr is the permittivity of the material, t is the layer thick- ness, and 1 is the skin depth of the conductor at the frequency of operation, which is given by 1 = (πf µ0σ)−1/2. The term σ refers to the conductivity of the metallic plane. To play this role, we propose to add a layer of Aluminium Nitride (AIN, ϵr = 8.6, lossless) as a common interface between all the chips and the back plane. This material is typically used as a heat spreader in chip packages [54] and would be compatible with the MS fabrication processes. We thus propose to fill the space between the MS back-plane and the chips (0.15 mm in this paper) with the AIN material. the results. The mean delay, not shown for brevity, increases significantly for the dielectric case as now signals propagate through a material with higher permittivity. As for the delay spread, Fig. 6(c) shows how the value for the dielectric- enhanced case rises by almost an order of magnitude with respect to the baseline. The worst-case delay spread reaches a value of 137.74 ps, leading to a coherence bandwidth of 7.24 GHz. At the other frequencies, we observed similar behaviors with different relative values. A. SIMULATION RESULTS Time domain analysis of the chip-layer communication scenario for three different frequencies: 60, 90, and 120 GHz. (a) Mean delay and (b) delay spread. follows a linear dependence with the distance, which suggests that a significant part of the energy is transported by the line-of-sight ray. The communication frequency is of minor relevance here. As for the delay spread, we cannot observe clear scaling trends with frequency or distance. In general, delay spread would increase with distance because the main ray has lower energy and reflections become significant. This behavior can be somehow inferred for f = 60 GHz. At higher frequencies, however, the behavior tends to differ In the time domain, we obtain the impulse response and calculate the mean delay and delay spread in the system. Figure 5 plots both metrics in each chip in a 5 × 5 array as a function of the distance. We first note that the mean delay 122936 122936 VOLUME 7, 2019 VOLUME 7, 2019 tercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces tion of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces ou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Met FIGURE 6. Effects of adding a dielectric layer to enhance propagation for the case f = 120 GHz. (a) Electric field distribution at the surroundings of the radiating chip without and with dielectric layer (top and bottom, respectively). (b) Path loss comparison. (c) Delay spread comparison. due to: (i) the directional radiation patterns and (ii) the fact that chips located in the center of the system are completely surrounded by other chips that could increase the number of relevant reflections. This justifies the higher delay spread at distances around 30–50 mm. In any case, the worst case delay is lower than 19.26 ps, which yields a coherence bandwidth over 51.92 GHz. B. ENHANCING INTER-CHIP PROPAGATION VIA SURFACE WAVES For instance, the path loss improvement is more modest at f = 60 GHz, around 6 dB in average, since the coupling to the dielectric layer is diminished due to its lower reactance. Figure 6(a) shows the electric field distribution at the inter-chip space at 120 GHz. The top chart illustrates how, as expected, most of the power radiates away from the metal- lic back plane without the dielectric layer. The bottom chart, on the other hand, demonstrates that the dielectric layer is able to bind the surface waves and reinforce propagation along the dielectric in the path between chips. C. DISCUSSION The results obtained in this section lead to several conclu- sions. First, the use of the chip layer for inter-cell wireless communication decouples the problem of antenna integration from the system perspective, but leaves it to the chip designers instead. The choice of the communication frequency needs to take into account the antenna placement and dimensions of the chip: the silicon die may act as a resonant cavity and modify the radiation pattern of the antenna, impacting on the expected path loss. Furthermore, the thickness of the silicon layer imposes a lower bound on the frequency of the communication as it determines the maximum practical length of the resonant monopole. Therefore, system architects may need to balance out performance gains arising for the To evaluate the improvement in terms of path loss, we obtained the channel attenuation with and without the dielectric layer for f = 120 GHz. As it is shown in Fig. 6(b), the path loss is reduced significantly. In average, the improve- ment is of 12.24 dB with maximum values around 30 dB, demonstrating that the dielectric layer can be an enabler of wireless communication in the chip layer. This reduction in losses, however, comes at the cost of a significant increase in the delay spread due to the leaky waveguide effect of the dielectric layer. To verify this, we repeated the time domain analysis with and without the dielectric layer and compared VOLUME 7, 2019 122937 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in t A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the L A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfac f < c0/(2d√εr). In our implementation we purposely design the dimension so that the frequency lies below the cut off and therefore single mode operation is forced. Since the EM energy is carried by the single TEM mode, the waveguide is naturally impedance matched with free-space; this allows the following approximation: We consider that the propa- gation in the 3D waveguide can be approximated by a 2D analogue where the monopoles are replaced by finite-size conducting scatterers, placed at the vertical positions of the antenna probes. IV. COMMUNICATION IN A DEDICATED PARALLEL PLATE WAVEGUIDE Another opportunity for the intercell communication chan- nel is the isolated layer depicted in Fig. 2(e). The channel is dedicated exclusively to transferring the signals between the communication nodes. It is implemented by introducing two additional metallic plates below the chip which form a parallel-plate metallic waveguide channel whose thickness, as explained later on, is specified by the desired frequency of operation. The space between the two metallic plates is empty or filled with a uniform dielectric material; here we assume that it is empty (air). In each chip we connect a probe antenna which extends in the parallel-plate waveguide space through a small perforation in the metallic plate below the chip, as shown in Fig. 2(e). The length of the wire antenna is assumed to be approximately equal to the height of the parallel-plate waveguide (actually a small gap to avoid short circuit is assumed) and it radiates omnidirectionally, i.e., a device that transmits or receives electromagnetic power isotropically in the horizontal xy-plane. The main advan- tage of the present configuration is that the communication channel is electromagnetically isolated from the rest of the system, that is, no electromagnetic coupling between the pro- cessor and metasurface operation and the communication is expected. Moreover, the parallel plates create a closed space where no energy leakage is allowed (the holes are electromag- netically small) and thus communication security is ensured beyond any doubt. Additionally, there are no obstacles in the propagation space, apart from the probe antennas themselves. For these reasons, this option offers robustness and design flexibility, although it requires additional fabrication effort and increases the overall volume of the unit cell. FIGURE 7. (a) Schematic of the TEM parallel waveguide 2D approximation, probe no.1 radiates. (b) and (c) Electric field intensity distribution at f = 25, 60 and 180 GHz when the emitter is (b) no. 1, in the corner, and (c) no. 13, in the middle, respectively. (d) Power fraction received at the node M when node N radiates, SMN, over the frequency range f = 25 to 200 GHz. Six cases of MN node pairs are schematically depicted in the insets. Red/blue is for transmitter/receiver. GURE 7. (a) Schematic of the TEM parallel waveguide 2D FIGURE 7. (a) Schematic of the TEM parallel waveguide 2D approximation, probe no.1 radiates. IV. COMMUNICATION IN A DEDICATED PARALLEL PLATE WAVEGUIDE (b) and (c) Electric field intensity distribution at f = 25, 60 and 180 GHz when the emitter is (b) no. 1, in the corner, and (c) no. 13, in the middle, respectively. (d) Power fraction received at the node M when node N radiates, SMN, over the frequency range f = 25 to 200 GHz. Six cases of MN node pairs are schematically depicted in the insets. Red/blue is for transmitter/receiver. The dedicated channel is custom-built to optimize wireless propagation. The metallic plates perfectly reflect the elec- tromagnetic waves and they can guide a discrete spectrum of Transverse Electric (TE) and Transverse Magnetic (TM) modes depending on the distance of the plates d, the dielectric permittivity of the filling materials εr, and the frequency of operation f . A distinctive feature of the parallel-plate waveg- uide is that it sustains the propagation of TEM (Transverse Electromagnetic) waves in which both the electric and mag- netic fields are perpendicular to the propagation direction. The TEM mode can be excited from zero frequency (DC) and is the only propagation mode supported by the waveguide up to the cut-off frequency of the first higher-order mode: C. DISCUSSION Each scatterer radiates 2D cylindrical waves in the surrounding space and diffracts the energy coming from the environment. The field radiated from the emitter and the diffracted field from the scatterers interfere creating destructive or constructive patterns in the waveguide. use of lower frequencies (larger antenna apertures with lower spreading losses) against the potential cost implications of increasing the size of the chips. Finally, it seems clear that the traditional tradeoff between path loss and delay spread is also apparent in this scenario: the addition of a dielectric layer improves the former but worsens the latter. It is in fact provable that the thickness of the dielectric can be used to tailor this tradeoff as, theoretically, thinner layers would lead to better delay spread (and worse path loss) figures while thicker layers would yield opposite results. A. SIMULATION RESULTS The 2D approximation allows us to solve for large areas and frequency spans which provides us with a qualitative evaluation of the propagation properties in a multiscatter- ing environment. A priori, we assume that the antennas are impedance matched in all the spectrum of interest and that only the TEM mode is excited, both effectively controlled by the height of the structure. We investigate the system of 5 × 5 nodes depicted in Fig. 7(a). Each antenna (scatterer) is a finite size copper cylinder of radius R = 0.12 mm. The distance between two neighbouring antennas, that is the 122938 122938 VOLUME 7, 2019 of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasur FIGURE 8. (a) A 5x5 grid of antennas where the emitter antenna is no. 1, in the corner. (b,c,d) Power transmission profiles, for frequency f = 50 to 70 GHz versus distance from the emitter; the colorbar refers to the power of the total field, emitted plus scattered, calculated along paths (b) #1, (c) #3 and (d) #5, as numbered in panel (a). These paths are selected so that all possible direction are included. pitch of the antenna grid, is equal to D = 12 mm, which is required for the metasurface operation at 5 GHz. In the 2D approximation we do not take into account the impedance characteristics of the antennas. The emitter is simulated as a field source that radiates omnidirectional electromagnetic waves. All the surrounding scatterers reflect the incoming wave. In this way we estimate the electric field intensity profile of the propagating waves in the presence of the reflect- ing obstacles. Figures 7(b,c) present the profile of the total radiated intensity at frequency f = 25, 60 and 180 GHz when the emitter is no. 1 and no. 13, respectively. The electromag- netic waves interfere either destructively or constructively producing patterns of high or low intensity corresponding to the bright and dark spots. Moreover, due to the symmetry of the configuration, the system presents a four fold symmetry, as evidently shown in the figure. This means that many com- munication pairs and communication paths have by definition identical properties. A. SIMULATION RESULTS As the communication quality depends both on the position of the antenna probes and the frequency, it is useful to evalu- ate the connection between separate nodes. This is achieved by estimating, in the position of the receiver, the power that can be captured by the multipath propagation coming from all directions. The total power accumulated in the position of the receiver M when N emits, PMN, is normalized by the total radiated power from the emitter P0. The system is reciprocal, that is, SMN = SNM. Figure 7(d) presents the power fraction received in the position M transmitted from emitter N over the frequency range of f = 25 to 200 GHz for node pairs schematically depicted in the insets. As observed in all cases, the received power remains on average the same for each pair in the entire frequency span. However, for nearly all cases, there are some frequency points where the received power drops. For example, for the case of the pair no. 7-no. 17 (panel vi) there appear three dips in the received power at around f = 45, 80 and 115 GHz. These points correspond to destructive wave interference. Moreover, we can observe the general tendency of the decreased received power with respect to the node-pair distance, i.e., for the pair no. 1-no. 21 (panel i) the average received power is −15 dB whereas for the pair no.1-no.6 (panel ii), the received power is on average −8 dB. FIGURE 8. (a) A 5x5 grid of antennas where the emitter antenna is no. 1, in the corner. (b,c,d) Power transmission profiles, for frequency f = 50 to 70 GHz versus distance from the emitter; the colorbar refers to the power of the total field, emitted plus scattered, calculated along paths (b) #1, (c) #3 and (d) #5, as numbered in panel (a). These paths are selected so that all possible direction are included. the transmission drops below −35 dB which means that these spots are unreachable for the transmitter. In the power profiles this is indicated by the blank (white) areas. Let us take for example the distribution in path #1 of Fig. 8(b); at the positions of the scatterers, |r| = 12, 24 and 36 mm, the field is zero given the presence of the perfect conductors. At frequency f = 65 GHz there is a zero-field area between element no. 3 and no. A. SIMULATION RESULTS The length of the antenna defines the frequency of operation, which is verified in the 3D simulation by the fact that at the resonance frequency where the antenna is matched, little or no radiation returns to the feeding port (S11 < −20 dB). The nominal resonant frequency of the antenna is that of the quarter-wave monopole antennas f = c0/(4L√εr) and for relative small dgap and L ≈d it is by definition smaller then the cut off fre- quency of the waveguide; single mode operation is ensured. In what follows we examine three frequency regimes, f = 60, 90 and 120 GHz; the monopole lengths where the probe is matched in our 3D implementation are L = 0.9, 0.6 and 0.45 mm, receptively. The calculated antenna gain in the azimuth plane is almost unitary (0 dB) and isotropic. L = d −2 dgap, where dgap is the gap between the antenna and the upper and lower plate and it is assumed much smaller than L. The monopole is excited by a discrete wire port of impedance equal to Z0 = 50 that feeds current to the rod and stimulates the radiation or receives radiation from the environment. The port is positioned between the ground (bottom plate) and the cylindrical rod. The length of the antenna defines the frequency of operation, which is verified in the 3D simulation by the fact that at the resonance frequency where the antenna is matched, little or no radiation returns to the feeding port (S11 < −20 dB). The nominal resonant frequency of the antenna is that of the quarter-wave monopole antennas f = c0/(4L√εr) and for relative small dgap and L ≈d it is by definition smaller then the cut off fre- quency of the waveguide; single mode operation is ensured. In what follows we examine three frequency regimes, f = 60, 90 and 120 GHz; the monopole lengths where the probe is matched in our 3D implementation are L = 0.9, 0.6 and 0.45 mm, receptively. The calculated antenna gain in the azimuth plane is almost unitary (0 dB) and isotropic. As a general trend we notice the mean delay increases lin- early with distance which is a feature of the line-of-sight ray transportation which is also observed in the chip communica- tion channel in Fig. 2(d). This is clearly observed assuming excitation from probe no. 1, shown in Fig. A. SIMULATION RESULTS 4, around |r| = 27 mm. This kind of intensity mapping can be calculated for every emitter, path and frequency. q y Using this 2D qualitative analysis as a guideline, we can select the operation frequency, optimum paths, probe posi- tions, etc., for the actual 3D implementation of the wireless communication channel in the software-defined HSF. The 3D implementation provides the quantitative evaluation of the communication through the time domain analysis and the assessment of the mean delay and the delay spread. The analysis is acquired from CST time-domain simulations. We choose to simulate the case of 5 × 5 probe antennas placed in a rectangular grid of pitch D = 12 mm. As men- tioned, the grid size is selected with respect to the metasurface operating at 5 GHz. Each 3D antenna probe is a single- wire antenna (monopole) placed vertically between the two copper plates. It is implemented by a cylindrical copper rod whose length L varies according to desired frequency of wire- less communication operation. The length of the antenna is directly connected to the distance of the plates, d, in particular Figure 8 presents the power transmission coefficient (equivalent to path loss, in this 2D approximation) with respect to the frequency and the distance of the emitter in the frequency range f = 50 to 70 GHz. In particular, in panels (b), (c) and (d) we assume that the element no. 1 radiates and then we calculate the transmission along paths #1, #3 and #5 that connect the position of element no. 1 with elements no. 5, no. 15 and no. 25, respectively, as shown in Fig. 8(a). For this calculation we assume that all the elements scatter the incoming field and we do not define a specific receiver. The general trend is that as the distance from the emitter increases the transmission drops. This stands in accordance with the 2π angular spread of the emitted energy and the power decay law, i.e. 1/r. Additionally, we observe that in some positions 122939 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the L Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfac FIGURE 9. Time domain analysis for the dedicated channel for three different frequencies, f = 60 GHz (green), 90 GHz (red) and 120 GHz (blue). A. SIMULATION RESULTS Top panels present the mean delay assuming that excitation comes from (a) probe no. 1, (b) probe no. 7 and (c) probe no. 13. Insets show the actual 3D simulated structure, the enumeration of the probes in the 5 × 5 arrangement and the selected radiating probe. Bottom panels show the calculated delay spread for excitation coming from (d) probe no. 1, (e) probe no. 7 and (f) probe no. 13. FIGURE 9. Time domain analysis for the dedicated channel for three different frequencies, f = 60 GHz (green), 90 GHz (red) and 120 GHz (blue). Top panels present the mean delay assuming that excitation comes from (a) probe no. 1, (b) probe no. 7 and (c) probe no. 13. Insets show the actual 3D simulated structure, the enumeration of the probes in the 5 × 5 arrangement and the selected radiating probe. Bottom panels show the calculated delay spread for excitation coming from (d) probe no. 1, (e) probe no. 7 and (f) probe no. 13. The evaluation of the isolated communication channel in the time domain is presented in Fig. 9. In particular we calculate the mean delay (top panels of Fig. 9) and the delay spread (bottom panels of Fig. 9) for three frequencies, f = 60, 90 and 120 GHz and assuming three different non symmetric alternatives as the radiating probe. In particular we examine the case when probe no. 1 [Fig. 9(a) and (d)], no. 7 [Fig. 9(b) and (e)] and no. 13 [Fig. 9(c) and (f)] feeds the communication. The selection of the three different positions at No. 1, No. 7, and No. 13 in the diagonal of the grid covers a large part of uniquely defined probe and communication paths and pairs in the four fold symmetric 5 × 5 system. L = d −2 dgap, where dgap is the gap between the antenna and the upper and lower plate and it is assumed much smaller than L. The monopole is excited by a discrete wire port of impedance equal to Z0 = 50 that feeds current to the rod and stimulates the radiation or receives radiation from the environment. The port is positioned between the ground (bottom plate) and the cylindrical rod. A. SIMULATION RESULTS 9(a), which is also the case that covers the maximum probe-to-probe distance (communication between probes no. 1 and no. 25). The linear trend of the mean delay is less obvious in Fig. 9(b) and the 122940 122940 VOLUME 7, 2019 VOLUME 7, 2019 of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Met case of radiating probe no. 7. In this case we also observe a smaller maximum probe-to-probe distance (communication between probes no. 7 and no. 25) and fewer points on the diagram as a result of multiple symmetric communication pairs (for example no.7-to-no.2 and no.7-to-no.6). The trend is even less clear in Fig. 9(c) which is also the point of the highest symmetry in the system. There, the maximum dis- tance becomes very small, actually minimum, (no. 13-no. 25) and we can only observe a general increase of the mean delay with distance. Moreover, in this case the symmetric communication pairs are the most numerous and, therefore, the calculated unique mean delay points are very few. Finally, we observe that in all cases the level of the mean delay is similar for the same distance values. the presence of multiple through-vias (four in each unit cell, connecting the chip with the four patches, see Fig. 2) imposes unavoidable obstruction and scattering. Moreover, it is noted that this layer has been specifically designed for the operation of the metasurface itself, for example, implementing a tunable absorber for 5 GHz impinging radiation. Consequently, all its geometric and EM parameters, e.g., the dielectric permittivity and thickness, the unit cell and patch sizes etc., have been accurately selected and cannot be modified for the intercell communication. For all the reasons outlined, the main parameter available for optimizing the performance in the metasurface channel is the length of the antenna, assumed, in its simplest form, as a monopole connected through the metallic ground plane to the controlling chip. In order to simplify the design and minimize crosstalk and leakage above the copper patches, one monopole antenna is placed in each unit cell, perpendicular to the ground plane and aligned below the center of one of the four patches. A. SIMULATION RESULTS This monopole antenna is a copper cylinder of 0.12 mm radius, same as the previous cases, which could be fabricated with a blind via through the ground plane and electroplated. Assuming a small gap of 0.1 mm between the ground plane and the feeding point of the monopole, its length cannot surpass 1.4 mm (when the blind via is drilled), as the thickness of the substrate dielectric is h = 1.575 mm. Moreover, it must be noted that the thickness of the monopole is expected to affect the performance of the antenna, and it cannot be arbitrarily thin due to fabrication limitations. The delay spread, on the other hand, exhibits a dis- tinct dependence on the excitation probe. As evident from Fig. 9(d), (e) and (f) the delay spread is significantly enhanced when the probe is away from the open termination of the structure. In fact, it is maximized in the case of no. 13 radi- ating probe which is located at the high symmetry point of the grid, surrounded by multiple densely-packed electromag- netic obstacles which lead to a high number of reflections and increased multipath scattering; this translates into the increase of the delay spread. B. DISCUSSION Communication in the dedicated parallel plate is the channel closest to a wired transmission. The features that attribute the wireless character is the obstacles and some multipath scattering. This channel is not the most efficient one in terms of volume and resources economy since it requires the addi- tion of an extra metallic plate. However, it is a stable solu- tion that allows the communication between distant nodes in the metasurface tile and can be readily designed for any selected frequency regime. Additionally, the communication channel is practically electromagnetically isolated from the outer world which means that it is the best solution in terms of security. In fact, a signal coming from the external envi- ronment could only couple to the layer through the probe vias which are extremely small thus practically eliminating coupling. Another parameter that could be, a priori, freely selected for optimized performance is the intercell communication frequency. In this work, we target 60 GHz, a band of increas- ing interest for mm-wave communications. However, we note that the natural resonance of the monopole antenna will be closer to 30 GHz; this is due to the presence of the ground plane and the patches plane, which form a quasi quarter- wavelength antenna environment so that fres = c0/(4h√εr), where h ≈1.5 mm is the thickness of the dielectric medium (or the maximum length of the antenna) and εr = 2.2 its permittivity. Note that increasing the intercell communication frequency above 60 GHz (λ0 = 5 mm) is not a viable option in this metasurface design for two reasons: firstly, because the gap between patches, set at 1.8 mm (approximately one fourth the 60 GHz free-space wavelength), will become electrically large, thus increasing the leakage losses; secondly, because the parallel-plate waveguide formed between the ground and the patches plane will become multimode, as detailed in Section IV, which constitutes a suboptimal propagation regime. A. SIMULATION RESULTS With all these technical aspects and design choices in mind, and without directly relying on mm-wave matching circuits for the antenna, we assume an S-parameter 50 -reference port between the ground plane and the monopole feed, shown with red in the base of the antenna in the inset of Fig. 10. Full-wave numerical simulations are conducted using CST Microwave Studio throughout this Section. We numerically calculate the monopole length where the antenna resonates or, similarly, where the amplitude |S11| parameter is mini- mized. A local optimal value for the monopole length was found near Lant = 1.2 mm, which leads to the |S11| spectrum depicted in Fig. 10, opening two well matched bands near 35 and 60 GHz. It must be noted that a proper resonance of the monopole, where Im{Zant} = 0 (zero reactance in the antenna input impedance), cannot be attained at 60 GHz for the given metasurface environment, so the |S11| ≈−15 dB value is deemed sufficient. The calculated antenna gain in the azimuth plane is approximately unitary (0 dB); a few beamwidth cancelling directions can be found towards the center of the unit cell where the four through vias act as reflectors, with extra gain in the opposite direction. The antenna is polarized primarily perpendicular to the ground plane. Concluding the design of the antenna, we verified that the coupling between the antenna feed port and both the chip ports and the Floquet port at the receiving side of the metasurface is negligible at its operation frequency (5 GHz); this eliminates cross-talk between the intercell communication channel and the main metasurface functionality. FIGURE 12. Field plots of the wave radiated from the 1.2 mm tall monopole antenna inside the metasurface layer at (a) 30, (b) 60 and (c) 90 GHz. The plots correspond to the amplitude of the vertically polarized E-field component, in the vertical cross-section plane passing through the axes of two neighbouring monopole antennas. and with a bandwidth decreasing as the unit-cell distance increases. A sharp drop in transmission takes place close to 80 GHz which is attributed to leakage between the copper patches, outside the metasurface dielectric. V. COMMUNICATION IN THE METASURFACE LAYER Amplitude of scattering parameter S11 for a monopole antenna of Lant = 1.2 mm, at its 50  feed port marked with red color in the inset, which depicts the cross-sectional side-view of a unit cell for the metasurface-layer intercell communication channel modeling. FIGURE 11. Transmission spectra in terms of S-parameter amplitude (in dB) for a 5 × 1 arrangement of unit cells where the transmitting antenna is in the first cell, as shown in the inset. FIGURE 10. Amplitude of scattering parameter S11 for a monopole antenna of Lant = 1.2 mm, at its 50  feed port marked with red color in the inset, which depicts the cross-sectional side-view of a unit cell for the metasurface-layer intercell communication channel modeling. FIGURE 11. Transmission spectra in terms of S-parameter amplitude (in dB) for a 5 × 1 arrangement of unit cells where the transmitting antenna is in the first cell, as shown in the inset. FIGURE 11. Transmission spectra in terms of S-parameter amplitude (in dB) for a 5 × 1 arrangement of unit cells where the transmitting antenna is in the first cell, as shown in the inset. V. COMMUNICATION IN THE METASURFACE LAYER It must be noted that a proper resonance of the monopole, where Im{Zant} = 0 (zero reactance in the antenna input impedance), cannot be attained at 60 GHz for the given metasurface environment, so the |S11| ≈−15 dB value is deemed sufficient. The calculated antenna gain in the azimuth plane is approximately unitary (0 dB); a few beamwidth cancelling directions can be found towards the center of the unit cell where the four through vias act as reflectors, with extra gain in the opposite direction. The antenna is polarized primarily perpendicular to the ground plane. Concluding the design of the antenna, we verified that the coupling between the antenna feed port and both the chip ports and the Floquet port at the receiving side of the metasurface is negligible at its operation frequency (5 GHz); this eliminates cross-talk between the intercell communication channel and the main metasurface functionality. Moving on to the frequency domain characterization of the intercell communication channel, we consider five unit cells FIGURE 11. Transmission spectra in terms of S-parameter amplitude (in dB) for a 5 × 1 arrangement of unit cells where the transmitting antenna is in the first cell, as shown in the inset. FIGURE 12. Field plots of the wave radiated from the 1.2 mm tall monopole antenna inside the metasurface layer at (a) 30, (b) 60 and (c) 90 GHz. The plots correspond to the amplitude of the vertically polarized E-field component, in the vertical cross-section plane passing through the axes of two neighbouring monopole antennas. and with a bandwidth decreasing as the unit-cell distance increases. A sharp drop in transmission takes place close to 80 GHz which is attributed to leakage between the copper patches, outside the metasurface dielectric. In order to visualize the performance of the monopole FIGURE 11. Transmission spectra in terms of S-parameter amplitude (in dB) for a 5 × 1 arrangement of unit cells where the transmitting antenna is in the first cell, as shown in the inset. FIGURE 12. Field plots of the wave radiated from the 1.2 mm tall monopole antenna inside the metasurface layer at (a) 30, (b) 60 and (c) 90 GHz. The plots correspond to the amplitude of the vertically polarized E-field component, in the vertical cross-section plane passing through the axes of two neighbouring monopole antennas. and with a bandwidth decreasing as the unit cell distance FIGURE 10. V. COMMUNICATION IN THE METASURFACE LAYER The last investigated option for the intercell communication channel is inside the dielectric layer of the metasurface, between the metallic back plane and the copper patches, as depicted in Fig. 2(f). The obvious advantage in this approach is the utilization of the metasurface landscape which naturally forms a waveguide channel for communication so that no additional metal plane is required, nor any consid- erable modification of the HSF architecture or performance. However, this environment hosts a number of characteristics which degrade the channel performance: Firstly, the dielectric medium itself is lossy; secondly, there are gaps between the patches therefore leakage to open space above the metasur- face is allowed when the gaps are electrically large; finally, Finally, it is worth noting that the guided modes propagat- ing between two penetrable metasurfaces can been formally studied as in Ref. [55]. That formulation can be used in the present context, by assuming that the surface impedance of one of the two metasurfaces is zero, thus modeling the uniform ground plane, while the other metasurface is actu- ally the patches plane of the HSF, penetrable by mm-wave radiation. 122941 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the L lamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces FIGURE 10. Amplitude of scattering parameter S11 for a monopole antenna of Lant = 1.2 mm, at its 50  feed port marked with red color in the inset, which depicts the cross-sectional side-view of a unit cell for the metasurface-layer intercell communication channel modeling. A. SIMULATION RESULTS With all these technical aspects and design choices in mind, and without directly relying on mm-wave matching circuits for the antenna, we assume an S-parameter 50 -reference port between the ground plane and the monopole feed, shown with red in the base of the antenna in the inset of Fig. 10. Full-wave numerical simulations are conducted using CST Microwave Studio throughout this Section. We numerically calculate the monopole length where the antenna resonates or, similarly, where the amplitude |S11| parameter is mini- mized. A local optimal value for the monopole length was found near Lant = 1.2 mm, which leads to the |S11| spectrum depicted in Fig. 10, opening two well matched bands near 35 and 60 GHz. VI. CONCLUSION This paper has examined the problem of intercell wireless communication in the complex landscape of intelligent meta- surface fabrics, where such communication is necessary to implement unique features such as autonomy, interconnectiv- ity, and distributed sensing and intelligence. The introduction of such integrated means of wireless communications is sup- ported by its natural broadcast capabilities, the ease of assem- bly, and the improved off-chip connectivity. We explored three possible propagation paths at mm-wave frequencies, a band that enables the integration of antennas within HSFs and theoretically avoids interference with the interaction of HSFs with external microwave sources. FIGURE 13. (a) Mean delay and (b) RMS delay spread extracted with time-domain simulation of a 5 × 5 arrangement of unit cells where the 60 GHz transmitting antenna is in one of the corner cells. Having quantified the path loss in the metasurface intercell communication channel, we conclude our analysis with the time domain metrics that offer an estimate of the coherence bandwidth. We assume a 5 × 5 arrangement of unit cells where the transmitting antenna is in one of the corner cells, similar to Fig. 7(a). The mean delay, τij and RMS delay spread, τ (i,j) rms , are acquired from CST time-domain simula- tions post-processed with Eqs. (6) and (7). The transmit- ted pulse bandwidth used for the calculations occupies a spectrum of ±20% around the central frequency, 60 GHz. The results are depicted in Fig. 13, where a 6 ps/mm and a 1.5 ps/mm trendline emerges for the mean delay and delay spread, respectively. The large deviation in the values of mean delay is in contrast with what was calculated in the previous communication channels, which is attributed to the structured geometry of the metasurface and multi-scatterer environment (four through-vias per unit cell). For the same reason, the maximum delay spread is rather large, exceeding 150 ps, which leads to a coherence bandwidth of only 7 GHz (10% of the operating frequency). Note that we limited the time-domain study to the 60 GHz band, as higher frequencies suffer from poor antenna matching and high path loss, as we evidenced in the frequency domain analysis. Our explorations have clarified the pros and cons of each alternative. The chip layer appears to be a natural choice since the antennas can be integrated on the controller chips and do not interfere with the metasurface operation. A. SIMULATION RESULTS Moving on to the frequency domain characterization of the intercell communication channel, we consider five unit cells in a row with a transmitting antenna placed in the first cell, and numerically calculate the transmission to the other cells, in terms of the scattering parameter amplitude |Si1|, where i = 2, 3, 4, 5. The resulting spectra are depicted in Fig. 11. The transmission to the adjacent neighbouring cell in 60 GHz is approximately |S21| ≈−20 dB and another 6 dB of path loss are accumulated with each added cell of lateral width (pitch) equal to 12 mm. The highest transmission band is in all cases near 60 GHz, well suited with the band where the antenna is well matched (exhibits low values of |S11|), In order to visualize the performance of the monopole antenna, we depict the radiated wave from one unit cell to its neighbouring one, at three different frequencies: 30, 60 and 90 GHz, in Fig. 12. The plots correspond to the amplitude of the E-field component polarized perpendicularly to the ground plane and the patches, in a vertical cross-section including two neighbouring unit cell antennas. Evidently, the optimal performance is at the design frequency of 60 GHz, where the field amplitude reaching the neighbouring cell is maximized. In both 30 and 90 GHz, the antenna radiates strongly outside the metasurface layer, which leads to the 122942 122942 VOLUME 7, 2019 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Met B. DISCUSSION lower transmission values (|S21|) observed in Fig. 11. This behaviour can be qualitatively explained by considering the waveguide formed between the ground plane and the patches plane [55], whose cut-off frequency is approximately 67 GHz as described in Section IV; optimal confinement in the meta- surface layer, and thus highest transmission, is attained for the frequency right below the cut-off, whereas confinement is reduced far below the cut-off (noting, also, that the patches plane is penetrable) and multimode regime is entered above the cut-off. Summarizing the analysis and design of the intercell com- munication in the metasurface layer, we have chosen to work with prescribed geometric and EM properties for the envi- ronment and design a simple low-cost monopole 60 GHz antenna in a perturbation approach, i.e., with the aim of minimizing the effect on the main metasurface operation, which is at 5 GHz. We then proceeded to evaluate the metrics of the channel model, namely the path loss and delay spread. Allowing a small amount of perturbation to the metasurface parameters, e.g. the dielectric thickness or the patch width, and/or freely choosing the intercell communication frequency band as fits best, and/or invoking microwave circuits to match |S11| at will, can lead to improved designs [31]. The main drawback in these approaches is the custom/non-standard ASICs (chips) required to compensate and even-out perfor- mance perturbations. Finally, arguably the optimal approach would be to ‘co-design’ the metasurface layer with both the main functionality and the intercell communication aspects factored in, right from the start; this could potentially lead an overall optimized performance at the cost of more resources and design iterations. FIGURE 13. (a) Mean delay and (b) RMS delay spread extracted with time-domain simulation of a 5 × 5 arrangement of unit cells where the 60 GHz transmitting antenna is in one of the corner cells. VI. CONCLUSION REFERENCES [20] A. M. Shaltout, V. M. Shalaev, and M. L. Brongersma, ‘‘Spatiotemporal light control with active metasurfaces,’’ Science, vol. 364, no. 6441, 2019, Art. no. eaat3100. [1] C. M. Soukoulis and M. Wegener, ‘‘Past achievements and future chal- lenges in the development of three-dimensional photonic metamaterials,’’ Nature Photon., vol. 5, pp. 523–530, Jul. 2011. [21] T. J. Cui, M. Q. Qi, X. Wan, J. Zhao, and Q. 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CONCLUSION Our analysis, however, yields a very large path loss of 40–50 dB at 60 GHz and up to 50–70 dB at 120 GHz, mainly caused by the lossy silicon at the chips. The introduction of a dielectric layer, originally employed for thermal and mechanical support, can decrease the path loss by around 10 dB. The second analyzed alternative consists of the inclusion of a dedicated layer for wireless inter-cell communication. This option yields a much lower path loss of 5–25 dB and effective protection to inter- ferences at all bands, but at the expense of (i) a relatively large delay spread in the order of 100 ps for a coherence bandwidth of 10 GHz and (ii) a higher volume and manufacturing cost. Finally, we showed that a promising path loss of 20–40 dB is achievable at 60 GHz without altering the HSF architecture by using the metasurface layer opportunistically as the prop- agation path. 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Abadal, ‘‘Channel characterization for chip-scale wireless communica- tions within computing packages,’’ in Proc. NOCS, 2018, Art. no. 20. 122945 VOLUME 7, 2019 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in t A. C. REFERENCES Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Met JULIUS GEORGIOU (M’98–SM’08) received the M.Eng. degree in electrical and electronic engineering and the Ph.D. degree from Imperial College London, in 1998 and 2003, respectively. For two years, he was the Head of micropower design with Toumaz Technology, a technology start-up company. In 2004, he joined the Johns Hopkins University, as a Postdoctoral Fellow, before becoming a Faculty Member of the Uni- versity of Cyprus, from 2005 onwards. He is an Associate Professor with the University of Cyprus. He is a member of the IEEE Circuits and Systems Society. He was a recipient of the Best Paper Award from the IEEE BioDevices 2008 Conference and the IEEE ISCAS 2011 International Symposium. In 2016, he received the 2015 ONE Award from the President of Cyprus for his research accomplishments. He was the Chair of the IEEE Biomedical and Life Science Circuits and Systems (BioCAS) Technical Committee and a member of the IEEE Cir- cuits and Systems Society Analog Signal Processing Technical Committee. He served as the General Chair of the 2010 IEEE Biomedical Circuits and Systems Conference. He was the Action Chair of the EU COST Action ICT-1401 on Memristors-Devices, Models, Circuits, Systems and Applications-MemoCIS. He was an IEEE Circuits and Systems Society Distinguished Lecturer, from 2016 to 2017. He is also is an Associate Editor of the IEEE TRANSACTIONS ON BIOMEDICAL CIRCUITS AND SYSTEMS and an Frontiers in Neuromorphic Engineering journal. IAN F. AKYILDIZ (F’97) is currently the Ken Byers Chair Professor in telecommunications with the School of Electrical and Computer Engi- neering, the Director of the Broadband Wire- less Networking Laboratory, and the Chair of the Telecommunication Group, Georgia Institute of Technology, Atlanta, USA. Since 2011, he has been serving as a Consulting Chair Professor with the Department of Information Technology, King Abdulaziz University, Jeddah, Saudi Arabia, and the Computer Science Department, University of Cyprus, since January 2017. He has been a Megagrant Research Leader of the Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, Russia, since May 2018. His current research interests include 5G wireless systems, nanonetworks, terahertz band communications, and wireless sensor networks in challenged environments. He is an ACM Fellow, in 1997. He received numerous awards from the IEEE and the ACM, and many other organizations. REFERENCES His h-index is 116 and the total number of citations is above 107 K as per Google scholar as of July 2019. SERGEI A. TRETYAKOV (M’92–SM’98–F’08) received the Dipl.Ing. (physicist), Ph.D., and D.Sc. degrees in radiophysics from Saint Petersburg State Technical University, Saint Petersburg, Russia, in 1980, 1987, and 1995, respectively. From 1980 to 2000, he was with the Radiophysics Department, Saint Petersburg State Technical Uni- versity. He is currently a Professor of radio science with the Department of Electronics and Nanoengi- neering, Aalto University, Espoo, Finland. He has authored or coauthored five research monographs and over 270 journal articles. His current research interests include electromagnetic field theory, complex media electromagnetics, metamaterials, and microwave engineer- ing. He has served as the Chairman for the Saint Petersburg IEEE Electron Devices/Microwave Theory and the Techniques/Antennas and Propaga- tion Chapter, from 1995 to 1998, the General Chair for the International Congress Series on Advanced Electromagnetic Materials in Microwaves and Optics (Metamaterials), from 2007 to 2013, and the President of the Virtual Institute for Artificial Electromagnetic Materials and Metamaterials (Metamorphose VI), from 2007 to 2013. ALBERT CABELLOS-APARICIO received the Ph.D. degree in computer science engineering from the Universitat Politècnica de Catalunya, in 2008, where he is currently an Assistant Profes- sor. He has coauthored over 80 research articles. He is the Co-Founder and the Scientific Director of the NaNoNetworking Center in Catalunya. He has been a Visiting Researcher with Cisco Systems and Agilent Technologies, and a Visiting Profes- sor with the KTH, Sweden, and the MIT, USA. His research interests include nanocommunications and software-defined networking. EDUARD ALARCÓN received the Ph.D. degree in electrical engineering from the Universitat Politèc- nica de Catalunya, in 2000, where he is currently an Associate Professor. He has coauthored over 400 scientific publications and eight book chap- ters. He holds 12 patents. His research interests include nanocommunications and wireless energy transfer. He was elected as an IEEE CAS Society Distinguished Lecturer, a member of the IEEE CAS Board of Governors, from 2010 to 2013, an Associate Editor of the IEEE TCAS-I, TCAS-II, JOLPE, and the Editor-in-Chief of JETCAS. ELEFTHERIOS N. ECONOMOU received the Ph.D. degree from the Physics Department, Uni- versity of Chicago, USA, in 1969, the Ph.D. degree from the Polytechnic of Grenoble, in 1994, and the Ph.D. degree from the University of Ioannina, in 2004. REFERENCES degrees from the School of Electrical and Computer Engineering, Aristotle University of Thessaloniki (AUTH), Greece, in 1994 and 1999, respectively, where he is currently a Professor of computational elec- tromagnetics and electromagnetic compatibility. He is also with the Foundation for Research and Technology Hellas (FORTH)-IESL. He has authored/coauthored three books, nine book chap- ters, more than 135 peer-reviewed journal articles, and 230 conference papers. His primary research interests include computational electromagnet- ics, metamaterials, graphene, real-world EMC/EMI problems, microwaves and antennas, and nanotechnology devices. He is the ICS member and an ACES member. FU LIU received the B.Sc. degree in applied physics from the China University of Mining and Technology, Xuzhou, China, in 2008, the M.Sc. degree in theoretical physics from Beijing Normal University, Beijing, China, in 2011, and the Ph.D. degree in physics from the City Univer- sity of Hong Kong, Hong Kong, in 2015. From 2014 to 2016, he was a Research Fellow of the School of Physics and Astronomy, University of Birmingham, Birmingham, U.K. Since 2017, he has been a Postdoctoral Researcher with the Department of Electronics and Nanoengineering, School of Electrical Engineering, Aalto University, Espoo, Finland. DIONYSIOS MANESSIS received the M.Sc. and Ph.D. degrees in materials science and engineering from the Stevens Institute of Tech- nology, NJ, USA, and project leadership certifi- cate degrees from Cornell University, NY, USA. He was a Technologist with Universal Instruments Corporation, NY, USA, and since 2001, he has been a Senior Technology Scientist with Fraun- hofer IZM in Berlin. He has published extensively in international conferences and peer-reviewed journals in his research fields. His main research interests include fine pitch flip chip and wafer level csp bumping, solder balling, materials selection for advanced packaging technologies, embedding processes for heteroge- neous integration of components in PCBs and optical PCBs, and large scale prototype manufacturing. CHRISTOS LIASKOS received the Diploma degree in electrical engineering from the Aristotle University of Thessaloniki (AUTH), Greece, in 2004, and the M.Sc. degree in medical infor- matics from the Medical School, AUTH, in 2008, and the Ph.D. degree in computer networking from the Department of Informatics, AUTH, in 2014. He is currently a Postdoctoral Researcher with the Foundation for Research and Technology Hellas (FORTH), handling the scientific and tech- nical co-coordination task of project VISORSURF. His research interests include computer networks, traffic engineering, and novel control schemes for wireless communications. VOLUME 7, 2019 122946 122946 A. C. REFERENCES He was a Professor with the Univer- sity of Virginia, from 1969 to 1981, the Uni- versity of Athens, from 1978 to 1981, and the Physics Department, University of Crete, from 1981 to 2007. He was the Founder (together with colleagues) of the FORTH and its first President, from 1983 to 2004. He is a Research Fellow of IESL-FORTH and an Emeritus Professor with the Physics Department, University of Crete. He has a strong research output on the properties of disordered systems, high Tc superconductors, amorphous semiconductors, and wave propagation in random media. In recent years, he has been focused mostly on the study of elastic and electromagnetic wave propagation in complex media, mainly photonic crystals, phononic crystals, and metamaterials. He is among the initiators of the field of phononic crystals. He has 271 publications in refereed journals and another 109 book chapters. He has written thirteen books. He has given many invited talks in conferences and at universities/institutes. He has been cited more than 21 405 times according to Google Scholar (13 408 citations to his articles according to the Web of Science) and 2549 to his book on Green’s functions. ANDREAS PITSILLIDES (M’90–SM’05) is cur- rently a Professor with the Department of Com- puter Science, University of Cyprus, where he is also the Head of the Networks Research Labora- tory. He is a Visiting Professor with the Univer- sity of Witwatersrand (Wits). He was previously at the University of Johannesburg. He has pub- lished over 270 refereed articles in flagship jour- nals, such as IEEE, Elsevier, IFAC, and Springer, international conferences, and books. His research interests include communication networks, the Internet- and Web-of-Things, smart spaces, intelligent surfaces and programmable wireless environments, and nanonetworking. 122947 VOLUME 7, 2019 A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in t A. C. Tasolamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the L lamprou et al.: Exploration of Intercell Wireless Millimeter-Wave Communication in the Landscape of Intelligent Metasurfaces MARIA KAFESAKI COSTAS M. SOUKOULIS received the Ph.D. degree from the Physics Department, University of Chicago, in 1978. He is currently a Senior Scien- tist with Foundation for Research and Technology Hellas (FORTH) and a Distinguished Professor of physics with Iowa State University. He is a Fellow of the APS, OSA, and AAAS. He has more than 400 publications in refereed journals. He has given more than 173 invited talks. His work on photonic band gaps (PBG), random lasers, plas- monics, graphene, metasurfaces, and nonlinear systems is well known and well received. He has organized three NATO ASI on PBGs. He was the Director of PECS-VI that took place in Crete, in June 2005. He also holds five patents concerning the potential applications of the photonic band gaps and left-handed materials. He recently received the senior Humboldt research award. He has been cited 57 000 times in SCI journals. He is the winner of the 2014 Max Born Award from the Optical Society of America for his creative and outstanding theoretical and experimental research in the fields of photonic crystals and left-handed metamaterials. He was included in the 2014 and 2015 Highly-Cited Researchers by Thompson-Reuters. In 2013, he won the McGroddy Prize for New Materials for the discovery of metamaterials from the American Physical Society. He has lead Research and Development projects supported by the EU, NSF, DOE, DARPA, the Office of Naval Research, MUTI-AFOSR, NATO, EPRI, and Ames Lab. MARIA KAFESAKI received the Ph.D. degree from the Physics Department, University of Crete, Greece, in 1997. She is currently an Associate Professor with the Department of Materials Sci- ence and Technology, University of Crete, and an Associate Researcher with the Institute of Elec- tronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH). She was a Postdoctoral Researcher with the Consejo Superior de Investigaciones Cientificas, Madrid, Spain, and in the IESL of FORTH, from 1997 to 2001. She has around 130 publications in refereed journals and conference proceedings (with 9200 citations). She has participated in various European projects as well as in the organization of many international conferences and schools. Her current research interests include the areas of electromagnetic wave propa- gation in periodic and random media, with emphasis on photonic crystals and metamaterials, especially negative refractive index materials. She is a fellow of the Optical Society of America. 122948 VOLUME 7, 2019

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Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion

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RESEARCH ARTICLE Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion Zirong Gu1, Fumiyasu Imai1, In Jung Kim2, Hiroko Fujita3, Kei ichi Katayama1, Kensaku Mori4, Yoshihiro Yoshihara3, Yutaka Yoshida1* 1 Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America, 2 Department of Ophthalmology and Visual Science and Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 3 RIKEN Brain Science Institute, Saitama, Japan, 4 Department of Physiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan * [email protected] * [email protected] OPEN ACCESS Cell adhesion molecules belonging to the immunoglobulin superfamily (IgSF) control synap- tic specificity through hetero- or homophilic interactions in different regions of the nervous system. In the developing spinal cord, monosynaptic connections of exquisite specificity form between proprioceptive sensory neurons and motor neurons, however, it is not known whether IgSF molecules participate in regulating this process. To determine whether IgSF molecules influence the establishment of synaptic specificity in sensory-motor circuits, we examined the expression of 157 IgSF genes in the developing dorsal root ganglion (DRG) and spinal cord by in situ hybridization assays. We find that many IgSF genes are express- ed by sensory and motor neurons in the mouse developing DRG and spinal cord. For in- stance, Alcam, Mcam, and Ocam are expressed by a subset of motor neurons in the ventral spinal cord. Further analyses show that Ocam is expressed by obturator but not quadriceps motor neurons, suggesting that Ocam may regulate sensory-motor specificity in these sen- sory-motor reflex arcs. Electrophysiological analysis shows no obvious defects in synaptic specificity of monosynaptic sensory-motor connections involving obturator and quadriceps motor neurons in Ocam mutant mice. Since a subset of Ocam+ motor neurons also express Alcam, Alcam or other functionally redundant IgSF molecules may compensate for Ocam in controlling sensory-motor specificity. Taken together, these results reveal that IgSF mole- cules are broadly expressed by sensory and motor neurons during development, and that Ocam and other IgSF molecules may have redundant functions in controlling the specificity of sensory-motor circuits. Citation: Gu Z, Imai F, Kim IJ, Fujita H, Katayama Ki, Mori K, et al. (2015) Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion. PLoS ONE 10(3): e0121550. doi:10.1371/journal.pone.0121550 Academic Editor: Michael A. Fox, Virginia Tech Carilion Research Institute, UNITED STATES Academic Editor: Michael A. Fox, Virginia Tech Carilion Research Institute, UNITED STATES Received: August 14, 2014 Accepted: February 3, 2015 Published: March 31, 2015 Copyright: © 2015 Gu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Gu et al. Introduction Members of the immunoglobulin superfamily (IgSF) are widely expressed in the invertebrate and vertebrate nervous systems and perform a variety of functions in the formation of neural circuits [1]. For example, IgSF members play important roles in axon growth and guidance to- wards their target regions [2,3]. In addition, recent studies reveal that IgSF molecules regulate synaptic specificity in the target zones through hetero- and homophilic interactions [4–7]. In C. elegans, heterophilic interactions between the IgSF molecules SYG-1 and SYG-2 are necessary for appropriate synapse formation between the HSNL motor neurons and adjacent target neurons and muscles [4,5]. SYG-1 is expressed by the HSNL neurons, whereas SYG-2 is expressed by guidepost cells that determine the subcellular localization of HSNL synapses [4,5]. In both syg-1 and syg-2 mutants, HSNL neurons do not synapse with proper targets, rath- er they form errant synapses with inappropriate targets [4,5]. In the formation of laminar-specific synapses in the chick retina, homophilic interactions of IgSF molecules are required for proper development. Synapses between retinal interneurons (amacrine and bipolar cells) and retinal ganglion cells (RGCs) are established in the inner plex- iform layer (IPL). The IgSF molecules sidekick1, sidekick2, Dscam, and Dscaml are expressed by non-overlapping a subset of amacrine, bipolar, and retinal ganglion cells, and engage in homophilic interactions [6,7]. Both gain-of-function and knockdown studies reveal that these homophilic interactions direct the formation of laminar-specific synapses in the chick IPL [6,7]. Thus, both hetero- and homophilic interactions of IgSF molecules contribute to synaptic specificity during development. The precise synaptic connections between proprioceptive sensory neurons and motor neu- rons present an ideal system for studying synaptic specificity in the mammalian central ner- vous system (Fig. 1A). Proprioceptive sensory neurons, whose cell bodies are located in the dorsal root ganglion (DRG), project axons to muscles peripherally, as well as centrally to the spinal cord (Fig. 1A). They are divided mainly into groups Ia and Ib [8]. Group Ia propriocep- tive afferents form monosynaptic connections with motor neurons in the ventral spinal cord (Fig. 1A) that project axons to the same or synergistic muscles, however, they rarely form con- nections with antagonistic motor neurons [9]. These specific connections appear to be formed in an activity-independent manner [10], suggesting that these connections are genetically determined. OPEN ACCESS This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the March of Dimes Foundation (5-FY09-106) and NINDS (NS065048). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 Introduction A recent study has shown that the general pattern of monosynaptic sensory-motor connec- tions is determined by the dorsoventral positions of various motor neuron pools in the ventral horn [11]. Finer connection specificity, however, is achieved through motor neuron-derived guidance molecules such as semaphorin3E (Sema3E). Sema3E is expressed in gluteus motor neurons and regulates specificity of monosynaptic sensory-motor connections through interac- tions with its receptor plexinD1 [12]. Since Sema3E is expressed by only a few of the roughly 50 different motor neuron pools found at limb levels in the spinal cord [12,13], additional mol- ecules likely participate in aiding motor pool specificity of sensory-motor connections during development. In this study, we looked at an array of IgSF molecules for their possible involvement in regu- lating sensory-motor specificity in the mouse. By performing a candidate gene screen, we ex- amined the expression patterns of 157 IgSF genes in the DRG and spinal cord during key stages in neural circuit development. Many of these genes, including Alcam, Mcam and Ocam were expressed by sensory and motor neurons in the DRG and spinal cord. We focused our analyses on the Ocam gene since Ocam showed transient strong expression in a subset of motor neurons at the embryonic stage when proprioceptive axons form synapses with motor neurons. We also found that Ocam is selectively expressed by obturator but not quadriceps 2 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 Expression of IgSF genes in the DRG and the spinal cord To identify the IgSF molecules that regulate sensory-motor specificity, we examined 157 genes encoding IgSF cell adhesion molecules [14] for their expression profiles in embryonic day (E) 14.5-E16.5 DRGs and spinal cords at lumbar levels. We used E14.5-16.5 embryos since sensory- motor connections are already detected in the lumbar region as early as E17.5 [9,15] suggesting that molecules involved in sensory-motor specificity would be expressed earlier by propriocep- tive sensory and/or motor neurons. Sensory-motor circuits are formed between presynaptic pro- prioceptive sensory neurons (marked by the expression of Pv, a specific proprioceptive marker [16,17]) (Fig. 1A, 1C), and postsynaptic motor neurons in the ventral spinal cord (identified by Islet-1) (Fig. 1A-1B). Fig. 1, S1, and S2 showed the expression patterns of representative IgSF genes at E15.5. CD83 was expressed by a subset of motor neurons in the ventral spinal cord as well as a subset of DRG sensory neurons (Fig. 1D-1E). Neurotractin was expressed by most spi- nal cord neurons, with particularly strong expression in motor neurons within the ventral spinal cord (Fig. 1F). Neurotractin was also strongly expressed by a subset of sensory neurons (Fig. 1F- 1G). Lrrn 2 was expressed by a subset of sensory and motor neurons (Fig. 1H-1I), while expres- sion of Lrrn 3 was restricted to a subset of motor neurons (Fig. 1J). Expression of Vstm5 was de- tected in the ventral spinal cord, with strong expression in a subset of motor neurons (Fig. 1L). Vstm5 was also expressed by a subset of sensory neurons (Fig. 1L-1M). Basigin was expressed by most spinal cord neurons, with motor neurons exhibiting the strongest expression (Fig. 1N). In addition, Basigin was expressed in tubular structures (likely endothelial cells) (Fig. 1N) and some DRG sensory neurons (Fig. 1N-1O). There was a gradient of SDR-1 expression in the spinal cord (Fig. 1P). SDR-1 was strongly expressed in the ventral spinal cord, and weakly expressed in the dorsal spinal cord (Fig. 1P). SDR-1 was also strongly expressed by a subset of DRG neurons and weakly expressed by other DRG neurons (Fig. 1P-1Q). Other IgSF genes (Camd4, Chl1, Dscaml1, Iglon5) were expressed in both the DRG and the spinal cord (S1 FigA-1L) while Pvrl3 and Bcam were expressed by a subset of sensory and motor neurons (S1 FigM–S1O and S2). IgSf Molecules and Sensory-Motor Specificity Fig 1. Expression of IgSFs in the developing DRG and spinal cord. (A) Diagram showing sensory-motor circuits in the spinal cord proprioceptive sensory neurons project axons to the ventral spinal cord to form monosynaptic connections with motor neurons innerva synergistic muscle, however, they do not form monosynaptic connections with motor neurons supplying antagonistic muscles. (B-Q) In Islet-1 (B), Pv (C), CD83 (D, E), Neurotractin (F, G), Lrrn-2 (H, I), Lrrn-3 (J, K), Vstm5 (L, M), Basigin (N, O), and SDR-1 (P, Q) on lumb from E15.5 wild-type embryos. doi:10.1371/journal.pone.0121550.g001 Fig 1. Expression of IgSFs in the developing DRG and spinal cord. (A) Diagram showing sensory-motor circuits in the spinal cord proprioceptive sensory neurons project axons to the ventral spinal cord to form monosynaptic connections with motor neurons innerva synergistic muscle, however, they do not form monosynaptic connections with motor neurons supplying antagonistic muscles. (B-Q) In Islet-1 (B), Pv (C), CD83 (D, E), Neurotractin (F, G), Lrrn-2 (H, I), Lrrn-3 (J, K), Vstm5 (L, M), Basigin (N, O), and SDR-1 (P, Q) on lumb from E15.5 wild-type embryos. doi:10.1371/journal.pone.0121550.g001 Fig 1. Expression of IgSFs in the developing DRG and spinal cord. (A) Diagram showing sensory-motor circuits in the spinal cord. Group Ia proprioceptive sensory neurons project axons to the ventral spinal cord to form monosynaptic connections with motor neurons innervating the same or a synergistic muscle, however, they do not form monosynaptic connections with motor neurons supplying antagonistic muscles. (B-Q) In situ hybridizations for Islet-1 (B), Pv (C), CD83 (D, E), Neurotractin (F, G), Lrrn-2 (H, I), Lrrn-3 (J, K), Vstm5 (L, M), Basigin (N, O), and SDR-1 (P, Q) on lumbar spinal cord sections from E15.5 wild-type embryos. doi:10.1371/journal.pone.0121550.g001 doi:10.1371/journal.pone.0121550.g001 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 3 / 19 IgSf Molecules and Sensory-Motor Specificity motor neurons in the ventral spinal cord at lumbar levels. Ocam mutant mice however, showed no obvious defects in motor neuron organization and central projections of proprioceptive sen- sory axons. In addition, electrophysiological analysis using intracellular recordings did not re- veal any noticeable defects in sensory-motor specificity in Ocam mutants. Since another IgSF molecule, Alcam is co-expressed with Ocam in a subset of motor neurons, it is possible that Alcam, or some other IgSF molecule(s), may compensate for the lack of Ocam functioning in the control of sensory-motor specificity. Expression of IgSF genes in the DRG and the spinal cord Moreover, since Alcam, Mcam and Ocam displayed more specific expression patterns in the spinal cord than other genes, we performed more detailed examinations of these genes at different develop- mental stages (see Fig.2–7). In summary, many IgSF genes are expressed by sensory and motor neurons at E15.5, a time just prior to the formation of sensory-motor connections, suggesting that IgSF proteins may participate in establishing these circuits. mRNA and protein expression of Alcam in the DRG and spinal cord Alcam (activated leukocyte cell adhesion molecule/CD166/SC1/BEN/DM-GRASP/Neurolin) is expressed in various regions of the nervous system [18–20]. Chick Alcam, SC1, has been shown to be expressed by sensory and motor neurons [21]. Alcam mutant mice show photore- ceptor ectopias, defects in retinal ganglion cell (RGC) axon fasciculation, and defects in RGC axon targeting in the formation of retinocollicular maps [19,22]. We examined the expression of Alcam in mice at E16.5, postnatal day 0 (P0), and P4 to determine whether Alcam might also 4 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity Fig 2. Expression of Alcam in the developing DRG and spinal cor. (A-I) In situ hybridizations for Alcam on lumbar spinal cord sections from E16.5 (A-C), P0 (D-F), and P4 (G-I) wild-type mice. Alcam was strongly expressed by a subset of sensory and motor neurons at E16.5. Alcam was ubiquitously expressed in the spinal cord at P0 and P4. d i 10 1371/j l 0121550 002 . Expression of Alcam in the developing DRG and spinal cor. (A-I) In situ hybridizations for Alcam on lumbar spi Fig 2. Expression of Alcam in the developing DRG and spinal cor. (A-I) In situ hybridizations for Alcam on lumbar spinal cord sections from E16.5 (A-C), P0 (D-F), and P4 (G-I) wild-type mice. Alcam was strongly expressed by a subset of sensory and motor neurons at E16.5. Alcam was ubiquitously expressed in the spinal cord at P0 and P4. doi:10.1371/journal.pone.0121550.g002 doi:10.1371/journal.pone.0121550.g002 be active during sensory-motor circuit development. In our analyses, Alcam was strongly ex- pressed in the floor plate at E16.5 (Fig. 2A) as reported previously [20]. In addition, Alcam was expressed moderately by many spinal cord neurons but strongly expressed in a subset of motor neurons at E16.5 (Fig. 2A-2B). The expression of Alcam in motor neurons was reduced from P0 (Fig. 2D-2E and 2G-2H). In addition to the spinal cord, Alcam was expressed by most DRG sensory neurons, but strongly expressed by a subset of these DRG neurons at E16.5, P0, and P4 (Fig. 2C, 2F, and 2I). be active during sensory-motor circuit development. In our analyses, Alcam was strongly ex- pressed in the floor plate at E16.5 (Fig. 2A) as reported previously [20]. In addition, Alcam was expressed moderately by many spinal cord neurons but strongly expressed in a subset of motor neurons at E16.5 (Fig. 2A-2B). IgSf Molecules and Sensory-Motor Specificity Fig 3. Alcam is expressed by sensory and motor neurons. (A-I) Transverse sections of the lumbar spinal cord and DRG at E16.5 (A-C) and P0 (D-I) w immunostained for Alcam, Pv, and ChAT. Alcam was strongly expressed by a subset of sensory neurons in the DRG (A-C). Some of the Alcam+ sensory neurons were Pv+ (arrows). (D-F) Alcam was also detected in sensory axons. Axons of Alcam+ and Pv−cutaneous sensory neurons terminated in the do horn, whereas axons of Alcam+ and Pv+ proprioceptive sensory neurons projected to the ventral horn. (G-I) A high power view of ventral spinal cord, show that some of Alcam+ and Pv+ axons were in close proximity to Alcam+ motor neurons. / Fig 3. Alcam is expressed by sensory and motor neurons. (A-I) Transverse sections of the lumbar spinal cord and DRG at E16.5 (A-C) and P0 (D-I) were immunostained for Alcam, Pv, and ChAT. Alcam was strongly expressed by a subset of sensory neurons in the DRG (A-C). Some of the Alcam+ sensory neurons were Pv+ (arrows). (D-F) Alcam was also detected in sensory axons. Axons of Alcam+ and Pv−cutaneous sensory neurons terminated in the dorsal horn, whereas axons of Alcam+ and Pv+ proprioceptive sensory neurons projected to the ventral horn. (G-I) A high power view of ventral spinal cord, showing that some of Alcam+ and Pv+ axons were in close proximity to Alcam+ motor neurons. doi:10.1371/journal.pone.0121550.g003 mRNA and protein expression of Alcam in the DRG and spinal cord The expression of Alcam in motor neurons was reduced from P0 (Fig. 2D-2E and 2G-2H). In addition to the spinal cord, Alcam was expressed by most DRG sensory neurons, but strongly expressed by a subset of these DRG neurons at E16.5, P0, and P4 (Fig. 2C, 2F, and 2I). Alcam protein expression at E16.5 and P0 was then determined using an anti-Alcam anti- body. The Alcam protein was strongly expressed in cell bodies of a subset of sensory neurons (Fig. 3A). Comparing Alcam expression with Pv, we found both Alcam+/Pv−and Alcam+/Pv+ populations (Fig. 3A-3C), demonstrating that Alcam is expressed by a subset of proprioceptive (Pv+) and cutaneous (Pv−) sensory neurons. In addition to Alcam expression in the cell bodies of proprioceptive sensory neurons, Alcam was also detected in the axons (Fig. 3D-3I). A subset of ChAT+ motor neurons also strongly expressed Alcam (Fig. 3D-3I). 5 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity Fig 4. Expression of Mcam in the developing DRG and spinal cord. (A-I) In situ hybridizations for Mcam on lumbar spinal cord sections from E16.5 (A- C), P0 (D-F), and P4 (G-I) wild-type mice. Mcam was strongly expressed by a subset of sensory and motor neurons at all stages. Expression of Mcam in the white matter was detected at P0 and P4 (D, E, G, and H). Fig 4. Expression of Mcam in the developing DRG and spinal cord. (A-I) In situ hybridizations for Mcam on lumbar spinal cord sections from E16.5 (A- C), P0 (D-F), and P4 (G-I) wild-type mice. Mcam was strongly expressed by a subset of sensory and motor neurons at all stages. Expression of Mcam in the white matter was detected at P0 and P4 (D, E, G, and H). Fig 4. Expression of Mcam in the developing DRG and spinal cord. (A-I) In situ hybridizations for Mcam on lumbar spinal cord sections from E16.5 (A- C), P0 (D-F), and P4 (G-I) wild-type mice. Mcam was strongly expressed by a subset of sensory and motor neurons at all stages. Expression of Mcam in the white matter was detected at P0 and P4 (D, E, G, and H). doi:10.1371/journal.pone.0121550.g004 doi:10.1371/journal.pone.0121550.g004 the nervous system is less defined, a recent study revealed that Mcam mutant mice show im- paired appetite, locomotor activity, and spatial learning [25]. We performed in situ hybridization assays for Mcam mRNA expression in the mouse DRG and spinal cord, and observed strong expression of Mcam in a subset of motor neurons at E16.5, P0, and P4 (Fig. 4A-4B, 4D-4E, and 4G-4H). In the DRG, Mcam was expressed by a sub- set of sensory neurons (Fig. 4C, 4F, and 4I). Since strong expression of Mcam in motor neurons was even detected at P0 and P4, Mcam may also be involved in synaptogenesis and the mainte- nance of sensory-motor connections. Expression of Mcam mRNA in the DRG and spinal cord Mcam (melanoma cell adhesion molecule/CD146), which was originally identified in human melanoma cells [23,24], has been extensively studied in tumors. Although the role of Mcam in PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 6 / 19 IgSf Molecules and Sensory-Motor Specificity Fig 5. Expression of Ocam in the developing DRG and spinal cord. (A-I) In situ hybridizations for Ocam on lumbar spinal cord sections from E16.5 (A-C), P0 (D-F), and P4 (G-I) wild-type mice. Ocam was strongly expressed by a subset of sensory and motor neuron at E16.5 (A, B). Ocam was ubiquitously expressed in the DRG and spinal cord at P0 and P4 (D-I). Fig 5. Expression of Ocam in the developing DRG and spinal cord. (A-I) In situ hybridizations for Ocam on lumbar spinal cord sections from E16.5 (A-C), P0 (D-F), and P4 (G-I) wild-type mice. Ocam was strongly expressed by a subset of sensory and motor neuron at E16.5 (A, B). Ocam was ubiquitously expressed in the DRG and spinal cord at P0 and P4 (D-I). doi:10.1371/journal.pone.0121550.g005 doi:10.1371/journal.pone.0121550.g005 In our in situ hybridization, we found that Ocam was strongly expressed by a subset of motor neurons in the ventral spinal cord at E16.5 (Fig. 5A-5B). By P4, the expression of Ocam in motor neurons had decreased (Fig. 5D-5E and 5G-5H). Ocam was also expressed in the DRG at E16.5, P0, and P4 (Fig. 5C, 5F, and 5I), but expression in the DRG was weaker than that in motor neurons at E16.5. The strong expression of Ocam in a subset of motor neurons at E16.5 suggests that Ocam is temporally and spatially well positioned to influence synaptic spec- ificity of monosynaptic sensory-motor connections. Expression of Ocam mRNA in the DRG and spinal cord Ocam/Ncam2/Rncam/mamFasII was initially identified as antigen Rb-8/R4B12 [26,27]. This IgSF molecule is expressed by a subset of olfactory and vomeronasal axons, and Ocam mutant mice exhibit defects in the establishment and maintenance of compartmental organization and the segregation of axodendritic and dendrodendritic synapses within glomeruli [28]. The in- volvement of Ocam in spinal circuits has never yet been shown. 7 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 Protein expression of Alcam, Mcam, and Ocam in motor neurons We then determined whether Alcam, Mcam, and Ocam proteins are expressed by a subset of motor neurons by examining their expression in a mouse model in which GFP labels all motor neurons. To generate this mouse line, we crossed CAG-CAT (CC)-EGFP (ccGFP) reporter mice [29] with Olig2-Cre mice in which Cre is expressed by motor neuron and oligodendrocyte pro- genitors [11,30]. Alcam, Mcam, and Ocam proteins were each expressed by a subset of GFP+ motor neurons (Fig. 6A-6F) with some subsets co-expressing both Alcam and Ocam (S5A-S5C 8 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity Fig 6. Differential expression of Alcam, Mcam, and Ocam proteins in a subset of motor neurons. (A-F) Transverse sections of the lumbar spinal cor from E15.5 Olig2-Cre; ccGFP embryos were immunostained for Alcam (A, B), Mcam (C, D), Ocam (E, F) and GFP (B, D, and F) expressions. All three IgS molecules were expressed by a subset of motor neurons (arrows). PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity Fig 7. Ocam was expressed by obturator but not quadriceps motor neurons. (A) Diagram showing Rho-Dex backfill experiments that label motor neurons supplying individual muscles. The boxed area indicates the ventral spinal cord with motor neurons. (B) Diagram showing motor neuron pools innervating different muscles in the lumbar spinal cord. (C) Immunostaining for Ocam (green) in lumbar spinal cord sections from E14.5 embryos with Rho- Dex (red) injections into Rf, Gr, or Ad muscles. Ocam was expressed by Gr and Ad but not Rf motor neurons. Fig 7. Ocam was expressed by obturator but not quadriceps motor neurons. (A) Diagram showing Rho-Dex backfill experiments that label motor neurons supplying individual muscles. The boxed area indicates the ventral spinal cord with motor neurons. (B) Diagram showing motor neuron pools innervating different muscles in the lumbar spinal cord. (C) Immunostaining for Ocam (green) in lumbar spinal cord sections from E14.5 embryos with Rho- Dex (red) injections into Rf, Gr, or Ad muscles. Ocam was expressed by Gr and Ad but not Rf motor neurons. doi:10.1371/journal.pone.0121550.g007 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 Fig 6. Differential expression of Alcam, Mcam, and Ocam proteins in a subset of motor neurons. (A-F) Transverse sections of the lumbar spinal cord from E15.5 Olig2-Cre; ccGFP embryos were immunostained for Alcam (A, B), Mcam (C, D), Ocam (E, F) and GFP (B, D, and F) expressions. All three IgSF molecules were expressed by a subset of motor neurons (arrows). Fig 6. Differential expression of Alcam, Mcam, and Ocam proteins in a subset of motor neurons. (A-F) Transverse sections of the lumbar spinal cord from E15.5 Olig2-Cre; ccGFP embryos were immunostained for Alcam (A, B), Mcam (C, D), Ocam (E, F) and GFP (B, D, and F) expressions. All three IgSF molecules were expressed by a subset of motor neurons (arrows). doi:10.1371/journal.pone.0121550.g006 doi:10.1371/journal.pone.0121550.g006 doi:10.1371/journal.pone.0121550.g006 Fig.). Mcam was not found to be co-expressed by any Ocam+ motor neurons (S5 FigD-S5F). Fi- nally, these IgSF proteins were enriched in the cell membrane (Fig. 6A-6F). Due to the distinct expression pattern and timing of Ocam expression in a subset of motor neurons (Fig. 5), we decided to further analyze Ocam for a potential role in establishing senso- ry-motor specificity. We first examined which motor neuron pools express Ocam at lumbar levels by injecting Rhodamine-conjugated dextran (Rho-Dex) into different hindlimb muscles of E14.5 embryos (Fig. 7A). We targeted the rectus femoris (Rf), adductor (Ad), and gracillis (Gr) muscles (Fig. 7B) since electrophysiological analysis has shown that obturator sensory af- ferents normally project to obturator motor neurons that innervate Ad and Gr muscles but not quadriceps motor neurons that control Rf muscle [9], providing a testable pair muscle for syn- aptic specificity. Immunohistochemistry for the Ocam protein using Rho-Dex labeled spinal cords revealed that Ocam expression was restricted to obturator (Ad and Gr) but not quadri- ceps (Rf) motor neurons (Fig. 7B-7C). 9 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 Normal motor neuron distribution and sensory axon projection in Ocam mutant mice The establishment of sensory-motor circuits requires the following sequential developmental processes: 1) differentiation of motor neurons and their migration to form stereotypic motor columns; 2) elaboration of motor neuron dendrites; 3) projection of proprioceptive axons into the ventral spinal cord; and 4) formation of specific connections between proprioceptive senso- ry and motor neurons. Therefore, we first examined whether sensory and motor neurons de- velop normally in Ocam mutant mice, prior to analyzing the synaptic specificity of obturator and quadriceps sensory-motor circuits in Ocam mutants. Motor neurons in the lumbar spinal cord can be divided into two columns, the medial motor column (MMC) and the lateral motor column (LMC). The motor neurons in the MMC innervate axial muscles, while neurons in the LMC innervate limb muscles [31]. The MMC and LMC are further divided into medial and lateral divisions, which can be identified by spe- cific markers [31]. Lhx3 and FoxP1 are expressed by medial MMC and LMC neurons, respec- tively, while Islet-1 is expressed by both MMC and medial LMC neurons [31–33]. The analysis of these markers at E12.5-E15.5 revealed no obvious differences in Lhx3+, Islet-1+, and FoxP1+ motor neuron numbers or distributions between control (Ocam+/−and Ocam+/+) and Ocam−/− mice (Fig. 8A-8G, 8J-8S, and S4). [9]We also examined the development of dendrites of Oca- mon Ad motor neurons in Ocam+/−and Ocam−/−mice. Dendritic trees of Ad motor neurons 10 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity Fig 8. Deletion of Ocam in mice does not affect the distribution of motor neurons or their dendritic patterning. (A-F) Transverse sections of E14.5 lumbar spinal cords from Ocam+/−and Ocam−/−embryos were immunostained for Islet-1 and DAPI. (G) Quantification of Islet-1+ motor neuron numbers in the LMC (dotted circle) at E14.5. The numbers of Islet-1+ motor neurons in Ocam−/−embryos (n = 4) were not significantly different from those in Ocam+/− embryos (n = 4) at E14.5. (H-I) Analysis of dendrite patterns of motor neurons in Ocam+/+ and Ocam−/−embryos by retrograde tracing from the Ad muscle E14.5. (J-Q) Transverse sections of E15.5 lumbar spinal cord from Ocam+/+ (n = 5) and Ocam−/−(n = 4) embryos were immunostained for Islet-1, Foxp1, a DAPI. (R-S) Quantification of Islet-1+ (R) and Foxp1+ (S) motor neuron numbers in the LMC at E15.5. The numbers of Islet-1+ and Foxp1+ motor neurons i Ocam−/−embryos were not significantly different from that of Ocam+/−embryos. IgSf Molecules and Sensory-Motor Specificity Fig 9. No obvious defects in central projections of proprioceptive (Pv+) axons in Ocam mutant mice. (A-D) Transverse sections of P0 lumbar spinal cords from Ocam+/+ (n = 4) and Ocam−/−mice (n = 4) were immunostained for Pv expression. (E) Quantification of Pv+ axon arbors in the ventral spinal cord showed no obvious difference between Ocam+/+ and Ocam−/−mice. Fig 9. No obvious defects in central projections of proprioceptive (Pv+) axons in Ocam mutant mice. (A-D) Transverse sections of P0 lumbar spinal cords from Ocam+/+ (n = 4) and Ocam−/−mice (n = 4) were immunostained for Pv expression. (E) Quantification of Pv+ axon arbors in the ventral spinal cord showed no obvious difference between Ocam+/+ and Ocam−/−mice. doi:10.1371/journal.pone.0121550.g009 Normal motor neuron distribution and sensory axon projection in Ocam mutant mice The graphs (G, R, S) represent the mean ± s.e.m. LMC, lateral motor column. Fig 8. Deletion of Ocam in mice does not affect the distribution of motor neurons or their dendritic patterning. (A-F) Transverse sections of E14.5 lumbar spinal cords from Ocam+/−and Ocam−/−embryos were immunostained for Islet-1 and DAPI. (G) Quantification of Islet-1+ motor neuron numbers in the LMC (dotted circle) at E14.5. The numbers of Islet-1+ motor neurons in Ocam−/−embryos (n = 4) were not significantly different from those in Ocam+/− embryos (n = 4) at E14.5. (H-I) Analysis of dendrite patterns of motor neurons in Ocam+/+ and Ocam−/−embryos by retrograde tracing from the Ad muscle at E14.5. (J-Q) Transverse sections of E15.5 lumbar spinal cord from Ocam+/+ (n = 5) and Ocam−/−(n = 4) embryos were immunostained for Islet-1, Foxp1, and DAPI. (R-S) Quantification of Islet-1+ (R) and Foxp1+ (S) motor neuron numbers in the LMC at E15.5. The numbers of Islet-1+ and Foxp1+ motor neurons in Ocam−/−embryos were not significantly different from that of Ocam+/−embryos. The graphs (G, R, S) represent the mean ± s.e.m. LMC, lateral motor column. doi:10.1371/journal.pone.0121550.g008 showed similar branching patterns when Rho-Dex was injected into Ad muscles of E14.5 Ocam+/+ and Ocam−/−embryos (Fig. 8H-8I). Taken together, these genetic analyses revealed that Ocam is not required for the proper differentiation, migration, and dendrite growth of motor neurons. We next determined the impact of Ocam deficiency on central projections of Pv+ proprio- ceptive sensory axons, and again found no obvious differences in the projections between Ocam+/+ and Ocam−/−mice at P0 (Fig. 9). These data suggest that proprioceptive sensory neu- rons also developed properly in Ocam−/−mice. 11 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 No obvious defects in specificity of monosynaptic sensory-motor connections in Ocam mutant mice To determine whether Ocam regulates the synaptic specificity of monosynaptic connections of obturator and quadriceps sensory-motor circuits, we performed intracellular recordings from motor neurons, identified by antidromic responses, in isolated P6–7 spinal cord preparations in wild-type and Ocam mutant mice (Fig. 10). We assessed the presence of monosynaptic in- puts in response to sensory afferent stimulation as previously reported employing the same cri- teria to define monosynaptic sensory-motor connections: a short onset latency and little variability/jitter (variance of < 0.2) in the onset latency from trial to trial [12,34–36]. Using these criteria, the stimulation of obturator sensory nerves evoked monosynaptic responses in obturator motor neurons but not in quadriceps motor neurons in recordings from wild-type mice (Fig. 10A, 10C, and 10I). Similarly, the stimulation of quadriceps sensory nerves evoked monosynaptic responses in quadriceps motor neurons but not in obturator motor neurons (Fig. 10E, 10G, and 10K). The set of latencies of obturator to obturator and quadriceps to quad- riceps connections was determined as <5.2 ms (monosynaptic range, gray bins in Fig. 10). These results were consistent with previously published observations [9]. Similar to wild-type mice, Ocam−/−mice showed monosynaptic connections between obturator sensory afferents and obturator motor neurons, as well as connections between quadriceps afferents and quadri- ceps motor neurons (Fig. 10B, 10F, 10I, and 10K). Moreover, we did not detect any ectopic 12 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 Discussion Development of synaptic specificity in sensory-motor circuits is precisely regulated in the spi- nal cord. Both motor neuron-independent dorsoventral tier-targeting and motor neuron-de- pendent mechanisms have been proposed to regulate sensory-motor circuit specificity [11,37]. Until now, using loss-of-function experiments, only Sema3E and its receptor plexinD1 have been shown to control motor pool specificity as motor neuron-derived molecules [12]. Attrac- tive or repellent molecules such as other semaphorins and ephrins may be involved in the es- tablishment of specific connectivities of sensory-motor circuits. IgSF molecules are also potential candidates for regulating sensory-motor circuit connections as they have been shown to control synaptic specificity in other regions of the nervous system [4–7,38,39]. In this study, we specifically examined IgSF molecules for their possible involvement in establishing sensory- motor specificity. We surveyed the expression profile of 157 IgSF genes in the DRG and spinal cord, and found that many IgSF genes are expressed by DRG sensory and motor neurons. Some genes were expressed by a subset of sensory and motor neurons, suggesting that the corresponding IgSF proteins could potentially contribute to the establishment of sensory-motor specificity in the developing spinal cord. Among the candidate IgSF genes, we focused on Ocam for mutant animal analysis based on the following observations: 1) Ocam is strongly expressed by a subset of motor neurons at E16.5 when proprioceptive sensory afferents reach their motor neuron targets (expression sub- sequently declines); and 2) Ocam is expressed by obturator but not the antagonistic quadriceps motor neurons. We examined whether Ocam regulates the synaptic specificity of obturator and quadriceps sensory-motor circuits using intracellular recording assays, however, we did not find any obvious defects in the specificity of these monosynaptic connections in Ocam mu- tants. One explanation could be that other IgSF molecules act redundantly with Ocam. Indeed, we found that Alcam was expressed by Ocam+ motor neurons (S5 Fig.). Therefore, analysis of double or triple mutant mice lacking multiple IgSF genes including Ocam and Alcam will likely shed new insights into the involvement of Ocam and other IgSF molecules in the establishment of sensory-motor specificity. In addition to Ocam, both Alcam and Mcam showed specific expression patterns in sensory and motor neurons (Fig. 2–4). The Alcam protein is expressed by a subset of motor neurons and proprioceptive sensory neurons. IgSf Molecules and Sensory-Motor Specificity motor neuron recording following Q nerve stimulation; and (G, H) Q motor neuron recording following Ob nerve stimulation. (I-L) Quantification of the latencies (I, K) and the variance (J, L) of the onset of monosynaptic EPSPs is shown. Gray bars in all panels indicate the monosynaptic latency windows. motor neuron recording following Q nerve stimulation; and (G, H) Q motor neuron recording following Ob nerve stimulation. (I-L) Quantification of the latencies (I, K) and the variance (J, L) of the onset of monosynaptic EPSPs is shown. Gray bars in all panels indicate the monosynaptic latency windows. doi:10.1371/journal.pone.0121550.g010 doi:10.1371/journal.pone.0121550.g010 monosynaptic inputs from obturator sensory afferents to quadriceps motor neurons or from quadriceps sensory afferents to obturator motor neurons in Ocam−/−mice as determined by la- tency and the variance of the onset latency (Fig. 10D, and 10H-10L). Therefore, in mice dele- tion of Ocam alone did not result in aberrant synaptic specificity of obturator and quadriceps sensory-motor circuits. IgSf Molecules and Sensory-Motor Specificity Fig 10. Specificity of monosynaptic sensory-motor connections is not altered in Ocam mutant mice. (A-H) Synaptic potentials recorded from individual motor neurons following muscle nerve stimulation from P6–7 Ocam+/+ (A, C, E, G) and Ocam−/−(B, D, F, H) mice. All traces shown are average responses from single motor neurons derived from 20 trials at 1Hz. Representative recordings are shown for the following motor neuron recording and ne stimulation pairs: (A, B) Ob motor neuron recording following Ob nerve stimulation; (C, D) Q motor neuron recording following Ob nerve stimulation; (E, F) Fig 10. Specificity of monosynaptic sensory-motor connections is not altered in Ocam mutant mice. (A-H) Synaptic potentials recorded from individual motor neurons following muscle nerve stimulation from P6–7 Ocam+/+ (A, C, E, G) and Ocam−/−(B, D, F, H) mice. All traces shown are average responses from single motor neurons derived from 20 trials at 1Hz. Representative recordings are shown for the following motor neuron recording and nerve stimulation pairs: (A, B) Ob motor neuron recording following Ob nerve stimulation; (C, D) Q motor neuron recording following Ob nerve stimulation; (E, F) Q Fig 10. Specificity of monosynaptic sensory-motor connections is not altered in Ocam mutant mice. (A-H) Synaptic potentials recorded from individual motor neurons following muscle nerve stimulation from P6–7 Ocam+/+ (A, C, E, G) and Ocam−/−(B, D, F, H) mice. All traces shown are average responses from single motor neurons derived from 20 trials at 1Hz. Representative recordings are shown for the following motor neuron recording and nerve stimulation pairs: (A, B) Ob motor neuron recording following Ob nerve stimulation; (C, D) Q motor neuron recording following Ob nerve stimulation; (E, F) Q PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 13 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 Mice All animals were treated according to institutional and National Institutes of Health guidelines, with the approval of the Institutional Animal Care and Use Committee at Cincinnati Chil- dren’s Hospital Medical Center. Embryos or postnatal wild-type mice from C57BL/6 mice were examined. The following mouse strains were used in this study: Ocam mutants; Olig2-Cre [11,30], and CAG-CAT-EGFP (ccGFP) mice [29]. The generation of Ocam mutant mice will be described elsewhere. IgSF library construction, RNA in situ hybridization screens, immunocytochemistry A library of 157 IgSF genes (Supplementary Table 1; [14]) were amplified by PCR (400–700bp) and cloned into the pGEM-T easy vector (Promega). T7 or Sp6 RNA polymerase (Roche) was used to synthesize anti-sense digoxigenin (DIG)-labeled probes for in situ hybridizations as previously described [40]. RNA in situ hybridization screens were performed on 16–20 μm cryosections according to standard protocols. CD83 (A45), and Mcam/CD146 (A46) were iden- tified in the first screen. Neurotractin (B3), Lrrn 2 (B31), Lrrn3 (B32), Vstm5 (B57), Basigin (B61), and SDR-1 (B62) were identified in the second screen. Ocam/Ncam2 was described pre- viously [26]. An additional seven IgSFs were cloned from E15.5 spinal cord cDNA using the following primers: Alcam F: CTGCATGAACTGAAAGCGACAC, Alcam R: CGGAGGCTCACGGAAACA Cadm4 F: TTCTTCATTTGACCCTACTCCC, Cadm4 R: TCCCATTTCCACGCCCTC; Chl1 F: GCGTGTCCAGAGGTTGAT, Chl1 R: GAGGGAAAGGTACATACAGAGT; Dscaml1 F: CACCTCACTCTGGACCCT, Dscaml1 R: TTTGTGCCCTGGCTTCAT; Iglon5 F: CTTAGCCACAGAGGAAGAAA, Iglon5 R: ATGGAGCAGGGAGAAACA; Pvrl3 F: CTTCAGCCGACAGTTCAG, Pvrl3 R: AGACATACCACTCCCTCC; B F CAGCAACAACGGAAACCC B R TCGTCGGCATCGTAATCC We used rabbit anti-parvalbumin (Pv) (Swant), rabbit anti-GFP (Molecular Probes), goat anti-human Alcam (R & D systems), goat anti-mouse Alcam (R & D systems), goat anti- Mcam/CD164 (R & D systems), mouse Anti-choline acetyltransferase/ChAT (Chemicon), goat anti-Islet1 (R & D systems), guinea pig anti-Foxp1 (kindly provided by Dr. Bennett G. Novitch, University of California, Los Angeles, Los Angeles, CA), rabbit anti-Lhx3 (kindly provided by Dr. Kamal Sharma, University of Chicago, Chicago, IL), and rabbit anti-Ocam antibodies [26]. Immunocytochemistry was performed as previously described [17]. IgSf Molecules and Sensory-Motor Specificity specificity. Future analysis of Alcam and Mcam mutant mice will be required in order to under- stand their roles in sensory-motor circuit development. In summary, our study presents the first detailed expression analysis of IgSF gene expression patterns in the developing DRG and spinal cord, opening the door for further characterization of their functions in the formation of sensory-motor circuits. Future analyses of mutant mice lacking IgSF genes will be necessary to reveal whether and how IgSF molecules regulate synap- tic specificity of sensory-motor circuits in the developing spinal cord. Discussion Since Alcam is expressed in the proprioceptive axon shafts as well as in axon terminals, it may regulate proprioceptive axon trajectory in addition to promoting sensory-motor specificity. Curiously, Mcam is not expressed by most other spinal cord neurons so its expression in a subset of motor neurons is intriguing. Mcam function is thus likely to be restricted to motor neurons and small populations of other neurons or glial cells in the spinal cord. Since Mcam expression is detected at P0 and P4, Mcam may be in- volved in synapse formation and maintenance of sensory-motor circuits in addition to synaptic 14 / 19 Intracellular recording Dissection of spinal cords and subsequent intracellular recodings were performed as described [9,12]. In brief, spinal cords along with peripheral nerves were removed from P6–7 pups and hemisectioned in artificial cerebrospinal fluid (aCSF) containing: NaCl (127mM), KCl (1.9mM), KH2PO4 (1.2mM), CaCl2 (2mM), MgSO4 (1mM), NaHCO3 (26mM) and D-glucose (20.5mM) with oxygenation (95% O2/5% CO2). Hemisected spinal cords were removed into an oxygenated bath containing aCSF for recordings. Using tightly fitting glass pipettes, obturator and quadriceps nerves were stimulated (10mA, S88X, SIU-C Grass technologies). Intracellular potentials were recorded (MultiClamp 700B, Digidata 1440A, Clampex 10, Molecular Devices) using glass micropipettes (90–180 MO) filled with 2M potassium acetate with 0.5% fast green and 300mM of lidocaine N-ethyl bromide (Sigma-Aldrich) to block the antidromic action po- tentials. Average synaptic potentials following nerve stimulation were derived from 20–60 re- petitive stimulations at 1Hz. Obturator or quadriceps motor neurons were identified by antidromic activation. Recordings were accepted only from neurons in which the resting mem- brane potential was lower than-40mV [9,12] Statistics All data are presented as mean ± s.e.m. All statistical analyses were performed in Graphpad Prism 6.0 using unpaired Student’s t—tests. P > 0.05 was considered as “not significant” (n.s.). Retrograde tracing experiments Rhodamine-conjugated Dextran (Rho-Dex; 3000MW, Invitrogen) was injected into particular muscles of E14.5 embryos, and then incubated in the presence of oxygen for 18 hours. Rho- Dex is transported from the muscles to the cell bodies of motor neurons Expression of Ocam Rhodamine-conjugated Dextran (Rho-Dex; 3000MW, Invitrogen) was injected into particular muscles of E14.5 embryos, and then incubated in the presence of oxygen for 18 hours. Rho- Dex is transported from the muscles to the cell bodies of motor neurons. Expression of Ocam 15 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity was identified by immunostaining on 16–20 μm cryosections and compared with Rho-Dex-ex- pression using rabbit anti-tetramethylrhodamine (Invitrogen). 200 μm vibratome sections were used to study motor neuron dendrites. Supporting Information S1 Fig. Expression of Cadm4, Chl1, Dscaml1, Iglon5, and Pvrl3 in the developing spinal cord and DRG. (A-Q) In situ hybridizations for the IgSF molecules Cadm4 (A-C), Chl1 (D-F), Dscaml1 (G-I), Iglon5 (J-L) and Pvrl3 (M-Q) on lumbar spinal cord sections from E14.5 (A, D, F, J, M), P0 (B, E, H, K, N), and P4 (C, F, I, L, Q) wild-type mice. (TIF) S1 Fig. Expression of Cadm4, Chl1, Dscaml1, Iglon5, and Pvrl3 in the developing spinal cord and DRG. (A-Q) In situ hybridizations for the IgSF molecules Cadm4 (A-C), Chl1 (D-F), Dscaml1 (G-I), Iglon5 (J-L) and Pvrl3 (M-Q) on lumbar spinal cord sections from E14.5 (A, D, F, J, M), P0 (B, E, H, K, N), and P4 (C, F, I, L, Q) wild-type mice. (TIF) S2 Fig. Expression of Bcam/CD239 in the developing DRG and spinal cord. (A-I) In situ hy- bridizations for Bcam/CD239 on lumbar spinal cord sections from E14.5 (A-C), E15.5 (D-F), and P0 (G-I) wild-type mice. Bcam was expressed by a subset of sensory and motor neurons at E14.5 and E15.5 (A-F) and ubiquitously expressed in the spinal cord at P0 (G-I). (TIF) S3 Fig. Specificity of the Ocam antibody. (A-F) Transverse sections of the E15.5 lumbar spi- nal cord from Ocam+/+ (A-C) and Ocam−/−(D-F) embryos were immunostained for Ocam ex- pression. Ocam was expressed by a subset of motor neurons in Ocam+/+ (A-C) but not in Ocam−/−embryos, demonstrating the specificity of this Ocam antibody. (TIF) S4 Fig. Deletion of Ocam in mice does not affect the distribution of motor neurons at g E12.5. (A-O) Transverse sections of E12.5 lumbar spinal cord from Ocam+/+ (A-H, n = 5) and Ocam−/−embryos (I-O, n = 4) were immunostained for Islet-1, Lhx3, and Foxp1. (P-Q) Quan- tification of Islet-1+ (P), and Foxp1+ (S) motor neuron numbers in the LMC. Quantification of Lhx3+ (Q) motor neurons in the MMC. The numbers of Islet-1+, Lhx3+, and Foxp1+ motor neurons in Ocam−/−embryos were not significantly different from those of Ocam+/- embryos. 16 / 19 PLOS ONE | DOI:10.1371/journal.pone.0121550 March 31, 2015 IgSf Molecules and Sensory-Motor Specificity The graphs (P-R) represent the mean ± s.e.m. MMC, medial motor column. LMC, lateral motor column. (TIF) The graphs (P-R) represent the mean ± s.e.m. MMC, medial motor column. LMC, lateral motor column. (TIF) S5 Fig. Alcam and Ocam are co-expressed by a subset of motor neurons. (A-F) Transverse sections of E16.5 lumbar spinal cord from wild-type embryos were immunostained for Alcam, Mcam, and Ocam expression. Alcam was co-expressed by certain subsets of Ocam+ motor neu- rons (A-C). Mcam and Ocam were expressed by different sets of motor neurons (D-F). (TIF) S1 Table. Library of IgSF genes. (XLS) S1 Table. Library of IgSF genes. (XLS) S1 Table. Library of IgSF genes. (XLS) Author Contributions Conceived and designed the experiments: ZG FI Y. Yoshida. Performed the experiments: ZG FI HF KK. Analyzed the data: ZG FI Y. Yoshida. Contributed reagents/materials/analysis tools: IJK HF KM Y. Yoshihara. Wrote the paper: Y. Yoshida. FI HF KK. Analyzed the data: ZG FI Y. Yoshida. Contributed reagents/materials/analysis tools: IJK HF KM Y. Yoshihara. Wrote the paper: Y. Yoshida. IJK HF KM Y. Yoshihara. Wrote the paper: Y. Yoshida. Acknowledgments We thank J. Sanes for providing the 157 IgSF genes. We also thank D. Ladle for comments on the manuscript. 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The Impact of COVID-19 on Public/Third-Sector Collaboration in the Italian Context

Sustainability

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Luigi Corvo 1, Lavinia Pastore 1,*, Marco Mastrodascio 2 and Luca Tricarico 3 Luigi Corvo 1, Lavinia Pastore 1,*, Marco Mastrodascio 2 and Luca Tricarico 3 1  Open Impact Research Spin off, Department of Management and Law, University of Rome Tor Vergata,  00133 Rome, Italy; [email protected] y g 2  Department of Management and Law, University of Rome Tor Vergata, 00133 Rome, Italy;  [email protected] 2  Department of Management and Law, University of Rome Tor Vergata, 00133 Rome, Italy;  [email protected] 3  Department of Business and Management, LUISS University, 00198 Rome, Italy; [email protected]  *  Correspondence: [email protected] 3  Department of Business and Management, LUISS University, 00198 Rome, Italy; [email protected]  *  Correspondence: [email protected] 3  Department of Business and Management, LUISS University, 00198 Rome, Italy; [email protected]  *  Correspondence: [email protected] *  Correspondence: [email protected] Abstract: The extent of the effects produced by the ongoing COVID‐19 pandemic on the collabora‐ tion between public administrations and the third sector is currently unclear. Undoubtedly, as in  any  other  organizations,  social  enterprises  and  non‐profit  organizations  have  been  severely  af‐ fected by the spread of COVID‐19, especially regarding their relationship with the public sector. Based on an analysis of 563 Italian third sector entities (ETSs) that responded to an online survey  launched in March 2020, this study aims to explore the current state and extent of the potential  change  in  the  collaboration  between  organizations  belonging  to  the  third  sector  and  the  Italian  public administration system in response to the COVID‐19 emergency. The results have shown that  only approximately one‐third of the organizations have been asked to jointly contribute with the  public sector to contain the negative effects of the pandemic. In other cases, spontaneous support  initiatives  have  been  undertaken  to  manage  the  crisis. The  findings  have  also  revealed that the  COVID‐19 pandemic has affected the internal operating and functioning mechanisms of the or‐ ganizations operating in the third sector. The study concludes with a forecast of the potential ex‐ acerbation of the difficulties currently faced by the third sector and with the provision of future  strategic paths to contain the health, social and economic effects of the pandemic. Citation: Corvo, L.; Pastore, L;    Mastrodascio, M.; Tricarico L. The    Impact of COVID‐19 on    Public/Third‐Sector Collaboration in  the Italian Context. Sustainability  2022, 14, 2228. https://doi.org/  10.3390/su14042228  Academic Editor: Ermanno C. Tortia Received: 17 December 2021 Accepted: 14 February 2022  Published: 16 February 2022 Keywords: third sector; pandemic; COVID‐19; public sector; public administration Academic Editor: Ermanno C. Tortia Citation: Corvo, L.; Pastore, L;    Mastrodascio, M.; Tricarico L. The    Impact of COVID‐19 on    Public/Third‐Sector Collaboration in  the Italian Context. Sustainability  2022, 14, 2228. https://doi.org/  10.3390/su14042228 1. Introduction g g g Based on the negative serious consequences generated by the spread of the corona‐ virus, this study aims to explore the current state and extent of the potential change in  the  collaboration  between  organizations  belonging  to  the  third‐sector  or  Enti del Terzo  Settore (ETS) (Pursuant to the Italian law (Legislative Decree No. 117 of 3 July 2017), ETS  are not‐for‐profit private organizations or entities that pursue civic, solidarity and social  benefits for the purpose of carrying out, exclusively or principally, activities of general  interest in an accountable and transparent way.) and the Italian public administration  system in response to the COVID‐19 emergency. However, it is doubtless that the COVID‐19 emergency is causing effects that, often  in total discontinuity compared to just a few months ago, will significantly determine  new health, relational, social and economic frameworks [1]. Among the many macro is‐ sues  that  may  impact  the  relationship  between  the  state  and  citizens  and  relations  among people, the focus of this article is mainly on the relationship of the state with the  area of the third sector and, therefore, of active, organized and supportive civil society. According to the literature, there are diverse advantages that can strengthen the collab‐ oration  between  governmental  and  nonprofit  organizations  [12]:  (1)  the  mutual  ex‐ change of soft and/or hard resources; (2) the improvement of the status quo due to col‐ lective effort and wisdom; (3) the increase in the legitimacy of the decision‐making pro‐ cess for the public bodies [15]; and (4) a reputational advantage for the non‐profit or‐ ganizations [16]. In this regard, a valid contribution is offered by Sancino and colleagues  (2018)  [17]  who  explored  how  the  diverse  forms  of  cross‐sectoral  collaboration  can  co‐create public value. p However, the extent of the effects produced by the ongoing COVID‐19 pandemic  on the collaboration between public administrations and the third sector is currently un‐ clear, and this article, framed within this branch of studies, endeavors to contribute to  further developing this under‐investigated stream of research. 1. Introduction In the history of humanity, crises and epidemics have not only resulted in violent  impacts, grief and heavy economic and social repercussions, they have often been con‐ sidered as a harbinger of essential changes [1]. Similar to both the black Plague in the XIV  century and the global financial crisis at the beginning of the third millennium, the 2020  COVID‐19, also called coronavirus, pandemic is leading all the countries ravaged by the  virus to rapidly carry out compelling strategies to contain the negative consequences of  the crisis in terms of global health and economic growth [2]. COVID‐19 has demonstrated  its strength by showing how fast it has been able to change the world, bringing about  deep implications for society [3]. Publisher’s  Note:  MDPI  stays  neu‐ tral  with  regard  to  jurisdictional  claims in published maps and insti‐ tutional affiliations. Publisher’s  Note:  MDPI  stays  neu‐ tral  with  regard  to  jurisdictional  claims in published maps and insti‐ tutional affiliations. Since the 2007/2008 international crisis, the socio‐economic situation both at national  and global levels has not been particularly prosperous [4]. In addition, Italy has been one  of  the  first  countries  severely  struck  by  the  coronavirus  to  have  generated  a  so‐ cio‐economic tsunami [4–7] and general discontent and negativity. In fact, according to  the Italian National Institute of Statistics, 75,891 people died from COVID‐19 in the pe‐ riod between February and 31 December 2020 [8]. At the time of writing this article, 15  April 2021, the number of deaths due to COVID‐19, unfortunately, has reached a peak of  115,088 in Italy and 2,960,777 globally [8], confirming the death toll of a natural disaster Copyright:  ©  2022  by  the  authors. Licensee  MDPI,  Basel,  Switzerland. This article is an open access article  distributed  under  the  terms  and  conditions of the Creative Commons  Attribution  (CC  BY)  license  (https://creativecommons.org/license s/by/4.0/). www.mdpi.com/journal/sustainability Sustainability 2022, 14, 2228. https://doi.org/10.3390/su14042228 Sustainability 2022, 14, 2228 2  of  17 2  of  17 as it has caused “acute collectively experienced events with sudden onset” [9] (p. 8) and  a disastrous reduction of resources [10,11]. This negative scenario has confirmed the ex‐ treme relevance of a timely provision of public services, in particular health care treat‐ ments and social care services, which are no longer a privilege of the public sector [12]. In fact, the delivery of many public services has progressively devolved to third sector  organizations collaborating with national governments [12–14]. 1. Introduction More specifically, based  upon the analysis of 563 social enterprises, non‐profit organizations and entities operat‐ ing  in  the  Italian  territory  that  responded  to  an  online  survey  launched  during  the  COVID‐19 outbreak (March 2020), this study aims to deepen the magnitude of the po‐ tential change in the collaboration between organizations belonging to the third sector  and the Italian public administration system in response to the national emergency trig‐ gered by the spread of COVID‐19. The article is organized as follows. The next section endeavors to highlight the the‐ oretical  roots  of  the  collaboration  between  third  sector  and  public  administration  sys‐ tems and how it has been developed throughout the years with a specific focus on the  Italian context. Then, after giving an account of the research methods implemented to  conduct this study, the results of the analysis are shown. The last section is dedicated to  the discussion, concluding remarks, future research paths and limitations of the study. 2. Research Background Prior to analyzing the negative effects of the national disaster of COVID‐19 [3] on  the relationship between organizations belonging to the third sector and the public ad‐ ministration system, a brief description of the third sector seems to be necessary. In this  regard, Iannello’s question What does third sector mean? (2020) seems to be challenging  to answer unambiguously. Sociologically, the third sector could be defined as a reality  with economic, social, political and cultural objectives that have nothing to do with the  aims of the market and profit, nor with the typical objectives of the public administra‐ tion [1]. Undoubtedly, the term “third sector” has now entered the common vernacular, Sustainability 2022, 14, 2228 3  of  17 3  of  17 together with other related terms such as fourth sector [18], private social, non‐profit,  social economy, third dimension and social enterprise. It is a neologism coined by soci‐ ological and economic investigation fueled by the influence of two distinct cultural and  ideological matrices: the North American line of thought of the third sector because of  the State’s or market’s failure and the European line of intermediate communities [19]. “Third sector” is therefore intended to indicate entities that are not the State, public in‐ stitutions or a for‐profit company operating in a private market. The origin of the third sector was originally investigated by Weisbrod (1972) [20],  who attempted to theorize the reasons for the birth of non‐profit organizations in a cap‐ italist economy. Years later, Salomon and Anheier (1998) [21] conceptualized six main  theories to explain the development of the sector in relation to specific socio‐economic  dynamics. More specifically: 1. Heterogeneity  theory:  an  unsatisfied  demand  for  public  or  quasi‐public  goods  in  situations of heterogeneity in demand leads to the emergence of non‐profit organi‐ zations. 2. Supply  theory:  non‐profit  organizations,  created  by  entrepreneurs  who  aim  to  maximize non‐monetary returns, constitute a reflection of the heterogeneity of de‐ mand. 3. Theory of trust: in conditions of information asymmetries, usually making moni‐ toring  more  expensive  and  suspecting  of  undeserved  profits,  the  constraint  on  non‐distribution of profit makes non‐profit organizations more trustworthy. 4. Welfare state theory: the industrialization process leads to the modern welfare sys‐ tem that displaces private organizations to non‐profits. 5. 2. Research Background In general, as observed in recent research conducted by Salamon and Sokolowski  (2016) [25], while in Anglo‐Saxon countries the concept of the non‐profit sector is linked  to  the  evolution  of  charities,  the  involvement  of  volunteers  and  the  constraint  of  non‐distribution of profits, in continental European countries and some countries locat‐ ed  in  Southern  Europe,  including  Italy,  the  non‐profit  sector  terminology  is  often  ac‐ companied by the third sector and the social economy, which represents a broader con‐ Sustainability 2022, 14, 2228 4  of  17 4  of  17 cept, as it includes also social cooperatives and mutual and other organizations that offer  goods and services to the market [25]. cept, as it includes also social cooperatives and mutual and other organizations that offer  goods and services to the market [25]. Overall, the relationship between third sector organizations and national govern‐ ments can be analyzed under three points of view [26]. The first point of view considers  the  relationship  under  a  financial  perspective,  more  specifically,  in  terms  of  funding  [27,28]. The second point of view focuses on interaction styles [29] while the third point  of view focuses on the relationship between the public and third sectors as service pro‐ viders that are able to develop effective synergies [18,30,31]. Regarding the first perspec‐ tive,  there  is  a  tight  dependency  of  third  sector  organizations  on  fund‐raising,  volun‐ teerism  and  public  subsidies,  allowing  them  to  exercise  the  allocative  and  integrative  function,  also  defined  as  the  twin‐function  [26]. Regarding  the  type  of  interaction  be‐ tween the public and third sectors, the unique political and administrative culture root‐ ed in each country shapes different national patterns of public–third sector interactions. Regarding the third perspective, organizations belonging to the third sector have been  actively involved in the provision of public services to meet the increasing demand of  needy individuals dealing with conditions of emergency. In fact, third sector involve‐ ment has changed the delivery of public services, and this, in turn, has changed the third  sector itself [32]. COVID‐19  has  clearly  demonstrated  that  unpredictable  events  are  able  to  deeply  defy societies and, above all, test the public sector to its extreme limits [33]. 2. Research Background In fact, the  spread of the virus has rapidly led to a public health crisis at a global level with strong  negative implications for national governments and for third sector organizations which  had already faced severe income shortfalls because of the 2007/2008 financial crisis and  the consequent economic recession [34]. Consequently, in response to the economic and  the following global fiscal crisis, many European Union (EU) governments had to im‐ plement austerity measures to enhance public expenditure performance [2], and organi‐ zations financed by public subsidies working in social welfare were hardly struck [35]. y p g y Many  scholars  have  emphasized  the  power  of  the  third  sector  in  response  to  COVID‐19,  which  shows  all  the  peculiarities  of  a  wicked  problem,  more  specifically,  complexity, open‐endedness and intractability [36,37]. For instance, Wakui [38] claims  that the third sector, as it neither belongs to the public sector nor the free market system,  can uniquely contribute to the fight against the pandemic. On the other hand, Naidoo  [39] highlights the fact that organizations belonging to the third sector are currently fac‐ ing a great challenge to survive due to the shrinking of the financial resources at their  disposal. In  fact,  the  majority  of  third  sector  organizations  only  rely  on  philanthropic  resources and fundraising resources collected through events and face‐to‐face donations,  which have been shut down due to the restrictions imposed by national governments  (lockdown) to contain the further spread of the virus. The social, political and economic disruptions caused by the COVID‐19 pandemic  have severely compromised the ability of third sector organizations to provide services  at  a  time  when  they  would  be  most  needed  [40,41]. However,  despite  the  limited  re‐ sources available,  the  third  sector  has demonstrated  the  ability to  provide  prompt  re‐ sponses to the physical, psychological and spiritual needs felt by indigent people during  the pandemic [42]. p The impact of COVID‐19 on third sector organizations, especially regarding their  ability to tackle the pandemic with the public sector, highly depends on local circum‐ stances and different national plans [34]. However, the extent of this relationship repre‐ sents a clear gap in the literature with only just‐emerging research shedding light on na‐ tional experiences. 2. Research Background Theory  of  interdependence:  due  to  lower  transaction  costs  (at  an  early  stage),  non‐profit  organizations  anticipate  the  government  in  supplying  public  utility  goods;  however,  due  to  the  “failures”  of  volunteering,  synergistic  relationships  with the sector are established over time. 6. Theory of social origins: the size and structure of the non‐profit sector reflect the  characteristics of the complex system of relationships, classes and social regimes in  which it is involved. 6. Theory of social origins: the size and structure of the non‐profit sector reflect the  characteristics of the complex system of relationships, classes and social regimes in  which it is involved. These  theories  represent  the  entire  spectrum  of  interpretative  possibilities  of  the  evolution  of  the  Italian  non‐profit  sector  [22]. In  particular,  the  theory  of  the  welfare  state appears particularly significant, as it allows us to verify the hypothesis put forward  by Weisbrod about the birth and development of these organizations (presence of areas  of dissatisfaction not covered by the offer of public goods and services). In Italy, follow‐ ing the crisis that occurred in 2007–2008 and the consequent decrease in investments and  public spending on social services, there has been a growth in the non‐profit sector [23]. Henry Mintzberg (2015) [24] participates in the current debate on third sector by in‐ troducing the concept of the plural sector, which is defined as that set of organizations  that, as they are not owned or controlled by either the state or private investors, can act  for the rebalancing of the society. Extending this theory, it could be affirmed that the so‐ cial  mission of  these  organizations  is  supported  by the  achievement  of  positive  social  and environmental impacts. These effects have the power of creating conditions to re‐ balance inequality and therefore return to what Weisbrod introduced; more specifically,  to aid unserved areas of dissatisfaction arising due to the inability of the public sector  and the for‐profit sector to meet all social needs. 3. Research Methods For any researcher of social science, the first major step is to select a paradigm and  method to conduct their research [43]. In this study, we overcame this barrier by firmly  deciding to engage in a qualitative approach. However, even though precise definitions  for qualitative research are rarely found in the literature [44], a steady point in this type  of research is represented by the close engagement of the researcher with the research  context where data are collected and analyzed [45] which means “getting closer to the  phenomenon studied” [46]. p In  their  Handbook  of  Qualitative  Methods,  Denzin  and  Lincon  [47]  argue  that  “while this increasing centrality [of qualitative research] might lead one to believe that  consensual standards have developed, this belief would be misleading” (1995: 417). In  fact, there is restless academic debate questioning the full validity of qualitative research  since “developing validity standards in qualitative research is challenging because of the  necessity  to  incorporate  rigor  and  subjectivity  as  well  as  creativity  into  the  scientific  process” (2001: 522) [48]. According to Aspers and Corte [46], qualitative research is an  iterative  process  in  which  improved  understanding  to  the  scientific  community  is  achieved  by  making  new,  significant  distinctions  resulting  from  getting  closer  to  the  phenomenon studied (2019: 139) [46]. This process can be implemented, as qualitative  researchers’ primary goal is to “make the facts understandable,” and often places less  emphasis  on deriving  inferences  or  predictions from  cross‐case  patterns  [49]. In  addi‐ tion, the qualitative method, in comparison with quantitative counterparts, allows the  researcher to be more creative both in terms of data collection and analysis [43]. y Among the qualitative research methods available, this study employs the basic or  fundamental  qualitative  description  method,  which  has  the  goal  of  qualitatively  de‐ scribing specific events experienced by individuals or groups of individuals (2012: 255)  [50]. The basic or fundamental qualitative description method allows researchers to in‐ terpret without compromising the neutral depiction of reality [51], which differentiates it  from  other  forms  of  qualitative  research  approaches,  such  as  grounded  theory  [52],  phenomenology [53,54] and ethnography [55], which basically aims to describe events. 2. Research Background This research is willing to contribute by investigating the extent to  which,  within  the  Italian context,  the  cooperation  between  organizations  belonging  to  the third sector and the public administration system has been changing in response to  the emergency induced by the spread of COVID‐19. 5  of  17 Sustainability 2022, 14, 2228 3. Research Methods To apply the  basic or fundamental qualitative description model, a web‐based questionnaire was sent  to third‐sector organizations across Italy in March 2020 and remained open until March  2021. We specifically chose Italy as the research context since all the authors are based in  Italy and have gained multi‐year experience within both the public and third sector re‐ search fields, which allowed us to construct our dataset. The  impact  of  COVID‐19  on  public/third‐sector  collaboration  was  evaluated  by  means of an ad hoc questionnaire (included at the end of the article) that was configured  through a mixed process, following the introductory fundamentals, research objectives  and experts’ judgments [58]. In addition, in order to test the robustness and reliability of  the questionnaire employed and, in particular, the internal consistency reliability of the  items included in the questionnaire, the Cronbach alpha (α) value was calculated (Table  1) giving a final value of 0.94, which is higher than the threshold of acceptance (set at  0.70) [59]. Therefore, the items included in the submitted questionnaire are highly relia‐ ble. Table 1. The Cronbach alpha test. Table 1. The Cronbach alpha test. Table 1. The Cronbach alpha test. No. of Respondents  No. of Items  (K)  Sum of Items Variance  (X)  Variance of the Total  Value per Respondent  (Y)  563  8  12.94  73.21  Cronbach’s alpha = 0.94  (K/K − 1) × (1 − (X/Y)) The questionnaire was composed of four main sections and a total of 38 questions  spread among multiple‐choice, five‐point Likert scale and, when a deeper investigation  was required, open‐ended questions were submitted. In the first section, basic descrip‐ tive information on the 563 third sector organizations and entities (ETSs) interviewed,  such as personnel structure (volunteers or non‐volunteers), geographical area of inter‐ vention  (municipal,  regional,  national  or  international),  legal  form  adopted  and  main  sector served such as health, education, religion, philanthropy and others, was collected. The second section of the survey covered the crux of the analysis by questioning the 563  third sector organizations and entities regarding the effect of COVID‐19 on their rela‐ tionship with the public administration system (PA). Furthermore, ETSs were asked if  they collaborated with the PA at a local, regional or national level during the pandemic  and,  if  so,  on  what  type  of  emergency  related  to  COVID‐19. They  were  also  asked  if  there was previous collaboration with the PA and if it was changed by the COVID‐19  emergency. 3. Research Methods On the other hand, through the implementation of the qualitative description method,  social researchers endeavor to provide a rich explanation of the occurrences described in  simply understood language [56] or, according to Caelli and Mill (2003), to discover and  understand a phenomenon, a process or the perspectives and worldviews of the people  involved [57]. The  reasons  behind  the  specific  choice  of  selecting  the  qualitative  description  method are twofold. Firstly, qualitative descriptive studies are “the least theoretical of  all the qualitative approaches to research. In addition, qualitative descriptive studies are  the  least  encumbered  studies,  compared  to  other  qualitative  approaches,  by  a  pre‐existing theoretical or philosophical commitment” [50]. As the aim of this research is  that of exploring the impact of one of the most unprecedented world‐wide catastrophic  events, the COVID‐19 pandemic, on the collaboration between public and third sector  organizations operating in the Italian territory, an approach that did not require a con‐ ceptual or highly abstract rendering of the data was needed. This explains our choice of  the qualitative description research method, as it is specifically adequate for this type of  first‐time event, according to Lambert (2012) [50]. Secondly, this research is the first at‐ tempt to descriptively analyze the consequences of COVID‐19 on the public–third sector  relationship. Therefore,  as  any  other  qualitative  approach  would  have  required  en‐ gagement with previous studies, which, at this stage, do not seem to be existing, qualita‐ tive descriptive research is the least “theoretical” of all of the qualitative approaches to  research as it is, in fact, purely data‐driven. Finally, the basic or fundamental qualitative  description research model does not require researchers to follow strict procedural steps. In fact, how the data are organized depends upon the researcher and how the data were  rendered (2012: 256) [50]. This gives an enormous opportunity to scientists to investigate Sustainability 2022, 14, 2228 6  of  17 6  of  17 a  research  field  under  unprecedented  circumstances  such  as  those  induced  by  the  COVID‐19 pandemic. a  research  field  under  unprecedented  circumstances  such  as  those  induced  by  the COVID‐19 pandemic. Overall, social scientists can implement numerous strategies to collect reliable data. One of the most utilized strategies involves surveys through structured questionnaires  that allow researchers to collect data from a large number of respondents. 3. Research Methods The third section of the survey was related to the activities carried by each  ETS to tackle the emergency. More specifically, they were asked the type of support they  provided, such as intensive care, prevention, diagnosis, family support, provision of ne‐ cessities,  etc. This  section  also  involved  whether  the  ETS  included  new  volunteers  in  their personnel, how many and how unpaid workers were recruited by the ETS. The last  section of the survey dealt with the consequences that COVID‐19 has had on the func‐ tioning mechanisms of ETSs. They were asked whether the pandemic has forced them to  change their methods of provision of services, such as digitalization, and to what extent  the  services  regularly  provided  have  changed,  in  both  qualitative  and  quantitative  terms, due to the emergency. In addition, ETSs were asked whether and, above all, to  what extent the public sector has retrenched public funding. 7  of  17 7  of  17 Sustainability 2022, 14, 2228 According to the latest report published by the Italian National Institute of Statistics  in  2021  [60],  there  is  a  total  bunch  of  362,634  third  sector  organizations  and  entities  (ETSs) operating in the Italian territory, of which 3566 were included in the initial da‐ taset used in this descriptive analysis. The Italian legal system provides for third sector  entities (ETSs) to sign up to specific registers in order to carry out activities of public in‐ terest and access certain benefits. The obligations imposed are based upon the legal form  chosen and  the  activities carried  out  by  the  third sector  entities. The final  list  of 3566  ETSs  included  in  the  dataset  was  generated  through  the  consultation  of  regional  ETS  lists and national third sector networks such as Legacoop, CNCA and Welfare Forum. We first launched the survey in March 2020, and it remained open for 12 months, until  March 2021. We specifically chose to keep the survey open to allow ETSs to freely an‐ swer during the difficult times caused by the COVID‐19 pandemic. In addition, all the  organizations involved were kept informed about the data collection and analysis dur‐ ing the process. 3. Research Methods More specifically, in June 2021, all respondents were sent a report with  the key findings of the research in Italian; an open access data visualization in powerBI  (powerBI data visualization was sent to the organizations involved in the study); and a  report explaining the next research steps that were going to be made. p p g p g g The  next  section  shows  the  results  of  the  qualitative  description  method  imple‐ mented on the final dataset. 4. Results This section explains the main findings stemming from the analysis of the four sec‐ tions  of  the  questionnaire  (see  Appendix  A). A  total  of  563  completed  questionnaires  (563  third  sector  organizations  and  entities)  were  collected  out  of  the  3566  originally  sent, generating a response rate of approximately 16%, distributed per legal form, type  of activity carried out and area of intervention (Table 2). y Concerning the significance of the sample, the confidence level is 95% and the con‐ fidence interval is 4.13 considering a standard deviation of 50%. Table 2. The respondents to the survey. Type of Activities    No. of Organizations  %  Civil protection/social care  162  28.8%  Unspecified    80  14.2%  Culture/sport/recreational activities  80  14.2%  International solidarity/cooperation  24  4.3%  Economic development/social cohesion    23  4.1%  Education/research  21  3.7%  Rights protection/political activities  20  3.6%  Environmental activities  19  3.4%  Philanthropic/voluntary activities  17  3.0%  Labor relations  1  0.2%  Religion  1  0.2%  TOTAL  563  100%  Legal Form  No. of Organizations  %  Volunteering organization  156  27.6%  Social cooperative  139  25.3%  Social promotion association  138  24.6%  Registered association  54  9.6%  Unregistered association  51  4.8%  Foundation    15  2.8%  Social enterprise (non‐social cooperative)  5  1.2%  Other forms  5  1.1% Table 2. The respondents to the survey. Sustainability 2022, 14, 2228 8  of  17 TOTAL  563  100%  Area of Intervention  No. of Organizations  %  Intermunicipal  165  29%  Municipal  136  24%  Regional    117  21%  National    86  15%  International  28  5%  Interregional  23  4%  TOTAL  563  100% As shown in Table 2, 12 types of different activities are carried out by the 563 or‐ ganizations included in the dataset. Almost half (n = 277, 49.2%) of the 563 organizations  analyzed  carry  out  activities  pertaining  to  either  the  health  or  social/civil  care  sector,  while cultural, sport and recreational activities are only carried out by 14% of the entire  list of organizations. In addition, 9 out of the total 12 categories of activities are conducted  by less than 5% each of the organizations included in the dataset, in line with the national  landscape. p Regarding the legal form adopted, three‐quarters (n = 433, 77.5%) of the organiza‐ tions included in the dataset adopted either a legal form of volunteering organization,  social cooperative or social promotion association, while other forms, such as founda‐ tions and non‐social cooperative enterprises, seem not to be favored as a legal form by the  representatives  of  the  third  sector. 4. Results Regarding  the  area  of  intervention,  organizations  predominantly operate at local level, more specifically, at municipal, intermunicipal and  regional levels, while interregional operations are only chosen by 4% of the 563 organi‐ zations included in this study. However, if we consider the top two categories of activi‐ ties, civil protection/social care and health, the area of intervention of the organizations  carrying out those activities seems to be slightly more equitably distributed, especially at  the national level, where almost 50% of the total number of organizations implements  civil protection/social care and health activities. Moreover, out of the 115 organizations  carrying out health activities, approximately half of them (47%) adopted the legal form of  volunteering  organization,  while  the  162  organizations  committed  to  conducting  civil  protection and social care largely chose the social cooperative legal form. To fully un‐ derstand the temporal evolution of the Italian third‐sector throughout the years, Figure 1  shows the number of organizations founded per year and included in the dataset. Figure 1. The number of organizations established per year. Figure 1. The number of organizations established per year. Sustainability 2022, 14, 2228 9  of  17 As shown in Figure 1, most organizations were established in the last 20 years. In  terms of the number of organizations funded per year, the peak was reached with 20  organizations  established  in  1986  and,  more  than  30  years  later,  in  2013. Overall,  the  growth of non‐profit organizations and entities operating in the Italian territory has been  fluctuating over the past thirty years, but progressively increasing if compared with the  period going from 1944 to 1984. p g g The results of the analysis conducted mainly focus on the organizations that were  asked to provide support to the Italian public administration system to fight against the  pandemic caused by the spread of the coronavirus (SARS‐CoV‐2). Interestingly, only 211  organizations (38%) were contacted by the public administration system in response to  the pandemic, and only 51 (9%) are carrying out health services. Compared to the large  extent  of  the  negative  effect  exercised  on  the  entire  nation  in  terms  of  the  number  of  deaths, the COVID‐19 outbreak seems to be a weak reason to ask for support from the  third sector by the Italian public administration system. 4. Results In addition, 133 out of 211 or‐ ganizations  were  contacted  by  local  administrations  (at  municipal  and  intermunicipal  levels) while only 5% (11 organizations) were contacted by public administrations at the  national level. From the analysis of the 211 organizations contacted by the Italian public admin‐ istration to contain the negative effects of the pandemic, 95% had previously collaborat‐ ed with public administrations at a local level while only 30% had done so at the nation‐ al  level. Therefore,  the  fight  against  the  pandemic  has  not  broadened  the  partnership  between the national public administration system and the third sector; it has merely con‐ firmed the solidity and continuity of precious voluntary collaborations at a local level. y y p y As confirmation of the constant help provided by the third sector to manage emer‐ gencies such as the current pandemic, only 7% (n = 39) of the 563 organizations included  in the dataset have not spontaneously undertaken activities to support the management  of the emergency. However, those organizations have been facing constant budget cuts,  which have substantially limited their ability to provide support. In fact, within the dataset  analyzed,  only  5%  of  the  organizations  have  been  either  publicly  funded  or  helped  through  donations  to  provide  support  for  the  COVID‐19  emergency. Therefore,  even  emergencies with the magnitude of a global disaster like the COVID‐19 pandemic seem  not to be satisfactory to reduce the constant financial cuts operated by the Italian national  public administration system. These budget reductions have led third sector organizations  to ponder changing their methods to provide services. Among these methods, the digital‐ ization of the organization, production and usage of services provided by the 563 organi‐ zations included in the dataset has been one of the effects induced by the emergency. However, the extent of the change towards the digitalization of services has been, in  fact,  less  adopted  than  it  was  expected. More  specifically,  on  a  five‐point  Likert  scale  with 1 as “none of the services has been converted in a digital modality” and 5 as “all the  services are digitally provided now”, the 563 organizations included in the data have  averagely selected 2.65, confirming only a partial change and not a radical variation in  the way services are provided during the pandemic. In  addition,  organizations  have  been  asked  to  rate  the  experienced  reduction  in  terms  of  the  services  provided  during  the  COVID‐19  crisis. 5. Discussions and Conclusions The COVID‐19 pandemic has caused a health, social and economic shock that has  few similar precedents in recent history [1,2]. The whole world has been substantially  blocked  in  its  mobility,  sociability  and  lifestyle  [61],  and  the  risk  to  which  billions  of  people have exposed themselves has put the social capital of territories under tremen‐ dous pressure, starting with the health system [7,8,10]. The effects of the crisis immediately became economic and social, and this required  an unprecedented intervention by governments, with a necessary rethinking of who are  to be considered public actors and their roles [2,3,6]. For decades, in fact, the debate on  public governance and on the need for a constellation of entities generating public value  has  tended  to  consider  articulated  ecosystems,  in  which  collaboration  between  public  and  private  entities  plays a  crucial  role  in  determining lasting  positive  impacts at  the  territorial level [12,14,17,62]. This  research,  therefore,  aimed  to  verify  whether,  in  a  moment  of  acute  crisis,  a  “collaborative instinct” has emerged on the part of public administrations [13,17]. If it is  true that the word crisis can also be interpreted as an opportunity to give rise to those  transformative changes that would not be able to be achieved under “normal” condi‐ tions, the collaborative possibility offered by the pandemic is unrepeatable: on the one  hand, due to the exceptional nature of the challenge to be faced, and on the other hand,  due to the extraordinariness of responses to be put in place through joint resources and  experimental synergies [3,4,6]. Three main points emerged from the analysis of 563 third sector organizations op‐ erating in the Italian territory during the COVID‐19 pandemic. Firstly, there has not been an increase in collaboration between the third and public  sectors in response to the crisis. In fact, only one‐third of the organizations were asked to  collaborate with the public sector to contain the negative effects of the pandemic. The  data showing the distribution of requests for collaboration with respect to the territorial  level are of particular interest: most (80%) were contacted by local administrations, fol‐ lowed by regional administrations and lastly by national administrations. This demon‐ strates how collaborative phenomena find a natural habitat in the levels of government  that are closest to social needs, highlighting the centrality of local administrations in cre‐ ating accessible governance opportunities for organizations operating in the area [12]. 4. Results Unsurprisingly,  organiza‐ tions had to consistently reduce the number of services regularly provided to comply  with reduced budgets (averagely rated nearly 4 in the 5‐point Likert scale). Overall, the  emergency seems to have negatively affected the capacity of the third sector to respond  to the crisis, primarily in terms of reduced financial resources; however, it has demon‐ strated its high resilience by continuing to provide constant support to the public sector  in fighting the pandemic. Lastly, organizations have been asked to forecast post‐crisis solutions and impacts. Regarding the former, co‐design and co‐planning represent the solutions highly recom‐ mended by the third sector. Both co‐designing and co‐planning are tools provided by  the new Italian Legislation of third sectors organizations (article ex. 55) that should in‐ Sustainability 2022, 14, 2228 10  of  17 crease the level of collaboration between ETSs and public administrations. Those solu‐ tions are therefore concrete ways in which public bodies and the third sector can work  together to pursue a shared purpose in sectors of general interest. This ought to be done  by maintaining full transparency of relations and the need to treat the subjects who de‐ velop  relations  with  the  public  administration  in  a  uniform  manner,  to  be  identified  through public tenders and based on criteria consistent with the objective to be pursued. Co‐planning is aimed at identifying the needs to be met, the interventions necessary for  this  purpose,  the  methods  of  realization  of  the same  and  the  resources available; it is  therefore the moment in which the third sector can fully participate in drafting public  policies by bringing its own point of view and perspective. Co‐design is instrumental in  the co‐planning process, as it aims to define and possibly carry out specific service or in‐ tervention projects aimed at satisfying defined needs based on co‐planning. p j y g p g Regarding the impacts that COVID‐19 will potentially have on the public admin‐ istration  systems  in  terms  of  investment  in  the  third  sector,  the  563  organizations  in‐ cluded in  the  dataset  have  shown  a  serious  concern about it  by  assigning an average  value of 3 out of 5, with 1 being “no negative impact will be generated from the pan‐ demic” and 5 being “a serious negative impact will be generated from the pandemic”. 5. Discussions and Conclusions The interesting aspect to be debated is how many of these collaborations are to be con‐ sidered effectively “new collaborations” and how many result as an extension of collab‐ orations already active with the same PA. From the joint analysis of the data referring to Sustainability 2022, 14, 2228 11  of  17 the territorial level of collaboration before and during the pandemic crisis, a rather sig‐ nificant picture emerges: the territorial level of collaboration before and during the pandemic crisis, a rather sig‐ nificant picture emerges: ‐ Compared to collaborations with the local PA pre‐pandemic among the ETS inter‐ viewed, 200 out of 211 declare that they have had collaborations. During the pan‐ demic crisis, of those 200, 170 were invited to extend collaboration to the responses  put in place to counter the current crisis. ‐ With respect to the collaboration with the regional PA, 149 ETS interviewees de‐ clare that they have collaborated pre‐pandemic with the regional PA and, of these,  66 have been hired to collaborate to counter the current crisis. ‐ With respect to the collaboration with the national PA, 146 ETS respondents declare  that they have collaborated pre‐pandemic and only 24 have been hired to collabo‐ rate with respect to the current crisis. ‐ With respect to the collaboration with the national PA, 146 ETS respondents declare  that they have collaborated pre‐pandemic and only 24 have been hired to collabo‐ rate with respect to the current crisis. The  overall  result  returns  a  substantial  narrowing  of  the  spaces  for  collaboration  between PA and ETS during the pandemic crisis at all levels of government. Secondly, although there has not been a quantitative increase in collaborative rela‐ tions between PAs and ETSs, where these were already present pre‐pandemic, an inter‐ esting qualitative evolution has been recorded. By qualitative evolution, we mean an in‐ creasingly active involvement of the ETS that is not limited exclusively to the service de‐ livery phase, but is aimed at the entire cycle of programming, design, management and  delivery of the service [3,4,12,14]. In other words, it was investigated whether the PAs  have valued the knowledge of the ETSs deriving from their recognized proximity to lo‐ cal needs. This evolution, certainly influenced by the nature of extreme urgency, has resulted  in the streamlining of administrative procedures and has brought out a perception of  greater  effectiveness  of  emergency  responses. 5. Discussions and Conclusions Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the  study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the  study. Data Availability Statement: The data are not publicly available, though the data may be made  available on request from the corresponding author. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. 5. Discussions and Conclusions Starting  from  these  considerations,  the  prospect  of  adopting  more  and  more  participatory  devices  (such  as  co‐planning  and  co‐design)  is  seen  as  an  excellent  opportunity  for  the  ETSs  to  manage  the  post‐emergency phase. Thirdly, third sector organizations have demonstrated high resilience, especially in  conditions of crisis. There has been a reformulation of activities regarding personal ser‐ vices and a  sudden  change  in  the financial situation. At  the  same  time,  organizations  have been subjected to an unexpected and extensive experimentation with digital ways  of working. From a pandemic response capacity point of view, the ETSs, despite these  adaptation  needs,  have  also  been  proactive  in  trying  to  organize  their  own  initiatives  beyond the provision of their services (when it has been possible to maintain or adapt  them). In the sample analyzed, 33% of organizations declared that they had organized  spontaneous initiatives to react to the pandemic crisis. Most of these activities were fi‐ nanced with the ETS promoters’ own economic resources (65%). Despite  the  reorganization  of  services  (and  digitalization  when  possible)  and  the  pro‐activity in promoting ad hoc initiatives, the ETSs overall declare a perception of a  reduction in the ability to affect social needs. The  563  organizations  included  in  the  dataset  showed  serious  concern  about  the  future  investments  operated  by  the  public  administration  system,  which  may  further  compromise the ability of the third sector to fully operate at all levels of society, from the  very local to the national and the international level, and make a significant and rapidly  increasing contribution to the health and well‐being of society, especially during nation‐ al and international emergencies such as the COVID‐19 pandemic. The considerations that emerged from the analysis are still preliminaries due to the  embryonic stage of the research project. The authors are aware that to support the early  evidence descriptively presented, a further and deeper investigation is needed. Therefore, the next steps of this research project are the following: Therefore, the next steps of this research project are the following: 1. Qualitative analysis of the open‐ended section of the questionnaire to explore spe‐ cific aspects of the dataset, such as those related to the services designed during the 1. 5. Discussions and Conclusions Qualitative analysis of the open‐ended section of the questionnaire to explore spe‐ cific aspects of the dataset, such as those related to the services designed during the Sustainability 2022, 14, 2228 12  of  17 pandemic;  the  qualitative  aspects  of  the  collaboration  established  between  public  administrations and other non‐profit organizations; and the future perspectives of  these organizations. 2. 2. The launching of a new version of the survey (in March 2022) to the same sample of  organizations previously investigated to assess how their conditions have changed  since the beginning of the pandemic. The new survey will be almost identical to the  one analyzed in this paper aside from potentially new elements that have emerged  from the qualitative analysis (point n. 1) that will help us to explore specific issues  such as the reliability of the services created during the first outbreak. 2. The launching of a new version of the survey (in March 2022) to the same sample of  organizations previously investigated to assess how their conditions have changed  since the beginning of the pandemic. The new survey will be almost identical to the  one analyzed in this paper aside from potentially new elements that have emerged  from the qualitative analysis (point n. 1) that will help us to explore specific issues  such as the reliability of the services created during the first outbreak. 2. The launching of a new version of the survey (in March 2022) to the same sample of  organizations previously investigated to assess how their conditions have changed  since the beginning of the pandemic. The new survey will be almost identical to the  one analyzed in this paper aside from potentially new elements that have emerged  from the qualitative analysis (point n. 1) that will help us to explore specific issues  such as the reliability of the services created during the first outbreak. 3. A quantitative analysis of the two surveys will be run at the end of 2022 to investi‐ gate the research hypotheses that emerged in this descriptive analysis and the ex‐ tent of change that occurred in the two‐year period. Author Contributions: Conceptualization, L.C. and L.P.; methodology, M.M.; data curation, L.P.;  writing—original draft preparation, L.P. and M.M.; writing—review and editing, M.M. and L.T.;  visualization, L.P.; supervision, L.C.; project administration L.P. All authors have read and agreed  to the published version of the manuscript. Funding: This research received no external funding. Appendix A The  recent  health  emergency  has  been  severely  challenging  the  resistance  of  the  third‐sector organizations and entities (ETSs). For us, it is very important to deepen the  point of view of these organizations to contribute to studies on the impact of the pan‐ demic and to the construction of new tools and to provide indications for policy makers. The questionnaire is aimed at all Third Sector Entities (ETSs), both those directly  involved in the management of the emergency and those suffering from severe reper‐ cussions. The research is conducted by the University of Rome “Tor Vergata,” the University  of Naples Federico II and by Open Impact. The data collected will be used anonymously  and integrated to respect the confidentiality of the individual body. p 1. Name of the organization *………………………………....................................................... 2. Email address............................................................................................................................. 3. Job position................................................................................................................................. 4. Number of employees............................................................................................................... 5. Number of volunteers............................................................................................................... 6. Establishment year.................................................................................................................... 7. Municipality (or area) where the main services are provided *…………………………. 8. Area or intervention *:    o Municipal  o Intermunicipal  o Regional  o Interregional  o National  o International p 1. Name of the organization *………………………………....................................................... 2. Email address............................................................................................................................. 3. Job position................................................................................................................................. 4. Number of employees............................................................................................................... 5. Number of volunteers............................................................................................................... 6. Establishment year.................................................................................................................... 7. Municipality (or area) where the main services are provided *…………………………. 8. Area or intervention *:    M i i l 13  of  17 Sustainability 2022, 14, 2228 9. Main sector (Italian National Institute of Statistics—INSTAT) *:  o Environment  o Social assistance/civil protection  o International cooperation and solidarity  o Culture, sport and recreation  o Philanthropy and promotion of volunteering  o Education and research  o Trade union relations and interest representation  o Religion  o Health  o Economic development and social cohesion  o Right protection and political activity  o Other    10. Legal form (please select one box only):  o Social cooperative (type A, type B, mixed, integrated)  o Social enterprise (non‐social cooperative)  o Association of social promotion  o Recognized association  o Unrecognized association  o Foundation  o Voluntary organization  o Network of Third Sector Body  o Other type of Third Sector Body o Voluntary organization o Network of Third Sector Body o Other type of Third Sector Body Section 2: Relations with the public administration system Section 2: Relations with the public administration system 11. Have you received a request for collaboration from the public administration  regarding the COVID‐19 emergency? * (please select one box only) o Yes  o No (skip to question 17) o Yes o Yes  o No (skip to question 17) o Yes  o No (skip to question 17) 12. In what area of the COVID‐19 emergency have you collaborated with the public  administration? o Emergency severe illness due to the high number of patients admitted to in‐ tensive and sub‐intensive care. o Emergency diagnosis due to the high demand for COVID‐19 tests to verify the  contagion of the population. o Prevention emergency due to the high demand for medical equipment neces‐ sary to reduce the risk of contagion (masks, gloves etc.). o Emergency support for the relatives/families of the infected, due to the need to  assist the families of patients in a state of isolation (with particular emphasis  on families who are in conditions of social fragility). o Emergency access to food and necessities. o Emergency access to food and necessities. o Other. 13. With which level of the public administration system did you collaborate? o National o Regional  o Local 14. Did you already collaborate with the public administration system? o At national level o At national level  o At regional level  o At local level o At regional level  o At local level 15. With respect to the ways in which the collaboration was established, you noticed that there was a change in the procedures: * 14  of  17 Sustainability 2022, 14, 2228 No changes in procedures 1–2–3–4–5 High variation of procedures  16. According to you * (select one box online) No changes in procedures 1–2–3–4–5 High variation of procedures o The collaboration has significantly increased emergency response capacity o The collaboration has slightly increased the capacity to respond to the emer‐ gency o The collaboration did not significantly increase to respond to the emergency Section 3: Third‐sector initiatives for the COVID‐19 emergency Section 3: Third‐sector initiatives for the COVID‐19 emergency Section 4: Effects of COVID‐19 on your organization 30. Have you noticed a reduction in the ability to intervene in comparison with the  services normally proved by your organization? *  No reduction 1 2 3 4 5 High reduction 31. Have you suffered budget reductions from the public administration due to the  COVID‐19 emergency? *  No budget reduction 1–2–3–4–5 High budget reduction 31. Have you suffered budget reductions from the public administration due to the  COVID‐19 emergency? * No budget reduction 1–2–3–4–5 High budget reduction 32. Have the restrictions imposed by the COVID‐19 emergency required a rethinking of  how your services are provided? * 32. Have the restrictions imposed by the COVID‐19 emergency required a rethinking of  how your services are provided? *  No 1–2–3–4–5 Deep rethinking y p No 1–2–3–4–5 Deep rethinking No 1–2–3–4–5 Deep rethinking 33. To what extent do you think that the application of the rules provided in the Code of  Contracts affects the activities carried out by your organization? *  Very negatively 1–2–3–4–5 Extremely positively 34. Co‐programming and co‐planning with public administrations are useful tools for  post‐emergency management. *  Not at all 1–2–3–4–5 Extremely useful 34. Co‐programming and co‐planning with public administrations are useful tools for  post‐emergency management. * Not at all 1–2–3–4–5 Extremely useful Not at all 1–2–3–4–5 Extremely useful 35. Is it possible to rethink, either fully or partly, the digitalization of the delivery of your organization’s services? o Yes, completely o Yes, partly o No, except for few specific aspects 36. Have the restrictions imposed by the COVID‐19 emergency led to a digitalization of  your services? * 36. Have the restrictions imposed by the COVID‐19 emergency led to a digitalization of  your services? *  Not at all 1–2–3–4–5 I managed to provide all services in a digital way Not at all 1–2–3–4–5 I managed to provide all services in a digital way 37. Do you think that the strong effort of the public administration (also in financial  terms) can have an impact on future investments by the public administration on  your organization? * 37. Do you think that the strong effort of the public administration (also in financial  terms) can have an impact on future investments by the public administration on  your organization? *  Not at all 1–2–3–4–5 Extremely impactful Not at all 1–2–3–4–5 Extremely impactful 38. Before you finish the survey, please remember that your opinion is very useful to us. Section 3: Third‐sector initiatives for the COVID‐19 emergency * o Through word of mouth o Through word of mouth o Via social media or advertising tools o Based on the required specialized skills o With the widest possible participation 15  of  17 15  of  17 Sustainability 2022, 14, 2228 26. Among the volunteers who participate permanently in the activities, there are also: * 27. As a result of the COVID‐19, has your organization recruited new volunteers? * 28. If yes, how many new volunteers are contributing to the organization’s activities? (Please insert a number) 29. In your own opinion, when the emergency phase is over: * o Fewer volunteers will be available o The same number of volunteers o More volunteers will be available o More volunteers will be available Section 4: Effects of COVID‐19 on your organization Section 3: Third‐sector initiatives for the COVID‐19 emergency 17. Has your organization undertaken spontaneous initiatives to support the  management of the emergency? * o Yes o No (skip to question 23) 18. The initiatives have been taken with respect to which type of emergency: * o Emergency severe illness due to the high number of patients admitted to in‐ tensive and sub‐intensive care. o Emergency diagnosis due to the high demand for COVID‐19 test to verify the  contagion of the population. o Prevention emergency due to the high demand for medical equipment neces‐ sary to reduce the risk of contagion (masks, gloves etc.). o Emergency support for the relatives/families of the infected, due to the need to  assist the families of patients in a state of isolation (with particular emphasis  on families who are in conditions of social fragility). g y o Emergency access to food and necessities. o Emergency access to food and necessities. 19. Briefly describe the type of initiative taken (max 100 words) 20. Was the initiative organized in collaboration with other Third Sector Entities (ETSs)? * o Yes, other ETSs have joined the initiative promoted by my organization. o Yes, my organization has joined the initiative promoted by other ETSs. o Yes, my organization has joined the initiative promoted by a network of ETSs  of which my organization is a member. o No, it was only conducted by my organization. o No, it was only conducted by my organization. o Other. o No, it was only conducted by my organization. o Other. o Other. 21. The economic resources necessary to face the activities related to the emergency have  been collected: * o From own resources o From own resources o Through dedicated donation campaigns o Through the financing of the public administration system  o Other 22. Enter the budget used for initiatives related to the COVID‐19 emergency (number in  euro). 23. Does your organization benefit from volunteers’ support? *  o Yes  o No (skip to question 30) 23. 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Nonprofit Organ. 1990, 1, 42–60. ent/third‐sector relationship in a comparative perspective: The cases of France and West Germany. Volunt. ofit Organ. 1990, 1, 42–60. p f g 27. Salamon, L.M. Partners in public service: The scope and theory of government‐nonprofit relations. In The Nonprofit Sector: A  Research Handbook; Powell, W., Ed.; Yale University Press: New Haven, CT, USA, 1987. 28. Salamon, L.M. The nonprofit sector at a crossroads: The case of America. Volunt. Int. J. Volunt. Nonprofit Organ. 1999, 10, 5–23. Sustainability 2022, 14, 2228 17  of  17 29. Lloyd, P.C. Voluntary associations and state agencies at the local level. In The Third Sector: Comparative Studies of Nonprofit  Organizations; Anheier, H., Seibel, W., Eds.; De Gruyter: Berlin, Germany, 1990; pp. 241–254. , g p y g J g , , 31. Weisbrod, B. The Nonprofit Economy; Harvard University Press: Cambridge, MA, USA, 1988. g p y g J g 31. Weisbrod, B. The Nonprofit Economy; Harvard University Press: Cambridge, MA, USA, 1988. 32. Brandsen, T.; Pestoff, V. Co‐production, the third sector and the delivery of public services: An introduction. Public Manag. Rev. 2006, 8, 493–501, doi:10.1080/14719030601022874. 33. Ansell, C.; Sørensen, E.; Torfing, J. The COVID‐19 pandemic as a game changer for public administration and leadership? The  need for robust governance responses to turbulent problems. Public Manag. Rev. 2020, 23, 1–12. ewhere  over  the  rainbow‐third  sector  research  in  and  beyond  coronavirus. Volunt. Sect. Rev. 2020,  11, 34. Macmillan,  R. Somewhere  over  the  rainbow‐third  sector  research  in  and  beyond  coronavirus. Vo 129–136. 35. Clifford, J.M. International Aid: The Flow of Public Resources from Rich to Poor Countries; Routledge: London, UK; New York, NY,  USA, 2017. 36. Head, B.W. Wicked problems in public policy. Public Policy 2008, 3, 101–118. 36. Head, B.W. Wicked problems in public policy. Public Policy 2008, 3, 101–118. p p p y y T. Coproduction during and after the COVID‐19 Pandemic: Will It Last? Public Adm. Rev. 2020, 80, 851–855. 37. Steen, T.; Brandsen, T. Coproduction during and after the COVID‐19 Pandemic: Will It Last? Public 38. Wakui,  M. References Phenomenological Research Methods; Sage Publications: Thousand Oaks, CA, USA, 1994. q y y g j p 52. Glaser, B.G.; Straus, A.L. The Discovery of Grounded Theory: Strategies for Qualitative Research; Adline 52. Glaser, B.G.; Straus, A.L. The Discovery of Grounded Theory: Strategies for Qualitative Research; Adline: New York, NY, USA, 1967. 53. Moustakas, C. Phenomenological Research Methods; Sage Publications: Thousand Oaks, CA, USA, 1994. 53. Moustakas, C. Phenomenological Research Methods; Sage Publications: Thousand Oaks, CA, USA, 1994. 54. Tricarico,  L. Is  community  earning  enough? Reflections  on  engagement  processes  and  drivers  in  two  Italian  energy  communities. Energy Res. Soc. Sci. 2021, 72, 101899. 55. Madison, D.S. Critical Ethnography: Method, Ethics, and Performance; Sage Publications: Thousand Oaks, CA, USA, 2011. 56. Sullivan‐Bolyai, S.; Bova, C.; Harper, D. Developing and refining interventions in persons with health disparities: The use of  qualitative description. Nurs. Outlook 2005, 53, 127–133. q p 57. Caelli, K.; Ray, L.; Mill, J. ‘Clear as mud’: Toward greater clarity in generic qualitative research. Int. 57. Caelli, K.; Ray, L.; Mill, J. ‘Clear as mud’: Toward greater clarity in generic qualitative research. Int. J. Qual. Methods 2003, 2, 1–13. 58. Rodríguez, G.; Gil, J.; García, E. Tradición y Enfoques en la Investigación Cualitativa. Metodol. Investig. Cual. 1996, 14. Available  online: https://bit.ly/3rOQML6 (accessed on 13 February 2022). y J g y g q J Q 58. Rodríguez, G.; Gil, J.; García, E. Tradición y Enfoques en la Investigación Cualitativa. Metodol. Investig. Cual. 1996, 14. Available  online: https://bit.ly/3rOQML6 (accessed on 13 February 2022). p y ( y ) 59. Field, A. Discovering Statistics Using SPSS; Sage Publications: Thousand Oaks, CA, USA, 2009. 60. ISTAT. Structure  and  Profiles  of  the  Nonprofit  Sector. 202 https://www.istat.it/it/files//2021/10/Report‐nonprofit‐2019.pdf (accessed on 30 June 2021). p p p p 61. Tricarico,  L.;  De  Vidovich,  L. Proximity  and  post‐COVID‐19  urban  development:  Reflections  from  Milan,  Italy. J. Urban  Manag. 2021, 10, 302–310. 61. Tricarico,  L.;  De  Vidovich,  L. Proximity  and  post‐COVID‐19  urban  development:  Reflections  from  Milan,  Italy. J. Urban  Manag. 2021, 10, 302–310. g 62. Tricarico, L.; Bitetti, R.; Leone, M.I. L’innovazione sociale nelle politiche urbane. Un caso studio nel contesto italiano. Territorio  2021, 96, 108–115, https://doi.org/10.3280/TR2021‐096010. g 62. Tricarico, L.; Bitetti, R.; Leone, M.I. L’innovazione sociale nelle politiche urbane. Un caso studio nel contesto italiano. Territorio  2021, 96, 108–115, https://doi.org/10.3280/TR2021‐096010.

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English

LEUKEMIA.REPORT OF A CASE SYMPTOMATICALLY CURED BY THE X-RAY.

JAMA

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p CONCLUSIONS. The lymphatic glands are not enlarged, but the inguinal glands are easily palpated. The thorax is of good size and symmetrical ; expansion fair. On per¬ cussion, one finds good resonance, the loAver lung boundaries normal and descending well; heart sounds are normal; the liver dullness extends to the edge of the ribs, and its thin edge is easily felt. Splenic dullness begins on the seventh rib in the mid-axillary line and extends two fingers' breadth below the navel in the left mammary line. To the right it extends one finger's breadth beyond the median line. On palpation the spleen is a hard, smooth, thick mass. No notch can be felt. The radial pulse is easily compressed, is moderately full and regular, Avith a rate of about 90. Temperature is 99. soft; panniculus thin. He is slightly anemic. The visible mucous membranes are of fair color; tongue is moist. There is slight edema of ankles and legs. The lymphatic glands are not enlarged, but the inguinal glands are easily palpated. The thorax is of good size and symmetrical ; expansion fair. On per¬ cussion, one finds good resonance, the loAver lung boundaries normal and descending well; heart sounds are normal; the liver dullness extends to the edge of the ribs, and its thin edge is easily felt. Splenic dullness begins on the seventh rib in the mid-axillary line and extends two fingers' breadth below the navel in the left mammary line. To the right it extends one finger's breadth beyond the median line. On palpation the spleen is a hard, smooth, thick mass. No notch can be felt. The radial pulse is easily compressed, is moderately full and regular, Avith a rate of about 90. Temperature is 99. tion, Avhere seven months ago a most pronounced case existed, yet only time Avili shoAV the permanency of the result. reaches the peritoneum, can not be too strongly empha¬ sized. In some cases the evidence of the early giving way of the obstruction may be sufficiently positive to admit of a diagnosis to that effect and an assurance of a mild attack that will not require an operation until "the interim." But this decision must rest on the most positive evidence after a painstaking study of the history and present condition of the patient. With such positive evidence furnished by Case 2, I believe that plan justifiable. But when in doubt, operate be¬ fore perforation has taken place. y y p y I submit this as a preliminary report, and will later collect other cases so treated, and will give a more detailed history of this one, with charts and plates showing the blood findings. p showing findings. Patient.—W. E., male, American, aged 30, a cigar maker, un¬ married; AA'as first examined July 1, 1903. His family history is negative, except that one sister has had tuberculosis. g , p History.—The patient has lived in Decatur all his life; his habits have been good, he does not use alcohol, but he is a moderate smoker. He has had the usual diseases of childhood, Avith good recovery in all. No history of malaria or syphilis. g y y yp Present Trouble.—The patient came to the office complain¬ ing of Aveakness, pain in the back and a feeling of Aveight and distress in the left side. Tavo years ago he had a fall, alighting on loAver ribs of left side, which compelled him to stop work for two days. He has had a slight cough for some time. For six months he has been troubled with palpitation, dyspnea and slight headaches. Lately he has noticed that his shoes fit tighter at night. He becomes very Aveak on exertion. p CONCLUSIONS. 1. Simple infection does not account for the sudden¬ ness of the attack nor early severity of the pathologic changes in acute appendicitis. changes appe d c t s 2. The evident interference with the blood supply is best accounted for by an increased intra-appendicular pressure. changes appe d c t s 2. The evident interference with the blood supply is best accounted for by an increased intra-appendicular pressure. g g y Examination.—The patient is moderately well nourished, but has a someAvhat cachectic appearance. His present Aveight is 169 pounds, which is his average summer weight; his expression is depressed; mind clear; frame medium sized; musculature pressure. 3. Simply injecting bacteria into the appendix will not produce appendicitis unless used in abnormal amounts and virulence. 4. Subperitoneal ligation of the appendix with a simple ligature without distention can not be made suf¬ ficiently permanent to produce a general infection of the appendix, typical of appendicitis in the human being. Splenomyelogenous leukemia. Photograph showing size of spleen efore treatment and after four months' treatment. appe d , typ ca appe d c t s be g 5. Experiments in dogs show that hydraulic pressure equal to the arterial tension maintained within the lumen of the appendix for a short time is promptly fol¬ lowed by typical appendicitis. by typical appendicitis. 6. The blood supply in an extremity may be cut off with impunity for hours; but in the appendix the ever- present bacteria at once begin an infection, their en¬ trance into the tissues being facilitated by the opening of normal and traumatic avenues by the very distention which cuts off the circulation. 7. The importance of making a complete diagnosis and prognosis during the first twelve hours of the at¬ tack is emphasized. emphasized. 8. This study suggests the possibility of infections or other lesions being produced in other hollow viscera, especially in the gall bladder, the stomach and in¬ testines by temporary overdistention. emphasized. 8. This study suggests the possibility of infections or other lesions being produced in other hollow viscera, especially in the gall bladder, the stomach and in¬ testines by temporary overdistention. Splenomyelogenous leukemia. Photograph showing size of spleen before treatment and after four months' treatment. soft; panniculus thin. He is slightly anemic. The visible mucous membranes are of fair color; tongue is moist. There is slight edema of ankles and legs. A CASE OF PSETJDOLEUKEMIA SUCCESS¬ FULLY TREATED WITH X BAYS. An œ-ray dermatitis of the splenic region has appeared once or twice, the outer layers of the skin desquamating, but by discontinuing treatment for a few days and using a dusting poAvder, no bad results have folIoAved. The skin over the exposed surfaces was rough at first, but after exfoliation has become smooth again. The hair and pigment have disappeared from the parts exposed. y g The patient came to me for treatment April 1, 1903. She presented all the symptoms above enumerated, with rapidly increasing nervous phenomena and steady loss of weight. All the treatment she had received had had absolutely no effect toAvard checking the disease. There had not been any definite diagnosis. Some had suggested entire removal of the diseased glands, but her parents Avould not consent. g , p Examination.—There were large glandular masses on the left side of the neck, anterior and posterior to the sterno- cleidomastoid muscles, the larger ones about the size of a lien's egg. They Avere movable under the skin, but bound down more or less to the deeper structures. The supraclavicular and epitrochlear glands Avere likewise affected. p g pp p p NoA-ember 9: Blood: leucocytes, 129,000; red blood corpus¬ cles, 4,124,000; hemoglobin, 85 per cent. Very little variation in size and shape of the red blood corpuscles and no nucleated forms Avere found. Myelocytes, 25 per cent.; polymorphonu- clear, 60 per cent. ; eosinophiles, 5 per cent. ; small lymphocytes, 1 per cent.; degenerates, 3 per cent. p g The spleen was someAvhat enlarged and tender to pressure. Temperature was 100 to 101 degrees. The hemoglobin index Avas 70 per cent. The differential leucocyte count Avas nega¬ tive. I failed to make a blood count. The pulse was 115 to 120. At no time did I detect the temperature loAver than 100 degrees or the pulse rate less than 102. g p December 6: Leucocytes, 44,360; red blood corpuscles, 4,360,000; hemoglobin, 85-90 per cent.; myelocytes, 20 per cent.; polymorphonuclear, 74 per cent.; eosinophiles, 1 per cent.; small lymphocytes, 2 per cent.; degenerates, 3 per cent. g p Treatment.—Under the use of FoAvler's solution ior three Aveeks the patient rapidly grew Avorse. April 20 I advised the use of the rays, and gave the first exposure, eight inches from the surface of the neck. The neck measured 13% inches over the most prominent portion. A CASE OF PSETJDOLEUKEMIA SUCCESS¬ FULLY TREATED WITH X BAYS. I made one exposure each day, of a duration of fifteen to twenty minutes. After the exposure on the second day, the patient complainea of feeling much worse. The temperature noAV registered 104.5, the high¬ est it had been at any time. The following morning it was again 102, and remained so until one-half hour after the ex¬ posure in the afternoon, Avhen it reached the same mark as on the preceding day January 9, 1904: Leucocytes, 17,825; hemoglobin, 90 per cent.; red count not taken. Myelocytes, 8 per cent.; poly¬ morphonuclear, 88 per cent.; basophiles, 2 per cent.; small lymphocytes, 4 per cent.; degenerates, 1 per cent. y p y g February 13: Leucocytes, 10,9S0; red blood corpuscles, 4,600,000; hemoglobin, 90 per cent.; polymorphonuclear, 86 per cent.; small lymphocytes, 8 per cent.; basophiles, 1 per cent.; transitional forms, 2 per cent; degenerates, 3 per cent. The albumin has entirely disappeared from the urine. The patient's weight to-day is 182 pounds, more than he has ever weighed before. Spleen can only be felt on deep palpation and on deep inspiration. Treatments are noAV reduced to every other day, the patient taking long Avalks on the intervening days. F b p g y Thinking there might be some latent malaria, I examined the blood, with negative results, and administered 12 grains of quinin per day for four days, but without any effect what¬ ever. After the third exposure, splenic tenderness Avas greatly increased, so that gentle percussion caused considerable pain. The patient now felt so miserable (to use her OAvn expression) that she kept her bed all day except at the treatment hour. April 24 her neck measured 12% inches and had visibly decreased in size. Tavo days later it measured 12 inches. The temperature Avas 103, the pulse 115. p g y Thinking there might be some latent malaria, I examined the blood, with negative results, and administered 12 grains of quinin per day for four days, but without any effect what¬ ever. After the third exposure, splenic tenderness Avas greatly increased, so that gentle percussion caused considerable pain. The patient now felt so miserable (to use her OAvn expression) that she kept her bed all day except at the treatment hour. p g g g y February 20: Leucocytes, 7,894; red blood corpuscles, 4,690,000; hemoglobin, 95 per cent. The accompanying table shows the remarkable reduction of leucocytes. A CASE OF PSETJDOLEUKEMIA SUCCESS¬ FULLY TREATED WITH X BAYS. Patient.—Miss L. N., aged 15, school girl. Family history: mother died from heart disease t\vo years ago ; father well ; 3 brothers, 2 well, 1 has chronic stomach trouble; 3 sisters, all Avell. No tubercular or syphilitic history. September 1 : The patient has returned to work. His gen¬ eral condition is improved; appetite good, Avith a gain in weight. The spleen has decreased in size and a notch is felt for the first time. Edema of ankles is less. Blood: leucocytes decreased to 58,000, with an increase in the red blood corpuscles to over 3,000,000. yp y History.—About five or six years ago she noticed that the glands on the left side of the neck became enlarged, but not painful. During the succeeding three years they increased slowly but gradually, and there were occasional sharp pains on left side of abdomen (site of spleen). These Avere of a lan¬ cinating character and of momentary duration. Certain nervous symptoms made their appearance: inability to con¬ centrate her thoughts on her studies, sleeplessness and pains in her legs, the latter causing some unsteadiness of gait. Her disposition likevvise suffered, changing from a bright cheerful tone to one, at times, bordering on melancholy. Two years ago the glands along both borders of the sternocleido muscle began to coalesce and to make more rapid progress in size. The supraclavicular glands also became prominent, and in due time also the axillary glands were involved. , , Encouraged by the results of treatment, Avhich had varied very little in the last tvvo months, and by Senn's article Avhich appeared at this time, it was decided to discontinue the iron and arsenic and to increase the frequency and distribution of the ¡u-ray exposures. These treatments Avere noAV given daily to the splenic region, the ends of the long bones, and to the sternum. In place of the iron and arsenic, one grain capsules of quinin, three times a day, were given as a placebo. q , y, g p October 4: The patient has not missed a day's AA'ork in the last month. The blood shows an increase in the number of leucocytes; the number of red blood corpuscles and the amount of hemoglobin, hoAvever, still show an increase. The spleen can be pushed to the edge of the ribs. A CASE OF PSETJDOLEUKEMIA SUCCESS¬ FULLY TREATED WITH X BAYS. OSCAR W. STEINWAND, M.D. Member American Association for the Advancement of Science. SELMA, CAL. A CASE OF PSETJDOLEUKEMIA SUCCESS¬ FULLY TREATED WITH X BAYS. OSCAR W. STEINWAND, M.D. Member American Association for the Advancement of Science. SELMA, CAL. LEUKEMIA. REPORT OF A CASE SYMPTOMATICALLY CURED BY THE X-RAY. Introduction.\p=m-\Acase of splenomedullary leukemia success- fully treated by the use of the Roentgen ray is reported by Senn.1 In looking up the literature at my disposal, which in- cludes the Index Medicus, I fail to find any further reports of cases thus treated. At the time of the appearance of Senn's report, the patient, whose history I give below, had been under x-ray treatment for seven weeks. p Examination of Fluids.—The urine gives a slight precipitate with heat and acid, and a few hyaline casts are found in the sediment. The blood examination, made by my associate, Dr. C. M. Jack, is as folloAvs: Red blood corpuscles, 2,600,000; leucocytes, 800,000; hemoglobin, 65 per cent.; specific gravity, 1,050. On staining with Ehrlich's triple and Janner's stains, the reds show a slight paleness, slight poikilocytosis and many nucleated forms. The differential count of the leucocytes gives : Polymorphonuclear, 40 per cent. ; myelocytes, 40 per cent.; eosinophiles, 8 per cent.; large mononuclear Avith baso- philic granules, 8 per cent.; degenerates, 4 per cent. From the time of the first recognition of this disease a fatal termination has always been expected, yet it is known that oc- casionally recovery occurs, or there is an apparent arrest, either spontaneously, or after the use of arsenic; hence in re- cording the effect of any treatment of leukemia the word "cure" must be used with caution. Although an examination of my patient to-day would show an entire absence of every symptom of the disease, both by physical and blood examina- 1. New York Medical Record, Aug. 22, 1903. 1. New York Medical Record, Aug. 22, 1903. ed From: http://jama.jamanetwork.com/ by a University of Michigan User on 06/18/2015 Downloaded From: http://jama.jamanetwork.com/ by a University of Michigan User on 06/18/2015 LEUC0CÏTES. TREATMENT. July 1.800,000.Arsenic and ¡r-ray. Aug. 1.160,000. Sept. 1. 58,000.X-ray alone. Arsenic discontinued. Oct. 4.106,000.X-ray alone, daily treatments. Nov. 9.129.000. Dec. 6. 44,000. Jan. 9. 17.825. Feb. 13. 10,980. Feb. 20. 7,894. Clinical Diagnosis.—Splenomyelogenous leukemia. Clinical Diagnosis.—Splenomyelogenous leukemia. Diagnosis. Splenomyelogenous Treatment.—The patient Avas informed of his condition and advised to stop Avork. I gave arsenic and iron internally and »-ray treatments to the splenic region twice a Aveek. ag os s Sp e o ye oge ous Treatment.—The patient Avas informed of his condition and advised to stop Avork. I gave arsenic and iron internally and »-ray treatments to the splenic region twice a Aveek. ay splenic region Course.—After one month the patient seems improved; feels stronger, and his appetite is better. On examination the spleen is apparently smaller, the right edge extending only to the sternum and the loAver edge on a line Avith the navel. The ankles are still swollen, but the blood shows a marked decrease in the number of leucocytes with a slight in¬ crease in the red blood corpuscles and in the amount of hemo¬ globin. Blood smears shoAV an apparent increase in the transi¬ tional forms AA'ith decrease of myelocytes. The normoblasts are markedly increased. Differential counting is unsatisfactory owing to the large number of forms lying on the borderline between the myelocytes and transitional forms. The urine still contains a trace of albumin. A CASE OF PSETJDOLEUKEMIA SUCCESS¬ FULLY TREATED WITH X BAYS. It shows the first reduction Avhen patient was taking both the arsenic and œ-ray treatment, then the in¬ crease in the white cells Avhen the arsenic Avas discontinued and the subsequent decrease to normal after »-ray treatments Avere given alone. p y p April 24 her neck measured 12% inches and had visibly decreased in size. Tavo days later it measured 12 inches. The temperature Avas 103, the pulse 115. 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https://www.researchsquare.com/article/rs-36184/latest.pdf

English

Bioclimatic zonation and potential distribution of Spodoptera frugiperda (Lepidoptera: Noctuidae) in South Kivu Province, DR Congo

BMC ecology

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Research article Keywords: Bioclimatic zone, Potential distribution, Spodoptera frugiperda, MaxEnt model, Environmental variables Posted Date: November 16th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-36184/v3 DOI: https://doi.org/10.21203/rs.3.rs-36184/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Ecology on November 30th, 2020. See the published version at https://doi.org/10.1186/s12898-020-00335-1. Page 1/23 Abstract Background: the fall Armyworm (FAW) Spodoptera frugiperda (JE Smith), is currently a devastating pest throughout the world due to its dispersal capacity and voracious feeding behaviour on several crops. A MaxEnt species distributions model (SDM) was developed based on collected FAW occurrence and environmental data’s. Bioclimatic zones were identified and the potential distribution of FAW in South Kivu, eastern DR Congo, was predicted. Results: Mean annual temperature (bio1), annual rainfall (bio12), temperature seasonality (bio4) and longest dry season duration (llds) mainly affected the FAW potential distribution. The average area under the curve value of the model was 0.827 demonstrating the model efficient accuracy. According to Jackknife test of variable importance, the annual rainfall was found to correspond to the highest gain when used in isolation. FAWs’ suitable areas where this pest is likely to be present in South Kivu province are divided into two corridors. The Eastern corridor covering the Eastern areas of Kalehe, Kabare, Walungu, Uvira and Fizi territories and the Western corridor covering the Western areas of Kalehe, Kabare, Walungu and Mwenga. Conclusions: This research provides important information on the distribution of FAW and bioclimatic zones in South Kivu. Given the rapid spread of the insect and the climatic variability observed in the region that favor its development and dispersal, it would be planned in the future to develop a monitoring system and effective management strategies to limit it spread and crop damage. Background The Fall Armyworm (FAW) Spodoptera frugiperda (J.E Smith 1797) is native to tropical and subtropical Americas [14, 20] and a major corn pest [30]. Its presence was first reported on the African continent in 2016 [20] and in Asia later on in 2018 [51, 52]. Whether FAW larvae is able to infest more than 80 crop species [18, 46], main damages were observed on grasses family (Poaceae) including corn, rice and sorghum [34]. Yield losses can reach up to 73% when 100% of the plants are infested with FAW [27]. According to Baudron et al. [5], maize infestation of 54.9% might have an impact on yield of approximately 12%. Due to its polyphagous feeding behavior and recent introduction in the African continent, FAW is expected to constitute a lasting threat to several important crops in African [20]. Studies on the behavioral characteristics of FAW strains in the Western Hemisphere indicated that two main strains, namely on rice and on maize, are able to mate with each other despite the existence of hybridization barriers [35, 38, 47, 50]. Both rice and maize strains can be found and collected from a single host plant species [29, 37, 47]. Given these characteristics, Nagoshi et al. [36] have even reported that the African infestation may represent a new hybrid population with potentially uncertain behavioral feeding characteristics to become a serious problem for Africa, including Democratic Republic of Congo (DRC). Page 2/23 The fall armyworm has only invaded areas that have a climate pattern similar to the native distribution, justifying the use of climatic Species Distribution Models (SDMs) for further predictive spreading [14]. In recent years, an increasing number of tools for spatial analysis of species distribution at different spatial scales have emerged [21, 28]. These tools have become increasingly popular in ecology. Particularly, The fall armyworm has only invaded areas that have a climate pattern similar to the native distribution, justifying the use of climatic Species Distribution Models (SDMs) for further predictive spreading [14]. In recent years, an increasing number of tools for spatial analysis of species distribution at different spatial scales have emerged [21, 28]. These tools have become increasingly popular in ecology. Particularly, niche distribution models were widely used in many ecological applications [41]. In fact, several methods of SDMs, also known as ecological niche modeling (ENM) have been developed [19]. Background In order to estimate the functional response related to various environmental variables [2], SDMs relate known locations of a species with their environmental characteristics, and then predict the potential geographical range of that species [17]. According to Westbrook et al. [56], the initiation and displacement patterns of insect migrations are dependent on these environmental factors. niche distribution models were widely used in many ecological applications [41]. In fact, several methods of SDMs, also known as ecological niche modeling (ENM) have been developed [19]. In order to estimate the functional response related to various environmental variables [2], SDMs relate known locations of a species with their environmental characteristics, and then predict the potential geographical range of that species [17]. According to Westbrook et al. [56], the initiation and displacement patterns of insect migrations are dependent on these environmental factors. Distribution of FAW has been investigated by Wang et al. [55] and Liu et al. [32] using SDM MaxEnt (Maximum Entropy). Also, the FAW distribution was modeled on a large scale using CLIMEX software integrating the species model “Wet tropical” [13]. Using similar software and two general circulation models (GCMs), Ramirez-Cabral et al. [49] assessed the climate change impact on future suitability for FAW expansion. Furthermore, Early et al. [14] used Species distribution models (SDMs) to forecast FAW global extent. However, FAW occurrence in South Kivu (Eastern DR Congo) has been reported by Cokola [10] but its distribution remains unknown. Several areas in South Kivu are favourable to FAW development according to suitable temperature, day length and precipitation during warm/wet season as provided by Abraham et al. [1]. Modeling potential distribution of species in relation to climatic conditions is an important tool to apply such as in South Kivu where FAW geographical distribution is still unknown. A FAW modeled proposal will be useful for further FAW monitoring and management in case of high scale infestations. Therefore, this study aims to determine bioclimatic zones and establish potential distribution of FAW in South Kivu, eastern Democratic Republic of Congo (DRC). Analysis of variable contributions The estimates of relative contributions of the environmental variables to the FAW MaxEnt model are presented (Fig. 5 and 6) showing that bio12 (Annual rainfall) played a major role in the FAW spread. Furthermore, the environmental variable with highest gain when used in isolation was bio12 (Annual rainfall) according to the Jackknife test of variable importance (Fig. 6). This environmental variable also decreases the most the gain while omitted, but also having the most useful information by itself and much more that is not available in the other variables. The bio12 variable was highly correlated with bio7 (Annual temperature range), bio13 (Rainfall of wettest month), bio16 (Rainfall of wettest quarter) and mi (Annual moisture index). Thus, it appears that these four variables also play a major role in the speed of FAW in South Kivu. Model performance In this study, from the ROC curves, AUC values were used to evaluate the performance of the MaxEnt model. Many studies showed that an AUC of high values leads to better results that significantly differed from the random predictions. The next picture is the receiver operating characteristic (ROC) curve showing the performance of the FAW MaxEnt model. The prediction accuracy of FAW MaxEnt model was found to be acceptable (AUC mean of 0.827 ± 0.033, Fig. 3) according to the identified evaluation criteria. The suitable areas of FAW in South Kivu province are divided into two corridors (Fig. 4): one covering eastern Kalehe, Kabare, Walungu, Uvira and Fizi territories and another the western areas of Kalehe, Kabare, Walungu and Mwenga territories, southern Shabunda and north-western Fizi territories. The most suitable areas for FAW in South Kivu are mostly located in bioclimatic zone 3. In bioclimatic zones 1 and 2, the probabilities of FAW occurrence are very low (medians below 0.063). As for bioclimatic zone 3, the probabilities of occurrence are relatively higher, with a median of 0.29. In South Kivu, FAW are most likely to be found in areas characterized by very high annual temperature range, longest dry season, very high annual moisture index. Furthermore, these zones are also characterized by very low rainfall (annually, in the wettest quarter, during the wettest month). Bioclimatic zones of the South Kivu province Three bioclimatic zones obtained by clustering using bioclimatic data were presented (Fig. 2). The respective characteristics (mean ± standard error) of each zone are given in Table 2. Zone 1 is mainly characterized by very high mean daytime temperature range and rainfall parameters (seasonality, duration for the wettest period, in the wettest quarter and annual values). Furthermore, it has very low temperature means (annual, for warmest and coldest quarters, for hottest month and potential evapotranspiration). Also, zone 2 is characterized by very high isothermal and specific rainfall conditions (during driest period, annually, for wettest quarter and moisture index for dry quarter). In addition, it is characterized by very short duration of dry season, very low temperature seasonality and annually, annual moisture index, mean daytime temperature range. Finally, zone 3 was characterized by very high annual temperature and for warmest quarter, longest dry season, very high annual moisture index. However, it Page 3/23 Page 3/23 was also characterized by very low annual rainfall and for wettest quarter, isothermality and moisture index of the dry quarter. Zones 1, 2 and 3 represented high, low and medium altitude areas respectively. M d l f was also characterized by very low annual rainfall and for wettest quarter, isothermality and moisture index of the dry quarter. Zones 1, 2 and 3 represented high, low and medium altitude areas respectively. Discussion The FAW is a tropical species mostly adapted to warmer parts of the New World [9]. In the current study, we modeled its distribution under tropical conditions in Eastern DR Congo. The existence of 3 bioclimatic zones for FAW was determined in South Kivu. One (zone 3) was found to correspond to the highest probability of FAW occurrence. Climate change has been reported to have different effects on insects, impacting directly their life cycles or indirectly their hosts and/or predators [3, 39]. However, the FAW may benefit from the climate change due to its polyphagous feeding behaviour, its phenotypic and genotypic plasticity [49]. Also, the adult migratory ability is one more adaptative trait to allow moving across regions to several miles (300 miles/generation in some years) [54, 57]. In an area such as South Kivu with an approximate surface area of 69,130 km2, the FAW migration would take place very quickly. Outbreaks of FAW are closely related to climate conditions and with good winter and spring conditions [49]. Cokola [10] noted that FAW incidence in South Kivu has been associated by temperature and rainfall. Moreover, study conducted by Liu et al. [32] founded that land-use was more important than climate factors, with larger potential distributions. In this study, among the 21 used bioclimatic variables, four of them influenced the potential distribution of FAW in the region. It is therefore seen that these four variables also play a major role in the spread of FAW in South Kivu. Wang et al. [55] modelled the distribution of FAW through MaxEnt with 19 bioclimatic variables related to temperature and humidity of which 10 influenced the FAW distribution. However, the FAW distribution may be influenced by other several non-climatic factors, such as host, natural enemy, management level and human activities [24], soil properties, land cover and agricultural management interventions (such as use of pesticides or fertilizers) [6]. This aspect need to be then incorporated into the model. Furthermore, it would also be important to model the FAW distribution by integrating local bioclimatic data into the model to minimize errors related to imported bioclimatic data. Soria-Auza et al. [53] reported that one of the least studied sources of uncertainty in species distribution modeling comes from the environmental data used to run the models, particularly the climate data, especially in the tropics, where comparatively few climatic stations are available. Response of variables to suitability The mean responses of variables to FAW habitat suitability over 100 replicate MaxEnt runs (red) and the mean +/- one standard deviation (blue, two shades for categorical variables) are presented. The bio12 (annual rainfall with less than 1600 mm) variable plays a major role in the FAW distribution according to the Jackknife test (Fig. 6). Furthermore, with a strong negative correlation with bio7 (annual temperature range), FAW also favours locations with high annual temperature. The probability of FAW occurrence is high in environments where (1) mean annual temperature (bio1) is comprised between 19°C and 23°C; (2) Page 4/23 Page 4/23 temperature seasonality (bio4) is less than 2.5 and (3) length of the longest dry season (llds) comprised between 2.5 and 4.5 (Fig. 7). temperature seasonality (bio4) is less than 2.5 and (3) length of the longest dry season (llds) comprised between 2.5 and 4.5 (Fig. 7). Discussion In the case of South Kivu province, however, it is difficult to obtain sufficient local bioclimatic data given the limited number of meteorological stations found in this region. The accuracy of prediction of FAW MaxEnt model showed high values of AUC (Fig. 3) confirming a good model performance [33]. Comparing our results with other studies, including Wang et al. [55], an excellent AUC was found. For instance, AUC often increases with the size of the study area because it contributes to include background points that have environmental characteristics greatly distant from the species requirement, resulting in artificial increase of SDM validation [4]. The suitable areas of FAW in South Kivu province are divided into two corridors (Fig. 4). The Eastern corridor covering the Eastern areas of Kalehe, Kabare, Walungu, Uvira and Fizi territories and the Western corridor covering the Western areas of Kalehe, Kabare, Walungu and Mwenga territories, southern Shabunda and north-western Fizi territories. Page 5/23 Page 5/23 Infestations are most prevalent in the first corridor. Differences in the FAW infestations within the said corridor, between the Ruzizi plain (low altitude) and Kabare (mid altitude) have been demonstrated [10]. According to the modeling realized by Early et al. [14], Sub-Saharan Africa, especially DR Congo, Gabon and Cameroon, appeared to have low suitability for FAW. Early et al. [14] explain that low suitability in these countries was more likely because of extensive forest cover. This is the case for example, here for Shabunda territory. However, this does not mean that pockets of the suitable habitats in the cited countries will not be severely affected, given the ability of the FAW to travel long distances [14]. Among the four environmental variables used as predictors in the FAW MaxEnt model, bio12 (annual rainfall) played a major role in the spread of FAW and contributed more to run the MaxEnt model (Fig. 6). With the Jackknife test for variable importance, the environmental variable exhibited highest gain when used in isolation with bio12 (annual rainfall). Day et al. [12] found that rainfall in the wettest periods and the coldest annual temperatures were important variables in FAW migration. The effects of rainfall on the distribution of FAW have been documented. For example, Early et al. [14] reported that rainfall have a negative impact on FAW larvae. Furthermore, a suitability map provided by Du Plessis et al. Discussion [13] demonstrated that natural rainfall and irrigation scenario were important variables in FAW distribution. The coldest annual temperature and the rainfall during the wettest three months were consistently identified by Early et al. [14] as the environmental variables that most affected FAW distribution. In this work, most suitable habitat for FAW was found in places where annual rainfall was less than 1600mm. According to Early et al. [14] and Nagoshi et al. [15], FAW was most commonly found in areas with very little forest cover, a minimum annual temperature of 18-26°C and with 500-700 mm rainfall in the three wettest months. Furthermore, given that variable bio12 is strongly negatively correlated with bio7 (annual temperature range), it seems clear that FAW also favours locations with high annual temperature. Temperature was the main environmental factor affecting the growth and reproduction of the FAW [8, 25]. FAW was most likely to be found in areas characterized by very high annual temperature range, very long duration of the longest dry season, very high annual moisture index, high maximum temperature of the hottest month and very high mean temperature of the warmest quarter. The probability of FAW occurrence is high in environments where mean annual temperature (bio1) is comprised between 19 °C and 23 °C. Du Plessis et al. [22] found that the development rate of FAW increased linearly with increasing temperatures between 18 and 30 oC. Additionally, Wang et al. [55] found that when the Mean Temperature of the Warmest Quarter varies between 19.15-29.73 °C, the existence probability of the FAW is higher. Among the four environmental variables used as predictors in the FAW MaxEnt model, bio12 (annual rainfall) played a major role in the spread of FAW and contributed more to run the MaxEnt model (Fig. 6). With the Jackknife test for variable importance, the environmental variable exhibited highest gain when used in isolation with bio12 (annual rainfall). Day et al. [12] found that rainfall in the wettest periods and the coldest annual temperatures were important variables in FAW migration. The effects of rainfall on the distribution of FAW have been documented. For example, Early et al. [14] reported that rainfall have a negative impact on FAW larvae. Furthermore, a suitability map provided by Du Plessis et al. [13] demonstrated that natural rainfall and irrigation scenario were important variables in FAW distribution. Discussion The coldest annual temperature and the rainfall during the wettest three months were consistently identified by Early et al. [14] as the environmental variables that most affected FAW distribution. In this work, most suitable habitat for FAW was found in places where annual rainfall was less than 1600mm. According to Early et al. [14] and Nagoshi et al. [15], FAW was most commonly found in areas with very little forest cover, a minimum annual temperature of 18-26°C and with 500-700 mm rainfall in the three wettest months. Furthermore, given that variable bio12 is strongly negatively correlated with bio7 (annual temperature range), it seems clear that FAW also favours locations with high annual temperature. Temperature was the main environmental factor affecting the growth and reproduction of the FAW [8, 25]. Temperature was the main environmental factor affecting the growth and reproduction of the FAW [8, 25]. FAW was most likely to be found in areas characterized by very high annual temperature range, very long duration of the longest dry season, very high annual moisture index, high maximum temperature of the hottest month and very high mean temperature of the warmest quarter. The probability of FAW occurrence is high in environments where mean annual temperature (bio1) is comprised between 19 °C and 23 °C. Du Plessis et al. [22] found that the development rate of FAW increased linearly with increasing temperatures between 18 and 30 oC. Additionally, Wang et al. [55] found that when the Mean Temperature of the Warmest Quarter varies between 19.15-29.73 °C, the existence probability of the FAW is higher. Conclusion In areas where investigations on FAW invasions remain limited, such as in the DR Congo, it is important to model its distribution and to detect areas with high infestation potential. Based on the obtained results, the South Kivu province is a favorable habitat for the development of FAW. However, given the rapid spread of the insect and the climatic variability observed in the region that favor its development and dispersal, it is necessary to pay particular attention to the management of this species now, in order to take effective measures and prevent its further spread. At the same time, effective and efficient Page 6/23 monitoring systems should be set up in its range to collect field data’s and improve further control of this pest. monitoring systems should be set up in its range to collect field data’s and improve further control of this pest. Study area and occurrence data collection This study focused on South Kivu in Eastern DR Congo, between 1º36’ and 5º South Latitude; 26º47’ and 29º20’ East Longitude. Biological data’s related to FAW occurrence were associated to locations with geo- referenced coordinates. Occurrence data of FAW were collected in Kalehe, Kabare, Walungu, Uvira, Fizi, Mwenga and Idjwi territories in collaboration with local farmers who observed FAW larvae and reported every related field in their localities. All suspected cases of FAW attacks were checked for confirmation through field surveys. To confirm that the larvae observed were indeed those of FAW, we had considered the morphological characteristics of FAW larvae as described by EPPO [16] and Sharanabasappa et al. [26]. Geographic coordinates of infested areas were selected only after positive FAW confirmation. Presence records were collected between February 2018 and September 2019 in 156 fields where FAW has been reported. Geographic coordinates on latitude and longitude in the WGS84 system were recorded using GPS Garmin 64s. The map representing the points of occurrence is illustrated in Fig. 1. Environmental variables In this study, we used elevation and potential evapotranspiration data’s combined with 19 bioclimatic variables. Altitude (Digital Elevation Model ASTERDEM) with 30m spatial resolution was obtained from USGS database (https://earthexplorer.usgs.gov) and the bioclimatic data’s were collected from the Africlim database (https://www.york.ac.uk/environment/research/kite/resources/). They were used to build the species distribution model in order to find the FAW suitable areas. Africlim provides high- resolution climate data’s for Africa. Bioclimatic data consisted of 21 environmental variables (Table 1) that were obtained from interpolations of monthly averages of precipitation and temperature taking into account climate data collected over long periods of time (1950 - 2000) [23]. The Africlim spatial database includes monthly grids of temperature and rainfall, deriving from bioclimatic summary variables such as moisture indices and dry season length. All environmental variables were in raster format with a 30 arc seconds resolution (0.93 km x 0.93 km ≈ 0.86 km2 at the equator). Both ArcGIS Desktop 10.6 and QGIS 3.10 were used to process the spatial data: data extraction to the South Kivu province extent, data management in geographic coordinates (datum: WGS84) and resampling all the raster layers to the same resolution for preparing the maps. Bioclimatic zonation Initially, all the environmental variables (n = 21) were clipped to have only spatial data corresponding to the extent of the South Kivu province. Then, geographic coordinates of the raster pixels centroids were used to extract the values for each variables corresponding to each pixel in order to produce a dataset to be used to delineate the bioclimatic zones. The generated bioclimatic dataset was used by processing the Page 7/23 Page 7/23 Principal Component Analysis (PCA) procedure of the FactoMineR [31] package of the R software version 3.5.3 [48]. Based on Kaiser's criterion, only the first 5 principal components were selected for further analysis. The loadings of pixels centroids on the first 5 principal components were then used to perform a hierarchical ascending clustering through the HCPC (Hierarchical Clustering on Principle Components) procedure of the FactoMineR package. Hierarchical clustering was realised using the Euclidean distance as the metric and Ward's aggregation method to determine the optimal number of clusters to be formed. The Kmeans procedure was then used to consolidate the obtained clusters. Clustering results were then imported into QGis 3.10 to produce a bioclimatic zone map of the South Kivu province. Principal Component Analysis (PCA) procedure of the FactoMineR [31] package of the R software version 3.5.3 [48]. Based on Kaiser's criterion, only the first 5 principal components were selected for further analysis. The loadings of pixels centroids on the first 5 principal components were then used to perform a hierarchical ascending clustering through the HCPC (Hierarchical Clustering on Principle Components) procedure of the FactoMineR package. Hierarchical clustering was realised using the Euclidean distance as the metric and Ward's aggregation method to determine the optimal number of clusters to be formed. The Kmeans procedure was then used to consolidate the obtained clusters. Clustering results were then imported into QGis 3.10 to produce a bioclimatic zone map of the South Kivu province. Selection of environmental predictors Prior to distribution modeling, all the environmental variables were subjected to a correlation test in order to select those susceptible to be used as predictors of the FAW distribution. Consequently, only variables with pairwise Pearson correlation coefficients falling under the interval of ]-0.75, 0.75[ were selected for modeling in order to control for multicolinearity problem in environmental predicators [58]. Species distribution modeling MaxEnt (Maximum Entropy) program 3.3.3 [43, 44] was used to establish current climate envelope for FAW natural occurrence in South Kivu. MaxEnt is a common species distribution modeling (SDM) tool used for predicting the distribution of a species from a set of records and environmental predictors [19]. The MaxEnt technique uses known occurrence locations (presence only data) and a set of gridded environmental layers to produce an output map of the predicted ecological niche of the species on a scale of 0 (lowest suitability) to 1 (highest suitability). MaxEnt is a modeling technique that measures entropy, a measure of ‘how much choice’ is involved in the selection of an event [44, 45]. MaxEnt is a general-purpose method for characterizing probability distributions from incomplete information. In estimating the probability distribution defining a species distribution across a study area, MaxEnt formalizes the principle that the estimated distribution must agree with everything that is known (or inferred from the environmental conditions where the species has been observed) but should avoid making any assumptions that are not supported by the data [44]. The approach corresponded to find the probability distribution of maximum entropy (a distribution that is most spread-out, or closest to uniform) subject to constraints imposed by the information available regarding the species observed distribution and related environmental conditions across the study area [44]. MaxEnt was presented as one of the highest performing SDM methods [7]. We ran 100 models, each trained to a randomly selected bootstrap process of the occurrence dataset. Prediction map from each model has been generated in order to calculate the mean prediction and standard deviation of each pixel. Model predictions were imported into ArcGis 10.6 to generate maps of the FAW occurrence probability in South Kivu. Assessment of variable contribution The Jackknife procedure was performed on climate variables to determine the major contributors to the prediction model. The model evaluation was completed by an assessment of the contribution of each variable used in the model based on Jackknife test. However, more detailed evaluation can be carried out during construction of the model by analyzing AUC obtained in different Jackknife test scenario. Then, AUC values obtained from a single variable or with the global models (from which a variable had been removed purposively) can be compared. The main goal in such situation is to identify which variable, when added or removed from the model, mainly modify the AUC value. In this study, the jackknife method was used to analyze the effects of environmental variables on model results in order to select dominant factors. Specifically, the process involves 3 independent steps: 1. Calculating the training gain for the model with only one variable. Higher training gain indicates that the variable has high prediction power and contributes greatly to species distribution; 1. Calculating the training gain for the model with only one variable. Higher training gain indicates that the variable has high prediction power and contributes greatly to species distribution; 2. Calculating the training gain for the model without a specific variable and analyzing the correlation between the removed variable and the omission error. If the removal of an environmental variable leads to a significant increase in the omission error, it indicates that the variable has a significant effect on the model's prediction; 3. Calculating the training gain for the model with all variables. Model evaluation Page 8/23 In this study, the Receiver Operating Characteristic (ROC) curve method was used to assess the model's performance [11, 40, 42]. One of the parameters used to evaluate predictive capacity of a model generated by MaxEnt is the area under the curve (AUC) or under the ROC curve. AUC can then be interpreted as the likelihood that a randomly selected point of presence is located in a raster cell with a higher probability of species occurrence than a randomly generated point [44]. The AUC is an effective threshold-independent index that can evaluate a model's ability to discriminate presence from absence (or background) occurrence. Also, the AUC is not affected by collinearity and spatiotemporal autocorrelation [11]. The closer AUC is to 1, the more predictive is the model. Random distribution has an AUC of 0.5. Overall value of AUC can be considered in evaluating the final model. AUC values of 0.5 - 0.7 indicate low accuracy, 0.7-0.9 useful applications and > 0.9 high accuracy [33]. Abbreviations AUC: Area under the curve; ROC: Receiver operating characteristic; MaxEnt: Maximum entropy; SDM: Species distribution model; HCPC: Hierarchical clustering on principle components; PCA: Principal component analysis; CLIMEX: Climate and population modeling software; DRC: Democratic Republic of Congo. Declarations Acknowledgments Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding This study was made possible with the financial support of Pain pour le Monde and Académie de recherche et d’enseignement supérieur (ARES). The funding body did not play any additional role in the design, collection, analysis, data interpretation, and/or writing of this study. Competing interests The authors declare that they have no competing interests. Acknowledgments Page 9/23 The authors acknowledge the help of Kazamwali Muzee Léon. The Université Evangélique en Afrique (UEA) is dully acknowledged through Professor Gustave Mushagalusa and Professor Katcho Karume, for manifold support to this work which had graciously been founded by the University project on human resource development for improvement of research and teaching quality funded by Pain pour le Monde (Project A-COD-2018-0383). Consent for publication Not applicable. Authorsʹ contributions FF and EBB planned and supervised the project. MCC and YM performed the experiments, analyzed the data, and contributed reagents/materials/analysis tools. NG, GBC, DMB and ANB analyzed the data and performed simulations. All authors contributed to the writing and revision of the final manuscript. All authors read and corrected the manuscripts. Ethics approval and consent to participate The research reported here was carried out in strict accordance with ethical guidelines and regulations in the standard operating procedures of the Université Evangélique en Afrique (UEA/Bukavu-DR Congo). Collection of fall armyworm occurrence data was approved with the approval reference number UEA/FACAGRO/KK/051/2018 and permitted by the Provincial Inspection of Agriculture, Livestock and Fisheries (Reference number: 18.0/0056/IPAPEL/SK/2018). References 1. Abrahams P, Beale T, Cock M, Corniani N, Day R, Godwin J, et al. Impacts and control options in Africa: preliminary evidence Note. 2017;18. Page 10/23 1. Abrahams P, Beale T, Cock M, Corniani N, Day R, Godwin J, et al. Impacts and control options in Africa: preliminary evidence Note. 2017;18. Page 10/23 Page 10/23 2. Austin MP, Belbin L, Meyers JA, Doherty MD, Luoto M. Evaluation of statistical models used for predicting plant species distributions: Role of artificial data and theory. 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Annu Rev Ecol Evol Syst. 2009;40:677–97. 18. Integrated management of the Fall Armyworm on maize: a guide for farmer field schools in Africa. 2018. 18. Integrated management of the Fall Armyworm on maize: a guide for farmer field schools in Africa. 2018. 19. Fourcade Y, Engler JO, Rödder D, Secondi J. Mapping species distributions with MAXENT using a geographically biased sample of presence data: a performance assessment of methods for correcting sampling bias. PLoS ONE. 2014;9:e97122. 20. Goergen G, Kumar PL, Sankung SB, Togola A, Tamò M. First Report of Outbreaks of the Fall Armyworm Spodoptera frugiperda (J E Smith) (Lepidoptera, Noctuidae), a New Alien Invasive Pest in West and Central Africa. PLoS ONE. 2016;11:e0165632. 20. Goergen G, Kumar PL, Sankung SB, Togola A, Tamò M. First Report of Outbreaks of the Fall Armyworm Spodoptera frugiperda (J E Smith) (Lepidoptera, Noctuidae), a New Alien Invasive Pest in West and Central Africa. PLoS ONE. 2016;11:e0165632. 21. Guisan A, Thuiller W. Predicting species distribution: offering more than simple habitat models. Ecol Letters. 2005;8:993–1009. 21. References Guisan A, Thuiller W. Predicting species distribution: offering more than simple habitat models. Ecol Letters. 2005;8:993–1009. 22. Du Plessis H, Schlemmer M-L, Van den Berg J. The effect of temperature on the development of Spodoptera frugiperda (Lepidoptera: Noctuidae). Insects. 2020;11:228. 22. Du Plessis H, Schlemmer M-L, Van den Berg J. The effect of temperature on the development of Spodoptera frugiperda (Lepidoptera: Noctuidae). Insects. 2020;11:228. 23. Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. Very high resolution interpolated climate surfaces for global land areas. Int J Climatol. 2005;25:1965–78. 23. Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. Very high resolution interpolated climate surfaces for global land areas. Int J Climatol. 2005;25:1965–78. 24. Hill MP, Hoffmann AA, McColl SA, Umina PA. Distribution of cryptic blue oat mite species in Australia: current and future climate conditions. Agr Forest Entomol. 2012;14:127–37. 24. Hill MP, Hoffmann AA, McColl SA, Umina PA. Distribution of cryptic blue oat mite species in Australia: current and future climate conditions. Agr Forest Entomol. 2012;14:127–37. 25. Hogg DB, Pitre HN, Anderson RE. Assessment of early-season phenology of the fall armyworm (Lepidoptera: Noctuidae) in Mississippi 1. Environ Entomol. 1982;11:705–10. 25. Hogg DB, Pitre HN, Anderson RE. Assessment of early-season phenology of the fall armyworm (Lepidoptera: Noctuidae) in Mississippi 1. Environ Entomol. 1982;11:705–10. 26. Sharanabasappa, Kalleshwaraswamy CM, Maruthi MS, Pavithra HB. Biology of invasive fall army worm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) on maize. Indian J Entomol. 2018;80:540. 27. Hruska AJ, Gould F. Fall Armyworm (Lepidoptera: Noctuidae) and Diatraea lineolata (Lepidoptera: Pyralidae): Impact of Larval Population Level and Temporal Occurrence on Maize Yield in Nicaragua. J Econ Entomol. 1997;90:611–22. 28. Jiménez-Valverde A, Lobo JM, Hortal J. Not as good as they seem: the importance of concepts in species distribution modelling. Diversity and Distributions. 2008;14:885–90. 29. Juárez ML, Schöfl G, Vera MT, Vilardi JC, Murúa MG, Willink E, et al. Population structure of Spodoptera frugiperda maize and rice host forms in South America: are they host strains? Entom Exp Appl. 2014;152:182–99. 30. Labatte JM. Modelling the larval development of Spodoptera frugiperda (J. E. Smith), (Lep., Noctuidae) on corn. J Appl Entomol. 1994;118:172–8. 30. Labatte JM. Modelling the larval development of Spodoptera frugiperda (J. E. Smith), (Lep., Noctuidae) on corn. J Appl Entomol. 1994;118:172–8. 31. Lê S, Josse J, Husson F. FactoMineR : An R package for multivariate analysis. J Stat Soft. 2008;25. doi:10.18637/jss.v025.i01. 31. References Lê S, Josse J, Husson F. FactoMineR : An R package for multivariate analysis. J Stat Soft. 2008; doi:10.18637/jss.v025.i01. 32. Liu T, Wang J, Hu X, Feng J. Land-use change drives present and future distributions of Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). Sci Tot Environ. 32. Liu T, Wang J, Hu X, Feng J. Land-use change drives present and future distributions of Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). Sci Tot Environ. Page 12/23 Page 12/23 2020;706:135872. 33. Manel S, Williams HC, Ormerod SJ. Evaluating presence-absence models in ecology: the need to account for prevalence: Presence-absence modelling. J Appl Ecol. 2002;38:921–31. 34. Montezano DG, Specht A, Sosa-Gómez DR, Roque-Specht VF, Sousa-Silva JC, Paula-Moraes SV, et al. Host Plants of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas. Afr Entomol. 2018;26:286–300. 35. Nagoshi RN. The fall armyworm triose phosphate isomerase (Tpi) gene as a marker of strain identity and interstrain mating. Ann Entomol Soc Am. 2010;103:283–92. 35. Nagoshi RN. The fall armyworm triose phosphate isomerase (Tpi) gene as a marker of strain identity and interstrain mating. Ann Entomol Soc Am. 2010;103:283–92. 36. Nagoshi RN, Goergen G, Plessis HD, van den Berg J, Meagher R. Genetic comparisons of fall armyworm populations from 11 countries spanning sub-Saharan Africa provide insights into strain composition and migratory behaviors. Sci Rep. 2019;9. doi:10.1038/s41598-019-44744-9. 36. Nagoshi RN, Goergen G, Plessis HD, van den Berg J, Meagher R. Genetic comparisons of fall armyworm populations from 11 countries spanning sub-Saharan Africa provide insights into strain composition and migratory behaviors. Sci Rep. 2019;9. doi:10.1038/s41598-019-44744-9. 37. Nagoshi RN, Meagher RL. Seasonal distribution of fall armyworm (Lepidoptera: Noctuidae) host strains in agricultural and turf grass habitats. Environ Entomol. 2004;33:881–9. 37. Nagoshi RN, Meagher RL. Seasonal distribution of fall armyworm (Lepidoptera: Noctuidae) host strains in agricultural and turf grass habitats. Environ Entomol. 2004;33:881–9. 38. Pashley DP, Martin JA. Reproductive Incompatibility between host strains of the fall armyworm (Lepidoptera: Noctuidae). Ann Entomol Soc Am. 1987;80:731–3. 38. Pashley DP, Martin JA. Reproductive Incompatibility between host strains of the fall armyworm (Lepidoptera: Noctuidae). Ann Entomol Soc Am. 1987;80:731–3. 39. Patterson DT, Westbrook JK, Joyce RJV, Lingren PD, Rogasik J. Weeds, insects, and diseases. Clim Ch. 1999;43:711-727. 40. Pearce J, Ferrier S, Scotts D. An evaluation of the predictive performance of distributional models for flora and fauna in north-east New South Wales. J Env Manag. 2001;62:171–84. 40. Pearce J, Ferrier S, Scotts D. References An evaluation of the predictive performance of distributional models for flora and fauna in north-east New South Wales. J Env Manag. 2001;62:171–84. 41. Peterson AT. Uses and requirements of ecological niche models and related distributional models. Biodiv Inform. 2006;3. doi:10.17161/bi.v3i0.29. 42. Peterson AT, Papeş M, Carroll DS, Leirs H, Johnson KM. Mammal taxa constituting potential coevolved reservoirs of filoviruses. J Mamm. 2007;88:1544–54. 43. Phillips SJ, Anderson RP, Dudík M, Schapire RE, Blair ME. Opening the black box: an open-source release of Maxent. 2017;40:887–93. 44. Phillips SJ, Anderson RP, Schapire RE. Maximum entropy modeling of species geographic distributions. Ecol Model. 2006;190:231–59. 45. Phillips SJ, Dudík M, Schapire RE. A maximum entropy approach to species distribution modeling. In: Twenty-first international conference on Machine learning - ICML ’04. Banff, Alberta, Canada: ACM Press; 2004. p. 83. doi:10.1145/1015330.1015412. 45. Phillips SJ, Dudík M, Schapire RE. A maximum entropy approach to species distribution modeling. In Twenty-first international conference on Machine learning - ICML ’04. Banff, Alberta, Canada: ACM Press; 2004. p. 83. doi:10.1145/1015330.1015412. 46. Prasanna BM, Huesing JE, Eddy R, Peschke VM (eds). Fall Armyworm in Africa: A Guide for Integrated Pest Management, First Edition. Mexico, CDMX: CIMMYT. 2018. 46. Prasanna BM, Huesing JE, Eddy R, Peschke VM (eds). Fall Armyworm in Africa: A Guide for Integrated Pest Management, First Edition. Mexico, CDMX: CIMMYT. 2018. 47. Prowell DP, McMichael M, Silvain J-F. Multilocus genetic analysis of host use, introgression, and speciation in host strains of fall armyworm (Lepidoptera: Noctuidae). Ann Entomol Soc Am. 2004;97:1034–44. 48. R Development Core Team. R statistical software (R Foundation for statistical computing: Vienna, Austria). 2018. Page 13/23 Page 13/23 49. Ramirez-Cabral NYZ, Kumar L, Shabani F. Future climate scenarios project a decrease in the risk of fall armyworm outbreaks. J Agr Sci. 2017;155:1219–38. 50. Schöfl G, Heckel DG, Groot AT. Time-shifted reproductive behaviours among fall armyworm (Noctuidae: Spodoptera frugiperda) host strains: evidence for differing modes of inheritance. J Evol Biol. 2009;22:1447–59. 51. Sharanabasappa D, Kalleshwaraswamy CM, Asokan R, Mahadeva Swamy HM, Maruthi MS, Pavithra HB, Hegde K, Navi S, Prabhu ST, Goergen G. First report of the fall armyworm, Spodoptera frugiperda(J E Smith) (Lepidoptera: Noctuidae), an alien invasive pest on maize in India. Pest Manag Hort Ecosyst. 2018;24(l):23– 52. Shylesha AN, Jalali SK, Gupta A, Varshney R, Venkatesan T, Shetty P, Ojha R, Ganiger PC, Navik O, Subaharan K, Bakthavatsalam N, Ballal CR. References Studies on new invasive pest Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) and its natural enemies. J Biol Control. 2018;32:145–51. 53. Soria-Auza RW, Kessler M, Bach K, Barajas-Barbosa PM, Lehnert M, Herzog SK, et al. Impact of the quality of climate models for modelling species occurrences in countries with poor climatic documentation: a case study from Bolivia. Ecol Model. 2010;221:1221–9. N. A Review of the Biology of the Fall Armyworm. Fla Entomol. 1979;62:82. 54. Sparks AN. A Review of the Biology of the Fall Armyworm. Fla Entomo 54. Sparks AN. A Review of the Biology of the Fall Armyworm. Fla Entomol. 1979;62:82. 55. Wang R, Jiang C, Guo X, Chen D, You C, Zhang Y, et al. Potential distribution of Spodoptera frugiperda (J.E. Smith) in China and the major factors influencing distribution. Glob Ecol Conserv. 2020;21:e00865. 56. Westbrook J, Fleischer S, Jairam S, Meagher R, Nagoshi R. Multigenerational migration of fall armyworm, a pest insect. Ecosphere. 2019;10. doi:10.1002/ecs2.2919. 56. Westbrook J, Fleischer S, Jairam S, Meagher R, Nagoshi R. Multigenerational migration of fall armyworm, a pest insect. Ecosphere. 2019;10. doi:10.1002/ecs2.2919. 57. Westbrook JK, Nagoshi RN, Meagher RL, Fleischer SJ, Jairam S. Modeling seasonal migration of fall armyworm moths. Int J Biomet. 2016;60:255–67. 57. Westbrook JK, Nagoshi RN, Meagher RL, Fleischer SJ, Jairam S. Modeling seasonal migration of fall armyworm moths. Int J Biomet. 2016;60:255–67. 58. Elith J, Kearney M, Phillips S. The art of modelling range-shifting species. Meth Ecol 2010;1:330–42. 58. Elith J, Kearney M, Phillips S. The art of modelling range-shifting species. Meth Ecol 2010;1:330–42. Table 2 Description of bioclimatic zones of South Kivu (Mean ± SE) Tables vironmental variables used to model Spodoptera frugiperda (FAW) distribution in South Kivu Page 14/23 Page 14/23 Environmental and bioclimatic parameters Code Units Mean annual temperature  (* 10) bio1 °C Mean daytime temperature range (monthly average) (* 10) bio2 °C Isothermality (bio1/bio7) * 100 bio3 - Temperature seasonality (standard deviation * 100) bio4 °C Maximum temperature of the hottest month (* 10) bio5 °C Minimum temperature of the coolest month (* 10) bio6 °C Annual temperature range (bio5-bio6) (* 10) bio7 °C Mean temperature of the warmest quarter (* 10) bio10 °C Mean temperature of the coldest quarter (* 10) bio11 °C Annual rainfall bio12 mm Rainfall during the wettest month bio13 mm Rainfall during the driest month bio14 mm Rainfall seasonality bio15 mm Rainfall in the wettest quarter bio16 mm Rainfall in the driest quarter bio17 mm Longest dry season duration llds - Annual moisture index mi - Moisture index of the dry quarter miaq - Moisture index of the wet quarter mimq - Potential evapotranspiration pet mm Elevation dem m Table 2 Description of bioclimatic zones of South Kivu (Mean ± SE) Page 15/23 Variables Zone 1 Zone 2 Zone 3 Global bio1 160.82 ± 19.96 227.28 ± 16.12 220.09 ± 18.06 210.53 ± 30.83 bio2 95.55 ± 4.53 106.89 ± 2.10 105.46 ± 4.75 103.93 ± 5.85 bio3 792.26 ± 39.18 857.40 ± 17.18 790.86 ± 35.53 812.91 ± 44.31 bio4 3.931 ± 0.80 2.99 ± 0.09 3.63 ± 0.80 3.48 ± 0.75 bio5 219.70 ± 23.05 288.97 ± 16.19 285.48 ± 18.98 273.40 ± 33.02 bio6 98.69 ± 16.75 164.26 ± 16.41 152.09 ± 17.09 145.34 ± 29.30 bio7 121.00 ± 9.754 124.70 ± 4.00 133.39 ± 4.15 128.06 ± 7.72 bio10 164.05 ± 20.37 229.63 ± 16.14 224.03 ± 18.06 213.81 ± 30.84 bio11 155.27 ± 20.02 223.59 ± 16.35 215.82 ± 18.31 206.19 ± 31.47 bio12 1893.89 ± 149.49 1940.80 ± 147.15 1563.16 ± 167.94 1753.17 ± 239.61 bio13 248.36 ± 27.76 235.78 ± 24.11 198.67 ± 16.41 220.80 ± 30.48 bio14 17.38 ± 8.03 55.42 ± 13.17 21.59 ± 10.16 31.81 ± 19.79 bio15 80.28 ± 11.02 61.72 ± 6.24 63.03 ± 6.53 66.07 ± 10.41 bio16 668.48 ± 65.06 668.73 ± 62.81 549.53 ± 50.97 612.44 ± 83.07 bio17 89.43 ± 35.89 198.67 ± 34.19 93.86 ± 38.22 127.25 ± 61.75 dem 2197.31 ± 348.68 847.65 ± 283.62 1145.35 ± 326.32 1259.45 ± 582.61 llds 2.69 ± 0.81 1.26 ± 1.13 3.29 ± 0.59 2.51 ± 1.23 mi 147.95 ± 23.30 118.39 ± 8.86 98.43 ± 12.58 114.91 ± 23.68 miaq 29.30 ± 13.16 50.70 ± 8.95 24.02 ± 9.79 33.81 ± 15.77 mimq 212.43 ± 35.75 160.83 ± 13.39 140.35 ± 15.53 161.54 ± 34.09 pet 1295.55 ± 100.38 1640.62 ± 65.71 1595.30 ± 92.60 1549.86 ± 155.42 Total area (km2) 11411.20 17293.40 30389.80 59094.40 SE: standard error SE: standard error Figures Page 16/23 igure 1 Occurrence records of fall armyworm (Spodoptera frugiperda) in South Kivu, DRC. Each point represents maize field in which fall armyworm larvae were detected and collected. This figure was created by the uthors using ArcMap version 10.6 (https://desktop.arcgis.com/fr/arcmap/) Figure 1 Figure 1 Occurrence records of fall armyworm (Spodoptera frugiperda) in South Kivu, DRC. Each point represents a maize field in which fall armyworm larvae were detected and collected. This figure was created by the authors using ArcMap version 10.6 (https://desktop.arcgis.com/fr/arcmap/) Page 17/23 Page 17/23 Figure 2 Bioclimatic zones of South Kivu. The zones are indicated in different colors on the m created by the authors using ArcMap version 10.6 (https://desktop.arcgis.com/fr/ar Figure 2 Bioclimatic zones of South Kivu. The zones are indicated in different colors on the map. This figure was created by the authors using ArcMap version 10.6 (https://desktop.arcgis.com/fr/arcmap/) Page 18/23 Page 18/23 Figure 3 Receiver Operating Characteristic (ROC) curve and Area Under the Curve (AUC) value of MaxEnt modeling (100 runs) Figure 3 Receiver Operating Characteristic (ROC) curve and Area Under the Curve (AUC) value of MaxEnt modeling (100 runs) Receiver Operating Characteristic (ROC) curve and Area Under the Curve (AUC) value of MaxEnt modeling (100 runs) Page 19/23 Page 19/23 Figure 4 Distribution of suitable areas of fall armyworm (Spodoptera frugiperda) in South K Figure 4 Distribution of suitable areas of fall armyworm (Spodoptera frugiperda) in South Kivu, DRC. This figure was created by the authors using ArcMap version 10.6 (https://desktop.arcgis.com/fr/arcmap/) Page 20/23 Fi 5 Figure 5 Contribution (A) and Permutation importance (B) of variables used as predictors in the fall armyworm (Spodoptera frugiperda) MaxEnt model. bio1: mean annual temperature; bio12: annual rainfall; bio4: temperature seasonality; llds: longest dry season duration Contribution (A) and Permutation importance (B) of variables used as predictors in the fall armyworm (Spodoptera frugiperda) MaxEnt model. bio1: mean annual temperature; bio12: annual rainfall; bio4: temperature seasonality; llds: longest dry season duration Page 21/23 Figure 6 Jackknife test of variables’ contribution in modeling Spodoptera frugiperda habitat suitability distribution in South Kivu: (A) without variable (B) with the variable only. bio1: mean annual temperature; bio12: annual rainfall; bio4: temperature seasonality; llds: longest dry season duration Jackknife test of variables’ contribution in modeling Spodoptera frugiperda habitat suitability distribution in South Kivu: (A) without variable (B) with the variable only. bio1: mean annual temperature; bio12: annual rainfall; bio4: temperature seasonality; llds: longest dry season duration Jackknife test of variables’ contribution in modeling Spodoptera frugiperda habitat suitability distribution in South Kivu: (A) without variable (B) with the variable only. bio1: mean annual temperature; bio12: annual rainfall; bio4: temperature seasonality; llds: longest dry season duration Page 22/23 Figure 7 Figure 7 Figure 7 Responses of variables to fall armyworm (Spodoptera frugiperda) habitat suitability. These curves show how each environmental variable affects the MaxEnt prediction. They also show how the predicted probability of presence changes as each environmental variable is varied, keeping all other environmental variables at their average sample value (Left side) or a MaxEnt model created using only the corresponding variable (Right side) Page 23/23 Page 23/23

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Gene Editing of Microalgae: Scientific Progress and Regulatory Challenges in Europe

Biology

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Gene Editing of Microalgae: Scientific Progress and Regulatory Challenges in Europe Andrew Spicer 1,* and Attila Molnar 2 ID 1 Algenuity, Eden Laboratory, Bedfordshire MK43 9ND, UK 2 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK; amolnar@exceed ed ac uk Andrew Spicer 1,* and Attila Molnar 2 ID Andrew Spicer 1,* and Attila Molnar 2 ID 1 Algenuity, Eden Laboratory, Bedfordshire MK43 9ND, UK 2 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-1234-765773 Received: 22 December 2017; Accepted: 1 March 2018; Published: 6 March 2018 Andrew Spicer 1,* and Attila Molnar 2 ID 1 Algenuity, Eden Laboratory, Bedfordshire MK43 9ND, UK 2 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3BF, U [email protected] * Correspondence: [email protected]; Tel.: +44-1234-765773 Received: 22 December 2017; Accepted: 1 March 2018; Published: 6 March 2018 1 Algenuity, Eden Laboratory, Bedfordshire MK43 9ND, UK 2 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK; l @ d d k Received: 22 December 2017; Accepted: 1 March 2018; Published: 6 March 2018 Abstract: It is abundantly clear that the development of gene editing technologies, represents a potentially powerful force for good with regard to human and animal health and addressing the challenges we continue to face in a growing global population. This now includes the development of approaches to modify microalgal strains for potential improvements in productivity, robustness, harvestability, processability, nutritional composition, and application. The rapid emergence and ongoing developments in this area demand a timely review and revision of the current definitions and regulations around genetically modified organisms (GMOs), particularly within Europe. Current practices within the EU provide exemptions from the GMO directives for organisms, including crop plants and micro-organisms that are produced through chemical or UV/radiation mutagenesis. However, organisms generated through gene editing, including microalgae, where only genetic changes in native genes are made, remain currently under the GMO umbrella; they are, as such, excluded from practical and commercial opportunities in the EU. In this review, we will review the advances that are being made in the area of gene editing in microalgae and the impact of regulation on commercial advances in this area with consideration to the current regulatory framework as it relates to GMOs including GM microalgae in Europe. Keywords: CRISPR; transgenic; GMO; GMM; gene editing; Cpf1; DSB; NPBT; SDN Keywords: CRISPR; transgenic; GMO; GMM; gene editing; Cpf1; DSB; NPBT; SDN biology biology Biology 2018, 7, 21; doi:10.3390/biology7010021 Keywords: CRISPR; transgenic; GMO; GMM; gene editing; Cpf1; DSB; NPBT; SDN 2. Definitions of GMOs and GMMs and Exemptions in Europe European Union (EU) legislation (Article 2 of EU Directive 2001/18/EC) [2] defines a genetically modified organism (GMO) as “an organism, with the exception of human beings, i which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination.” Genetically Modified Microorganisms are often referred to as GMMs. Directive 2009/41/EC [3] defines microorganisms as “any microbiological entity, cellular or non-cellular, capable of replication or of transferring genetic material, including viruses, viroids, animal and plant cells in culture”. This would include all microalgae, encompassing both eukaryotic and prokaryotic (cyanobacteria) microalgae. As such, under this broad definition, many experimentally produced organisms fall under this umbrella. This includes the following organisms: 1. All organisms where a ‘transgene’ or more than one transgene encoded by DNA has been integrated stably into the genome (nuclear, mitochondrial, or other genome such as chloroplast) such that it is inherited by offspring or daughter cells—this includes cisgenes (gene is from the same species or a closely related species) and transgenes (a foreign gene from a completely unrelated species or a synthetic, artificially designed gene for a specific function). For the sake of this paper, the broad term ‘transgene’ will be inclusive for the above genes (i.e., cis and transgenes); 2. All organisms where an artificial chromosome has been introduced into the organism such that it is replicated alongside the natural chromosomes and stably inherited in offspring and/or daughter cells; 3. All organisms where a native gene has been stably mutated by non-natural, laboratory means—including the introduction of nucleotide substitutions, deletions, rearrangements, duplications, insertions, or inactivation by any one of the following methods: 3. All organisms where a native gene has been stably mutated by non-natural, laboratory means—including the introduction of nucleotide substitutions, deletions, rearrangements, duplications, insertions, or inactivation by any one of the following methods: • UV- or radiation-induced mutagenesis; • Chemical mutagenesis; • Gene editing by site-directed nucleases including Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas), Transcription • Gene editing by site-directed nucleases including Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas), Transcription Activator-Like Effector Nucleases TALENs), and Zinc Finger Nucleases (ZFNs) • Site-specific recombinases, integrases, or meganucleases. It might be surprising to the average reader to learn that organisms derived from chemical or UV/radiation mutagenesis in a laboratory would be broadly included under the GMO umbrella in Europe. 1. Introduction Most of the food or feed that has been consumed by humans, cattle, and pets comes from plants, animals, fungi, and even microorganisms grown, bred, and improved by mankind over thousands of years. Through the centuries, by using different breeding methods, we have managed to select the best-performing individuals and strains of crop plants, animals, fungi, etc., with enhanced growth rates and productivities, and improved nutritional composition and health. As a general rule, the improved organisms arose from naturally occurring variations in the genetic make-up of those plants, animals, fungi, etc., or from natural partial or full genome duplications or breeding (crosses) between closely related organisms. For the past 90 years, radiation-induced changes in genomes [1] have been used as a way to accelerate the development of improved organisms for exploitation by man. Since the 1970s, it has also become possible to modify the genetic make-up of living cells and organisms using biotechnology-based techniques, usually called genetic engineering. In general, genetically engineered crops or animals are produced through modification of the genetic material by introduction of a specific piece of DNA called a transgene (generally considered to be a gene derived from a different, unrelated species, such as a bacterial gene introduced into a higher plant, or even a synthetic gene) to give the organism a new property (e.g., resistance to a disease or a specific herbicide, improvement of food quality, or increased crop productivity). www.mdpi.com/journal/biology www.mdpi.com/journal/biology Biology 2018, 7, 21; doi:10.3390/biology7010021 Biology 2018, 7, 21 2 of 13 Organisms produced in this way are called “genetically modified organisms” (abbreviated commonly as GMOs). It follows that food and feed that contain or consist of such GMOs, or are produced from GMOs, would be “genetically modified (GM) food or feed”. Organisms produced in this way are called “genetically modified organisms” (abbreviated commonly as GMOs). It follows that food and feed that contain or consist of such GMOs, or are produced from GMOs, would be “genetically modified (GM) food or feed”. The development of modern molecular approaches to genetically improving organisms is necessitating a reconsideration of the definition and risk assessment process associated with the current GMO regulatory framework in Europe but also globally. In this review we will consider the current regulatory framework within which gene-edited algae still fall and also summarise and highlight advances in technology development with recommendations on steps that should be taken. 3. Risk Assessments: Relative Risks are Key The risk assessment debate is most developed around and within applications of GMOs in food and feed but specific regulations and risk assessment requirements also apply to application within other sectors including areas such as fuels, cosmetics, personal care, and household products. The European Food Standards Agency (EFSA) states that identification, characterisation, and handling of risk(s) associated with GMOs should follow a structured approach, consisting of three interconnected elements: risk assessment, risk management, and risk communication [4]. All three elements need to be considered and documented. Risk assessment is a scientific exercise. Principles for risk assessment of GMOs are described in Directive 2001/18, Annex II, supplemented by Commission Decision 2002/623/EC [2,5]. This is related to production or application of GMOs in a ‘deliberate release’ scenario such as outdoor cultivation or sale of a GMO or GMO-derived product for human or animal consumption. The alternative to ‘deliberate release’ is contained use. The GMO Regulations define contained use as 'any activity in which organisms are genetically modified or in which such organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which specific containment and other protective measures are used to limit their contact with the general public and the environment’. The contained use of genetically modified microorganisms is regulated by Directive 90/219/EEC [6], as amended by Directive 98/81/EC [7]. Most of the controversy and debate around the production and use of GMOs is limited to risk assessments associated with deliberate release. An extensive overview of risk assessment procedures is provided by the Scientific Committee of the EFSA [8]. The risk assessment involves generating, collecting, and assessing information on a given GMO in order to determine its potential impact on human and/or animal health and the environment compared with the nonmodified organism from which it is derived. To carry out the risk assessment, sufficient scientific and technical data must be available to arrive at qualitative and/or quantitative risk estimates. In general, a risk assessment includes a consideration of the parental organism including its associated biology, likelihood of spread in the environment and any barriers that may act to limit this spread, the nature of the genetic modification, and the presence of the GMO or GMM and its derivatives, including DNA, in the final food or feed product. 2. Definitions of GMOs and GMMs and Exemptions in Europe In practice, however, organisms produced through chemical or UV/radiation mutagenesis are considered to be excluded or given an exemption with regard to regulations that consider the risk assessments that need to be undertaken for controlled release or utilisation of such organisms. This exemption is made as organisms produced in this way generally have a long-standing safety record. Random mutation occurs spontaneously in nature and is the basis for the genetic variation we see within species. The laboratory-driven mutagenesis techniques of UV/radiation and chemical mutagenesis do not result in the introduction of any new genetic material into the organism. These methods effectively accelerate what would happen in nature over years or centuries; they 3 of 13 Biology 2018, 7, 21 create mutants, but no foreign gene is added. This is an important distinction that we will come back to with regard to a similar consideration that should be applied to gene-edited organisms where no foreign gene has been added to the genome. 3. Risk Assessments: Relative Risks are Key GMMs used for food and feed purposes can be differentiated on the basis of their use in (i) GMOs deliberately released into the environment, according to Directive 2001/18/EC [2], and used as food or feed or contained in food or feed; (ii) GMOs deliberately released into the environment, according to Directive 2001/18/EC [2], and used for the production of food or feed; (iii) GMOs used for the production of food or feed under ‘contained use’. For uses under (i) and (ii), a full risk assessment according to Regulation 1829/2003 [9] in combination with Directive 2001/18/EC is required and is covered by this guidance. With regard to uses as in (iii), i.e., GMOs used for food or feed production under containment, this guidance covers the assessment of the final product to be used as food or feed for placing in the market, while taking into account the characteristics of the GMO, but does not cover the production process as performed under containment. Regulation 1829/2003 specifically covers traceability of the GMO including the methods to be used. While most methods rely upon detection of the GMO using nucleic-acid-based approaches, some consider detection of phenotypic traits as well as protein [10]. Risk assessments associated with GMOs are related primarily to the risk of damage that the modified organism may produce to the environment, humans, or animals, including the risk of the ‘transgene’ spreading by gene transfer to other organisms or the risk of the GMO itself spreading as an aggressive or invasive organism outside of its primary site of production. Indeed, most of the risk assessment process considers the relative harm that could be caused either by the altered organism retaining a new undesirable or hazardous property that is attributed to the modified DNA or the Biology 2018, 7, 21 4 of 13 ‘transgenic’ DNA spreading to other organisms including microorganisms, higher plants, or even animals. Notable examples of transgenes where significant risk could be considered are transgenes encoding herbicide resistance, antibiotic resistance, or enhanced growth under natural or agricultural field conditions. Mutations produced by chemical or UV/radiation mutagenesis are random and often a cell or organism produced in this way contains multiple independent mutations at different sites within the genome. 3. Risk Assessments: Relative Risks are Key Due to the random nature of these mutations, it could be considered that any or all of these mutations could occur naturally; they are not specifically engineered—they are organisms (mutants) where mutagenesis (called classical or random mutagenesis since very long ago) has allowed the selection of desirable phenotypes by the breeder. As such, there is no transgene that would be the subject of a risk assessment, although one could argue that any of the mutated native genes could be associated with a potential or hypothetical—even if not directly measurable—risk with regard to acquisition of a new trait, including altered growth properties or relative resistance to a specific herbicide. A comparison of the relevant methods for genetic modification and the associated risk assessments and exemption status is summarised in Table 1. 5 of 13 Biology 2018, 7, 21 Table 1. What is a genetically modified organism (GMO)? Types of modifications in broad terms, the nature of the change to the genome, risk management considerations, exemptions that might be granted with regard to classification as GMO or not in the EU, and any rulings of non-GMO from outside Europe are noted. Type of Modification Change to Genome Risk Management Exemptions Granted (EU) Ruling of Non-GMO Outside Europe? Transgene Stably integrated foreign gene. Spread of foreign transgene or modified organism in the environment. Risk of harm of transgene product in food chain. NO NO Cisgene Stably integrated gene: from same species or closely related. Spread of modified gene or organism in the environment. Risk of harm of cisgene product in food chain. NO YES Artificial chromosome Stably inherited artificial chromosome. Spread of artificial chromosome, genes contained therein, or modified organism in the environment due to acquired trait. Risk of harm of transgene or cisgene products in food chain. NO NO Chemical-, UV-, or radiation-induced mutagenesis Single nucleotide changes, small deletions—usually many/organism— changes in native genes only. No foreign or artificial gene integrated. Changes only to native DNA but often multiple genetic hits in the genome. Genetic changes can change phenotype including relative growth rate and survivability. YES YES Recombinase- or integrase-driven change Partial deletions, inversions, duplications, rearrangements or insertions. Recombinase or integrase stably or transiently expressed. No foreign gene where recombinase or integrase transiently expressed: mods to native genes only. Risk of spread of stably integrated recombinase or integrase. Spread of modified organism. 4. Gene Editing: What Is It? Why Is It So Controversial? 4. Gene Editing: What Is It? Why Is It So Controversial? Gene editing refers to a group of techniques where specific genetic modifications are designed into the native genome of a given organism by application of one of a suite of approaches (see below) that permit the creation of a cell or entire organism with precise genetic modifications in native gene sequences without the need to integrate transgene DNA into the genome. The gene editing approaches considered herein rely upon directing a molecular tool to a specific selected site in the genome to create a double-strand break (DSB) in the DNA sequence. The cell re-joins cut DNA predominantly without reference to the original DNA sequence. This mechanism is error prone and can result in random mutations (nucleotide insertions or deletions) at the site of DNA repair. If the targeted DNA is within a gene that encodes a protein, this can generate mutations that prevent the production of a functional protein. Although at much lower frequency, precise DNA editing can also be induced if DNA is provided as a repair template via the homology-directed DNA repair pathways. Thus, the gene editing technology is ideal for producing novel genetic variants in a sequence-specific manner. All of these methods are grouped under a common set of approaches referred to as Site-Directed Nucleases (SDN) because they rely upon directing a specific DSB in the DNA, created by a nuclease, to a desired site within a complex genome. Sometimes, these methods are collectively called New Plant Breeding Techniques (NPBT) [13] when used to refer to crop plants, or Oligonucleotide Directed Nucleases (ODNs) when used in a more general context of selective mutagenesis, as compared with spontaneous or induced random mutagenesis—the main current technology for cultivar or strain improvements. The first approach that gained attention with regard to established gene editing was the application of protein domains called zinc fingers (DNA binding proteins) fused to nucleases [14] (DNA cleavage proteins), creating zinc finger nucleases or ZFNs. The zinc finger domains direct pairs of proteins to a specific DNA sequence, with the protein complex assembling at the desired site in the genome to effectively form a pair of molecular scissors to create a DSB at that site. 3. Risk Assessments: Relative Risks are Key NO YES Gene edit: transgene-driven change (stable or transient) Subtle mutation to native genes. Stable transgene-driven = conventional transgenic; transient transgene: (DNA) or RNA-driven = no integrated transgene. No foreign gene where gene editing nuclease is transiently expressed. Risk of spread of transgene or modified organism. Changes to native genes. NO YES [11,12] Gene edit: Ribonucleoprotein (RNP)-driven change Precise mutation to native genes only (some of target mutagenesis measurable but minimal). No foreign gene. Risk of spread of modified organism. More precise genetic changes than chemical, radiation, or UV mutagenesis. NO YES [11,12] Biology 2018, 7, 21 Biology 2018, 7, 21 6 of 13 4. Gene Editing: What Is It? Why Is It So Controversial? The next approach that increased specificity and precision of the DSB process was the discovery and application of proteins referred to as TALEs (Transcription Activator-Like Effectors) [15] fused to nuclease domains to create so-called TALENs, again to direct a set of molecular scissors to a specific genomic target sequence to create a precise genome change at that site. TALENs increased the capacity and opportunities to apply gene editing across diverse platforms including within commercial organisations, in the clinic, and in domesticated/herd animals [16]. The true step-change in gene editing arose, however, through the discovery of [17] and ultimate application of the CRISPR/Cas [18] system, in which a common endonuclease (Cas9 most usually, but other similarly acting proteins such as Cpf1 (also known as Cas12) have now been identified and successfully applied) is directed to a desired genomic DNA target through the use of a short, easy-to-engineer RNA sequence referred to as a guide sequence. CRISPR/Cas has made gene editing technology readily accessible to clinicians, researchers, and breeders. Indeed, it has already been applied successfully within a molecular medicine context in the clinic in humans [19–21], and modified fungi [22] and plants [23] created using CRISPR/Cas have now been approved for production for human consumption [24]. The advent of these advanced genome modification technologies has created a stir of activity globally within regulatory bodies that work within the various economies of the world to assess, rule, and oversee safe application of technology, particularly ruling on those technologies that introduce products into the human or animal food chain. This includes organisations such as EFSA, the US Department of Agriculture’s Animal and Plant Health Inspection Service (APHIS), the German Federal Office of Consumer Protection, and even the European Court of Justice. Indeed, a proposed regulatory framework has been put forward to consider the relative risk assessments that should be or could be applied to crops or organisms produced through gene editing [11]. Furthermore, the Norwegian Biotechnology Advisory Board has made recommendations along the lines that a new three-tier system could be envisaged where reporting, risk assessment, and regulation are applied at increasing levels [25]. 4. Gene Editing: What Is It? Why Is It So Controversial? Biology 2018, 7, 21 Biology 2018, 7, 21 7 of 13 Because of the subtle and specific nature of the mutations that can be created and the fact that a foreign transgene does not need to be integrated into the genome and is, therefore, not stably inherited, it has been strongly argued that such organisms produced by gene editing including plants, fungi, animals, and microorganisms should not be considered under the current broad umbrella definition of GMO [13,24]. Indeed, the ability to distinguish if an organism has been produced through gene editing or is, in fact, a naturally occurring variant organism carrying a desired mutation, or one produced by conventional mutagenesis methods, is essentially impossible given any technology. This goes against EU legislation that requires that GMOs be identifiable and traceable using suitable detection methods [9,10]: this directive stems from the assumption that was made when the GMO definition was originally put into action in the early 2000s, when it was widely assumed that all GMOs would result from integration of a transgene into a genome—a transgene that could then be detected using available detection methods. In short, we believe that the current EU definition of GMO and the directives associated around risk and relative safety of organisms produced through conventional transgenesis cannot be applied in the same way to organisms generated through gene editing where a modification in a native gene is the only introduced modification. We also believe that there is no rational reason why an organism created in this manner would be considered firmly under the GMO umbrella while an organism made by chemical or UV/radiation mutagenesis would be considered exempt from the GMO umbrella and its associated regulations. Indeed, all such variants are not fundamentally different from the spontaneous mutations that arise in nature in crop plants and animals all the time and have been the basis for traditional crop and animal selection and improvements. In many regards, gene editing could be seen as genetic engineering taken to the height of its possible precision. Indeed, using the CRISPR/Cas system, human genetic disease is already being treated in the clinic [24]. 4. Gene Editing: What Is It? Why Is It So Controversial? It is perhaps ironic then that while this technology has been approved for application in humans for molecular medicine [24], plants, fungi, or microorganisms created using the same level of precision and the same approach have, for the most part, not been ruled upon within Europe with regard to their use in food and feed production. Specifically, while some national bodies [26] have made recommendations that SDN-modified organisms where no foreign transgene has been integrated into the genome should not be considered GMOs, the EU has not made any specific ruling (see below). This includes an official EU position on any organism including micro or macroalgae or even bacteria (including cyanobacteria) produced through gene editing approaches. Recently, however, a formal opinion from an advocate general in the European Court of Justice suggests that crops and drugs created using powerful gene editing techniques such as CRISPR/Cas9 might not need to be regulated by the strict European Union rules that govern genetically modified organisms (GMOs) [27]. 5. Gene Editing in Algae: Current State of the Art Genetic modification techniques in algae are largely restricted to laboratory exercises and not to algae produced commercially for GMO-derived products sold in today’s market. Genetically modified algae mostly include a handful of strains, with most studies carried out on the laboratory model green microalga, Chlamydomonas reinhardtii. Overall, approximately 20 different microalgal species including cyanobacteria have been successfully genetically modified to date. A smaller number of macroalgae have also been genetically modified [28]. In the vast majority of cases, these modifications would fall under the broad and classical GMO view where a transgene has been integrated into the nuclear or, in some cases, the chloroplast genome of the given algal species. As such, all these micro and macroalgae would fall broadly under the GMO umbrella. There has been some debate as to whether or not GM algae fall under Genetically Modified Microorganisms (GMMs) or Genetically Modified Plants, or even both categories, and questions as to how this might impact any risk assessments that would need to be performed. EFSA reviewed this area and commented as follows: “According to our experience, the current European legislative framework for GMOs and for food/feed products of microbial origin 8 of 13 Biology 2018, 7, 21 (whether they are GM or not) would cover GM microalgae sufficiently.” Regardless of the category of organism, the regulations considered herein relate to GMOs broadly and would include both GMMs and GM Plants. With regard to production of GM algae beyond labscale, contained use—often double-contained or restricted-access greenhouse—is considered to be a safe working model at the current time, although outdoor trials have gone ahead and have been reported in the USA [29]. g g p It is only very recently that any progress has been made with gene editing in algae. The first reported successful application of a gene editing approach in a microalga was achieved in the marine diatom, Phaeodactylum tricornutum, by a team at Cellectis in France using TALENs [30]. More recently, application of CRISPR/Cas9 and CRISPR/Cpf1 have been successfully reported in Phaeodactylum tricornutum [31], Thalassiosira pseudonana [32], Chlamydomonas reinhardtii [33–37], and Nannochloropsis oceanica [38]. Different methods have been employed to edit the genome of alga species (Figure 1). Some use transgenes as means of stable expression of CRISPR/Cas. 5. Gene Editing in Algae: Current State of the Art Others introduce preassembled active ribonucleoprotein (RNP: enzyme + guideRNA) complexes by electroporation (Figure 1) or take a hybrid approach where Cas9 is produced from a transgene and other editing components are delivered directly to the nucleus [39]. While transgenes may be used to initially deliver the CRISPR reagents, they are not needed once the genome has been edited. Since they are located elsewhere in the genome, transgene-free, genome-engineered progenies can be recovered by mating/breeding. Intriguingly, direct delivery of CRISPR/Cas9 or CRISPR/Cpf1 RNPs completely bypasses transgenesis. Why is this important? It is important as it means that, not only is there no foreign transgene integrated into the resultant algal cell’s genome, but that a foreign transgene was never introduced to effect the desired change—this was, rather, effected by the use of an active protein–RNA complex that was introduced into the cell, and subsequently targeted the desired change to the nuclear genome [35,37]. The half-life of active RNPs is around 24–48 h, greatly reducing any off-target (unwanted) mutations that have already been shown to be very low or virtually nonexistent in plants [40]. Thus, RNPs are considered as safe and efficient molecules to induce the desired mutations without any trade-offs. The mutations induced by Cas9- or Cpf1-mediated DNA cleavage arise by the cell’s natural process for repairing DNA, which can occur under natural conditions, such as after DNA damage by sunlight. The resulting mutations are random insertions/deletions (Figure 1). In contrast, templated DNA repair—single-stranded DNA (ssDNA) ([37], see Figure 1) and double-stranded DNA (dsDNA)—can result in nucleotide-specific precise DNA replacement. However, bigger insertions such as tags or selection markers may be considered as transgenes depending on the size of integrated DNA. g y g p g g While it is early days in the application of gene editing approaches to algae, it is likely to become a standard procedure that can be applied to most algal species where DNA, RNA, or protein can be delivered into the cell. This will include microalgae, cyanobacteria, and macroalgae. The power of gene editing lies within the ability to create desired subtle changes to genomes. In microalgae, for instance, inactivation or changes in specific genes that impact the metabolic output including oil composition, protein composition, cell wall composition, motility, nutrient uptake or utilisation, photosynthetic efficiency, and numerous others would be expected to be transformational to the industry. 5. Gene Editing in Algae: Current State of the Art It can be noted that these genome changes would also be achievable through natural or accelerated (that is, induced) mutagenesis and selection for the desired phenotypes if achievable, or by using extensive bioprospecting and genomic sequencing to identify specific underlying genotypes. New gene editing and sequencing technologies greatly facilitate and accelerate the creation of such new variants through such selective mutagenesis, compared with traditional random—and more laborious and uncertain—methods. 9 of 13 Biology 2018, 7, 21 Figure 1. Gene editing approaches in microalgae. Relative frequency range of successful mutations reported from each approach are provided. RNP = ribonucleoprotein; gRNA = guideRNA; dsDNA = double-stranded DNA; ssDNA = single-stranded DNA. Transgene-based expression [31–33,38]; Direct delivery, RNPs made outside cell [34,35]; Direct delivery, RNPs and dsDNA made outside cell [36]; Direct delivery, RNPs and ssDNA made outside cell [37]. Figure 1. Gene editing approaches in microalgae. Relative frequency range of successful mutations reported from each approach are provided. RNP = ribonucleoprotein; gRNA = guideRNA; dsDNA = double-stranded DNA; ssDNA = single-stranded DNA. Transgene-based expression [31–33,38]; Direct delivery, RNPs made outside cell [34,35]; Direct delivery, RNPs and dsDNA made outside cell [36]; Direct delivery, RNPs and ssDNA made outside cell [37]. The potential positive impact of the application of these strain engineering approaches has been highlighted in the final report of the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium, a $48.6 million Department Of Energy (DOE) and private-investor-funded three-year project in the USA [41]. This closeout report concluded that the first primary area of cost reduction in a push towards sustainable and scalable algal commodity products was in the area of new strain development, and that when naturally bioprospected microalgal strains are “combined with genetically modified (GMO) versions of the strain, the cost of algal ‘biocrude’ would be reduced by 85%”. The majority of the genetic targets identified through this body of work were native genes with mutations, therefore made feasible by gene editing. 6. Council- and Country-Specific Rulings and Recommendations What is happening at the moment within and outside of the EU regarding organisms created using gene editing? In the USA, the US Department of Agriculture (USDA) has ruled that the regulations should relate to the product or the organism itself rather than the process. It is tending to rule in favour of gene-edited plants and organisms with regard to approval for growth or entry into the food chain for animals, fungi and plants, or clinical practice for humans. This is the case for a number of other countries outside of Europe, where many are now moving towards a case-by-case consideration process [12]. This is in contrast to the EU and the UK, where current policy and GMO guidelines continue to include plants, animals, fungi, and microorganisms modified by gene editing under the GMO umbrella. It is not that surprising to note, then, that the majority of the commercial and clinical applications or developments of gene editing technologies are happening outside of the EU. This is in spite of the fact that the majority of the initial and even recent innovations around gene editing are credited to groups based in the EU. This includes TALENs and CRISPR/Cas. Thus, economic development in this area is stalled within the EU across all markets and sectors. Companies, in general, are not investing in research and development around this area in the EU [43]. How important is the current EU non-ruling in this area? There are current trade and political talks going on between the USA and EU, the results of which could have a substantial impact on trading relationships with regard to food and feed entering the EU marketplace. Economically, the impact of these rulings should not be understated. Several EU countries have proceeded ahead of any official EU or ECHJ (European Court of Human Justice) decision and have made rulings in favour of a non-GMO label being applied to gene-edited crops. These countries include Sweden and Finland, with the Netherlands and Germany also approaching a decision in favour of a non-GMO label. All these countries that are moving early with regard to making national recommendations have stated that they would defer to EU rulings if and when that is made. Acknowledgments: We thank members of the European Algal Biomass Association Steering Committee and Scientific Advisory Committee for review of the content of this article. Attila Molnar is a Chancellor’s Fellow at the University of Edinburgh. Alga genome-editing research in the Molnar lab was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) PHYCONET Proof of Concept Fund, Grant PHYCPoC-31. 5. Gene Editing in Algae: Current State of the Art The second area that was identified where significant improvements could be made in the economics of the overall process was in the area of cultivation—specifically, the “development of a new open pond cultivation system, the Aquaculture Raceway Integrated Design (ARID), which uses little energy, extends the growing period, improves productivity, and provides a 16% cost reduction.” Hundreds of millions of dollars and euros have been invested over the past 30 years in an attempt to move microalgal platform technologies towards economic and sustainable scalable models to impact society and the environment as we move towards solving the challenges of an increasing population and reducing resources. For microalgal commodity products, including food and feed but also fuels and fuel additives, low cost, outdoor production, and strain improvements established by approaches including non-GMO directed evolution and gene editing of specific target genes are likely to be key solutions that will be applied in an additive or collective manner. Unlike niche-focused industries, the algae industry is a global market with broad impact. The algae industry has experienced high levels of competition due to profitability struggles, commodisation, and high exit costs. The emerging algae industry is still, however, an opportunity, with large potential fueled by drivers of increasing population, demand for fuel and food, and the 10 of 13 10 of 13 Biology 2018, 7, 21 pressing need for sustainable solutions to address climate change. Transparency Market Research [42] valued the 2015 global algae biomass market at $608 million with projections of 7.39% Current Annual Growth Rate (CAGR) to $1.14 billion in 2024 and total algae production at 27,552 tonnes by 2024; the global algal biomass production capacity underpins global markets of $1.38 billion and $616 million for algae oils and proteins, respectively, with CAGRs of 4.3% and 6.27% projected by 2025 and 2022. Despite the current size, growth, and market potential, the algae industry is underachieving and should be hitting market sizes and production levels of at least an order of magnitude higher. g p g g Gene editing approaches will be part of the solution as described above in increasing profitability via improved productivity of strains and improvements in harvestability, processability, and other desirable traits. We caution that the precautionary principle, sometimes heavily applied within regulatory frameworks to novel technologies, can stifle innovation and commercial application if not weighed appropriately against benefits and scientific knowledge. References 1. Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the Deliberate Release into the Environment of Genetically Modified Organisms and Repealing Council Directive 90/220/EEC. Official Journal of the European Communities. Available online: http://eur-lex.europa. eu/legal-content/EN/TXT/?uri=celex:32001L0018 (accessed on 16 February 2018). 1. Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the Deliberate Release into the Environment of Genetically Modified Organisms and Repealing Council Directive 90/220/EEC. Official Journal of the European Communities. Available online: http://eur-lex.europa. eu/legal-content/EN/TXT/?uri=celex:32001L0018 (accessed on 16 February 2018). g y 2. Broad, W.J. Useful Mutants Bred with Radiation. Available online: http://www.nytimes.com/2007/08/28/ science/28crop.html (accessed on 19 December 2017). 2. Broad, W.J. Useful Mutants Bred with Radiation. Available online: http://www.nytimes.com/2007/08/28/ science/28crop.html (accessed on 19 December 2017). 3. Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the Contained Use of Genetically Modified Micro-Organisms. Official Journal of the European Union. Available online: http: //eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex%3A32009L0041 (accessed on 16 February 2018). 3. Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the Contained Use of Genetically Modified Micro-Organisms. Official Journal of the European Union. Available online: http: //eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex%3A32009L0041 (accessed on 16 February 2018). 4. EFSA Panel on Genetically Modified Organisms (GMO). Scientific Opinion on Guidance on the risk assessment of genetically modified microorganisms and their derived food and feed products. EFSA J. 2011, 9, 54. [CrossRef] 4. EFSA Panel on Genetically Modified Organisms (GMO). Scientific Opinion on Guidance on the risk assessment of genetically modified microorganisms and their derived food and feed products. EFSA J. 2011, 9, 54. [CrossRef] 5. Directive 2002/623/EC, Commission Decision of 24 July 2002 Establishing Guidance Notes supplementing Annex II to Directive 2001/18/EC of the European Parliament and of the Council on the deliberate release into the Environment of Genetically Modified Organisms and Repealing Council Directive 90/220/EEC Official Journal of the European Communities. Available online: http://eur-lex.europa.eu/legal-content/ EN/TXT/?uri=CELEX:22007D0127 (accessed on 16 February 2018). 5. Directive 2002/623/EC, Commission Decision of 24 July 2002 Establishing Guidance Notes supplementing Annex II to Directive 2001/18/EC of the European Parliament and of the Council on the deliberate release into the Environment of Genetically Modified Organisms and Repealing Council Directive 90/220/EEC Official Journal of the European Communities. Available online: http://eur-lex.europa.eu/legal-content/ EN/TXT/?uri=CELEX:22007D0127 (accessed on 16 February 2018). 6. 6. Council- and Country-Specific Rulings and Recommendations One possibility is a total redefinition of the concept of a GMO and the risks and regulations associated therein, such as a definition and risk assessment process that work together on a product or case-by-case basis, considering the pros, cons, and risks, and following a responsible innovation evaluation process [44]. We believe that there is great practical and scientific necessity and a time imperative for moving expeditiously, as both scientific and commercial advances do not allow further hesitation. We would argue strongly along with many others that gene-edited organisms where subtle changes in native genes have been made, where no transgene DNA has been integrated, and particularly where no foreign transgene has been introduced into the cell, should be considered outside the currently restrictive and outdated GMO regulatory umbrella. Acknowledgments: We thank members of the European Algal Biomass Association Steering Committee and Scientific Advisory Committee for review of the content of this article. Attila Molnar is a Chancellor’s Fellow at the University of Edinburgh. Alga genome-editing research in the Molnar lab was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) PHYCONET Proof of Concept Fund, Grant PHYCPoC-31. 11 of 13 Biology 2018, 7, 21 Author Contributions: Andrew Spicer was responsible for the core text of the proposal including consideration of regulation and regulatory process. Attila Molnar contributed to the microalgal genome editing section and produced Figure 1. Conflicts of Interest: The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to publish the results. References Directive 90/219/EEC Council Directive 90/219/EEC of 23 April 1990 on the Contained Use of Genetically Modified Micro-Organisms. Official Journal of the European Communities. Available online: http://eur-lex. europa.eu/legal-content/EN/TXT/?uri=CELEX:31990L0219 (accessed on 16 February 2018). 7. Council DIRECTIVE 98/81/EC of 26 October 1998 amending Directive 90/219/EEC on the Contained use of Genetically Modified Micro-Organisms. Official Journal of the European Communities. Available online: http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:31998L0081 (accessed on 16 February 2018). 8. EFSA Scientific Committee. Scientific Opinion on Risk Assessment Terminology. EFSA J. 2012, 10, 2664. [CrossRef] 7. Council DIRECTIVE 98/81/EC of 26 October 1998 amending Directive 90/219/EEC on the Contained use of Genetically Modified Micro-Organisms. Official Journal of the European Communities. Available online: http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:31998L0081 (accessed on 16 February 2018). y g J p http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:31998L0081 (accessed on 16 February 2018). 8. EFSA Scientific Committee. Scientific Opinion on Risk Assessment Terminology. EFSA J. 2012, 10, 2664. [CrossRef] 8. EFSA Scientific Committee. 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Reduction of oxidative-nitrosative stress underlies anticataract effect of topically applied tocotrienol in streptozotocin-induced diabetic rats

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RESEARCH ARTICLE Nurul Alimah Abdul Nasir1*, Renu Agarwal1, Siti Hamimah Sheikh Abdul Kadir1, Sushil Vasudevan1, Minaketan Tripathy2, Igor Iezhitsa1,3, Aqil Mohammad Daher4, Mohd Ikraam Ibrahim1, Nafeeza Mohd Ismail1 Nurul Alimah Abdul Nasir1*, Renu Agarwal1, Siti Hamimah Sheikh Abdul Kadir1, Sushil Vasudevan1, Minaketan Tripathy2, Igor Iezhitsa1,3, Aqil Mohammad Daher4, Mohd Ikraam Ibrahim1, Nafeeza Mohd Ismail1 Nurul Alimah Abdul Nasir1*, Renu Agarwal1, Siti Hamimah Sheikh Abdul Kadir1, Sushil Vasudevan1, Minaketan Tripathy2, Igor Iezhitsa1,3, Aqil Mohammad Daher4, Mohd Ikraam Ibrahim1, Nafeeza Mohd Ismail1 1 Center for Neuroscience Research, Faculty of Medicine, Universiti Teknologi MARA Sungai Buloh Campus, Sungai Buloh, Selangor, Malaysia, 2 Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, Puncak Alam, Selangor, Malaysia, 3 Research Institute of Pharmacology, Volgograd State Medical University, Volgograd, Russia, 4 Department of Community Medicine, Faculty of Medicine and Defence Health, National Defence University of Malaysia, Sungai Besi Camp, Kuala Lumpur, Malaysia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * [email protected] OPEN ACCESS Cataract, a leading cause of blindness, is of special concern in diabetics as it occurs at ear- lier onset. Polyol accumulation and increased oxidative-nitrosative stress in cataractogen- esis are associated with NFκB activation, iNOS expression, ATP depletion, loss of ATPase functions, calpain activation and proteolysis of soluble to insoluble proteins. Tocotrienol was previously shown to reduce lens oxidative stress and inhibit cataractogenesis in galactose- fed rats. In current study, we investigated anticataract effects of topical tocotrienol and pos- sible mechanisms involved in streptozotocin-induced diabetic rats. Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Diabetic rats were treated with vehicle (DV) or tocotrienol (DT). A third group consists of normal, non-diabetic rats were treated with vehicle (NV). All treatments were given topically, bilaterally, twice daily for 8 weeks with weekly slit lamp monitoring. Subsequently, rats were euthanized and lenses were subjected to estimation of polyol accumulation, oxidative-nitrosative stress, NFκB acti- vation, iNOS expression, ATP levels, ATPase activities, calpain activity and total protein lev- els. Cataract progression was delayed from the fifth week onwards in DT with lower mean of cataract stages compared to DV group (p<0.01) despite persistent hyperglycemia. Reduced cataractogenesis in DT group was accompanied with lower aldose reductase activity and sorbitol level compared to DV group (p<0.01). DT group also showed reduced NFκB activa- tion, lower iNOS expression and reduced oxidative-nitrosative stress compared to DV group. Lenticular ATP and ATPase and calpain 2 activities in DT group were restored to nor- mal. Consequently, soluble to insoluble protein ratio in DT group was higher compared to DV (p<0.05). In conclusion, preventive effect of topical tocotrienol on development of cata- ract in STZ-induced diabetic rats could be attributed to reduced lens aldose reductase activ- ity, polyol levels and oxidative-nitrosative stress. These effects of tocotrienol invlove Citation: Abdul Nasir NA, Agarwal R, Sheikh Abdul Kadir SH, Vasudevan S, Tripathy M, Iezhitsa I, et al. (2017) Reduction of oxidative-nitrosative stress underlies anticataract effect of topically applied tocotrienol in streptozotocin-induced diabetic rats. PLoS ONE 12(3): e0174542. https://doi.org/ 10.1371/journal.pone.0174542 Introduction Cataracts are one of the common causes of visual impairment in diabetic subjects [1, 2]. In patients with diabetes, cataract has an early onset and develops 2 to 5 times more frequently [3–5]. Development of cataractous opacities in diabetics is a consequence of alterations in sev- eral metabolic pathways due to long standing hyperglycemia that culminate into increased lens oxidative and nitrosative stress and impaired functions of enzymes that maintain lens water and electrolyte homeostasis [6, 7]. Among the hyperglycemia-triggered metabolic changes, polyol pathway in which aldose reductase (AR) is the first enzyme involved, is widely investigated [8, 9]. AR converts glucose into sorbitol, a polyol, and utilizes NADPH. Highly active polyol pathway depletes NADPH, which is also required as cofactor for synthesis of reduced glutathione (GSH), hence reducing GSH synthesis [10]. Additionally, non-enzymatic glycation of antioxidant enzymes impairs their function and further contributes to ROS generation and oxidative stress. Increased avail- ability of ROS promotes its reaction with nitric oxide (NO) to form peroxynitrite (ONOO-), a potent reactive nitrogen species. This reaction occurs almost five times faster compared to the neutralization rate of superoxide radicals by superoxide dismutase (SOD), hence favoring ONOO- formation [11]. In lenticular cells, excessive NO formation results from increased inducible nitric oxide synthase (iNOS) expression [12], secondary to activation of nuclear fac- tor kappa B (NFκB) [13, 14]. ONOO- also causes sustained NFκB activation by favouring its release from its complex with inhibitory kappa B (IκB) [15]. High level of NO associated with increased iNOS expression have been observed in cataractous lenses [16,17]. Increased oxidative stress also causes cellular ATP depletion due to mitochondrial dysfunc- tion [18–21]. Cellular ATP loss and non-enzymatic glycation result in impaired function of ATPases such as Na+ K+ ATPase and Ca2+ ATPase [22,23]. Na+ K+ ATPase regulates sodium, potassium and water homeostasis in the lens, and its reduced functions lead to intracellular accumulation of sodium and water [24]. Similarly, impaired functions of plasma membrane Ca2+ ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), which regulate the intracellular calcium homeostasis [25], lead to high intracellular calcium and extremely high calcium level has been detected in cataractous lenses compared to normal lenses [26]. High intracellular calcium activates the calcium-dependent cysteine protease, cal- pain, which was shown to promote crystallin proteolysis, leading to opacification of the lens [27–29]. Editor: Reza Yousefi, Shiraz University, ISLAMIC REPUBLIC OF IRAN Editor: Reza Yousefi, Shiraz University, ISLAMIC REPUBLIC OF IRAN Received: September 4, 2016 Accepted: March 10, 2017 Published: March 28, 2017 Copyright: © 2017 Abdul Nasir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by Ministry of Higher Education, Government of Malaysia, under the grant no 600-RMI/RAGS 5/3 (39/2014). Competing interests: The authors have declared that no competing interests exist. Copyright: © 2017 Abdul Nasir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by Ministry of Higher Education, Government of Malaysia, under the grant no 600-RMI/RAGS 5/3 (39/2014). Competing interests: The authors have declared that no competing interests exist. 1 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 Anticataract effect of tocotrienol in streptozotocin-induced rats reduced NFκB activation, lower iNOS expression, restoration of ATP level, ATPase activi- ties, calpain activity and lens protein levels. reduced NFκB activation, lower iNOS expression, restoration of ATP level, ATPase activi- ties, calpain activity and lens protein levels. reduced NFκB activation, lower iNOS expression, restoration of ATP level, ATPase activi- ties, calpain activity and lens protein levels. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 Study design A pilot study was initially performed to evaluate effect of vehicle on the lenticular transparency in STZ- induced diabetic rats. The vehicle later was used to formulate tocotreinol microemul- sion for further studies. Animals were divided into three groups (n = 6 per group). Group 1 received sodium citrate buffer intraperitoneally (NP) whereas groups 2 (SP) and 3 received intraperitoneal STZ. Post 48 hours of STZ injection, group 3 (VP) was treated with a single 10 μl drop of vehicle topically, bilaterally, twice daily for 8 weeks. Anterior segment imaging was done weekly to assess the lenticular changes. Blood glucose and body weight were also monitored weekly. For the main experimental study, animals were divided into three groups (n = 39; 78 eyes per group). Group 1 consisted of rats that received sodium citrate buffer intraperitoneally. These rats were treated with vehicle (NV). Animals in groups 2 and 3 received intraperitoneal STZ and were treated with vehicle (DV) and 0.03% microemulsion of tocotrienol (DT), respec- tively. All treatments started 48 hours postintraperitoneal injections and were given topically in a volume of 10 μl, bilaterally, twice daily for 8 weeks. The choice of 0.03% concentration of tocotrienol was based on our previous study [31]. Anterior segment imaging was done once before intraperitoneal injections as a baseline and, subsequently, weekly post-STZ/ sodium citrate buffer injection. Blood glucose level and body weight were recorded weekly during the experimental period. After eight weeks of treat- ment, the animals were sacrificed with overdose of intraperitoneal ketamine (250 mg/kg) and xylazine (50 mg/kg). The lenses were carefully dissected out and stored at -80˚C until further analysis. The lenses were processed for estimation of lens polyol contents, oxidative-nitrosative stress, NFκB signaling pathway, ATP contents and ATPase activities, calpain 2 activity and proteins levels. Generally, each lens was homogenized in 1 ml of 50 mM cold phosphate buff- ered saline (PBS, pH 7.4, with 1 mM EDTA), unless stated otherwise. The homogenate was centrifuged at 890 g for 15 min. Supernatant was separated and used for estimation of the bio- chemical parameters. All estimates were done in duplicate. Animals Animal handling and all procedures were performed in line with ARVO statement for the use of animals in ophthalmic and vision research as well as local institutional ethical guidelines by Animal Care & Use Committee (ACUC), Faculty of Medicine of Universiti Teknologi MARA under approval number ACUC-9/13. Male Sprague-Dawley rats weighing 150–200 g were obtained from Laboratory Animal Care Unit (LACU) of Universiti Teknologi MARA and maintained under standard laboratory conditions at 12 hours light/dark cycle. Animals were caged individually and given food and water ad libitum. All animals were subjected to systemic and ophthalmic examination and those found normal were included in the study. Since dia- betic animals had polyuria, bedding was changed at least twice a day to ensure cleanliness and hence minimize the risk of infection. All animals were monitored daily for their wellbeing. Introduction Crystallins are the proteins essential for lens transparency and change in their struc- ture leads to opacification of the lens [30]. Increased oxidative stress also causes cellular ATP depletion due to mitochondrial dysfunc- tion [18–21]. Cellular ATP loss and non-enzymatic glycation result in impaired function of ATPases such as Na+ K+ ATPase and Ca2+ ATPase [22,23]. Na+ K+ ATPase regulates sodium, potassium and water homeostasis in the lens, and its reduced functions lead to intracellular accumulation of sodium and water [24]. Similarly, impaired functions of plasma membrane Ca2+ ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), which regulate the intracellular calcium homeostasis [25], lead to high intracellular calcium and extremely high calcium level has been detected in cataractous lenses compared to normal lenses [26] High intracellular calcium activates the calcium dependent cysteine protease cal Ca2+ ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), which regulate the intracellular calcium homeostasis [25], lead to high intracellular calcium and extremely high calcium level has been detected in cataractous lenses compared to normal lenses [26]. High intracellular calcium activates the calcium-dependent cysteine protease, cal- pain, which was shown to promote crystallin proteolysis, leading to opacification of the lens [27–29]. Crystallins are the proteins essential for lens transparency and change in their struc- ture leads to opacification of the lens [30]. Our previous studies have shown that topical application of the microemulsion of tocotrie- nol, a vitamin E analog, delays the onset and progression of galactose-induced cataract in rats and the maximum anticataract efficacy was observed at a concentration of 0.03%. Further- more, this anticataract effect was associated with reduced lenticular oxidative and nitrosative stress [31]. However, the mechanisms underlying these effects of tocotrienol remain unclear. Hence, in this study we used streptozotocin (STZ)-induced rat model of diabetes, which is a closer representation of human diabetic cataract, to investigate the effects of topical application of tocotrienol on lenticular polyol pathway, NFκB activity, expression of iNOS, oxidative stress PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 2 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats levels, ATP, ATPases, calpain 2 activities and protein levels. We correlated these effects of toco- trienol with changes in lenticular oxidative-nitrosative stress and progression of cataract. Anterior segment imaging Anterior segment imaging was done using Hawkeye Portable Slit Lamp (Optotek Medical, Ljubljana, SI) equipped with digital camera (Pentax Optio, S60, Denver, CO). Tropicamide 1% (Alcon Laboratories, Inc., Fort Worth, TX)) was topically applied 1 minute prior to imaging for mydriasis. Animals were lightly restrained with hand to get a still lens image. Lenticular changes were graded as described by Suryanarayana et al. [32] with slight modification. Accordingly, changes were categorized into 4 stages: Stage 0—normal lenses; Stage 1 –minimal opacity at the centre of lens; Stage 2 –patchy appearance of opacity both in the centre and periphery of lens; Stage 3—uniform opalescence all over the lens; Stage 4 –mature cataract with nuclear opacity (Fig 1). The grading of cataractous changes was done independently by three observers that were unaware of the grouping of animals. Quantification of lens polyol contents and AR activity Extent of polyol accumulation in lenses was estimated by measuring lens D-sorbitol concen- tration using Colorimetric Assay Kit (Biovision, CA, USA) and AR activity using sandwich- ELISA kit (Elabscience, Wuhan, China) as per manufacturer’s instructions. Microemulsion formulation of tocotrienol Microemulsion formulation of tocotrienol was prepared as described previously by Nasir et al [31]. Briefly, Kolliphor P188 (Sigma Aldrich, St. Louis, MO) was added into double distilled PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 3 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats water to create an aqueous phase. Annatto tocotrienol, which contained 90% of δ-tocotrienol, 10% of γ-tocotrienol and no tocopherol, was a gift from American River Nutrition, Inc. (Had- ley, MA). Tocotrienol was added into Miglyol 812 (AXO Industry, Wavre, BE) to create an oily phase. The oily phase was then added into the aqueous phase under moderate agitation. Subsequently, the particle size was reduced using ultrasound sonicator (Fisher Scientific, FB120, Hampton, NH) for 40 minutes at the setting of 80% amplitude with cycles of 50 sec- onds on and 20 seconds off. After sonication, sorbitol and disodium edetate (EDTA, 0.1%) (Fisher Chemical, Hampton, NH) were added as isotonizing agent and stabilizer respectively. Vehicle microemulsion was formulated as above without the addition of tocotrienol in the oily phase. Induction of diabetes with STZ Animals were fasted overnight and then were given intraperitoneal injection of 65 mg/kg STZ in sodium citrate buffer (10 mmol/L, pH 4.5). Forty-eight hours post-STZ injection, blood was collected from tail vein for determination of blood glucose level using Accu Chek Performa glucometer (Roche Diagnostic, Basel, CH). Animals with blood glucose level more than 20 mmol/L were included for further study. Control rats were similarly injected with sodium cit- rate buffer that was used as vehicle for STZ. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 Estimation of the lenticular NFκB expression and activation Expression of NFκB was first visualized by immunohistochemistry (IHC). For quantitative estimation of the extent of NFκB activation, we measured cytoplasmic levels of phosphorylated IκB-α (pIκB-α) in the cytoplasmic extract and NFκB p65 in the nuclear extract. For immunohistochemistry, lenses were directly immersed in 10% buffered formalin after washing with PBS and then were processed for paraffin embedding. Subsequently, eyes were sectioned at a thickness of 4 μm with rotary microtome. Tissue sections were deparaffinized with xylene and alcohol and then antigen retrieval was done by heating the sections in 10 mM citrate buffer (pH 6.0) for 8 min at 95˚C. Staining was done using Pierce1 Peroxidase Detec- tion Kit (Pierce Biotechnology, Rockford, IL, USA). After antigen retrieval, tissues were incu- bated with hydrogen peroxide for 30 minutes and washed. Then, non-specific binding of PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 4 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats Fig 1. Retroillumination anterior segment photographs showing progression of cataract from stage 0 to stage 4 in STZ-induced diabetic rats. A: Stage 0. B: Stage 1. C: Stage 2. D: Stage 3. E: Stage 4. https://doi.org/10.1371/journal.pone.0174542.g001 Fig 1. Retroillumination anterior segment photographs showing progression of cataract from stage 0 to stage 4 in STZ-induced diabetic rats. A: Stage 0. B: Stage 1. C: Stage 2. D: Stage 3. E: Stage 4. https://doi.org/10.1371/journal.pone.0174542.g001 antibody was blocked using blocking buffer for 30 minutes and incubation was done with pri- mary antibody (NFκB p65 rabbit polyclonal antibody, 1:100, Pierce Biotechnology, Rockford, IL, USA) for 30 minutes. After washing, tissue sections were incubated with horseradish per- oxidase (HRP) conjugated secondary antibody (Ready-to-Use Goat anti rabbit/mouse anti- body, Dako, Glostrup, Denmark) for another 30 minutes. The immunostaining was performed using 3,3’-diaminobenzidine (DAB)/ metal and peroxide mixture for 10 minutes. Tissue sections were then counterstained with hematoxylin for 2 minutes, dehydrated and mounted. All washes in this procedure were done three times for 3 minutes each with TBS mixed with 10% Tween 20 (TBST), whereas antibodies were diluted in blocking buffer. For negative controls, tissue sections were incubated with TBST. antibody was blocked using blocking buffer for 30 minutes and incubation was done with pri- mary antibody (NFκB p65 rabbit polyclonal antibody, 1:100, Pierce Biotechnology, Rockford, IL, USA) for 30 minutes. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 Estimation of iNOS expression Quantitative estimation of iNOS protein expression was done using commercially available sandwich-ELISA kit (Elabscience, Wuhan, China) containing wells pre-coated with antibody specific to iNOS, as per manufacturer’s instructions. All estimations were done in duplicate. In addition, gene expression for iNOS mRNA was assessed using qPCR. Lens tissue was placed in RNAlater Stabilization Solution directly after dissection to stabi- lize and protect cellular RNA. RNA extraction and purification was done using GeneJET RNA purification kit (Thermo Scientific Inc., Rockford, IL). The extracted RNA was then cleaned up to remove any genomic DNA present using RapidOut DNA Removal Kit (Thermo Scien- tific Inc., Rockford, IL). RNA concentration was determined using Nanodrop Spectrophotom- eter 1000 (Thermo Scientific Inc., Rockford, IL), whereas, RNA quality was assessed using Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA) based on the RNA integ- rity number (RIN) read by Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Inc., Santa Clara, CA). Then, cDNA synthesis was performed using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific Inc., Rockford, IL). Briefly, 1μg RNA was added to 5X Reac- tion Mix (contained reaction buffer, dNTPs, oligo (dT)18 and random hexamer primers), reverse transcriptase, RNase inhibitor and nuclease free water to make a solution volume up to 20 μl. The solution was gently mixed and centrifuged for 1 minute and then incubated at 25˚C for 10 minutes, followed by 50˚C for 15 minutes and lastly at 85˚C for 5 minutes. y y iNOS gene expression was measured in relation to expression of two reference genes; glyc- eraldehyde- 3-phosphate dehydrogenase (GADPH) and β- actin. The primers used were; 50- CACGGCAAGTTCAACGGCACAG-30 and 50-ACGCCAGTAGACTCCACGACAT-30 for GADPH, 50-ACTCTTCCAGCCTTCCTTC-30 and 50-ATCTCCTTCTGCATCCTGTC-30 for β- actin, 50- CTTGGAGCGAGTTGTGGATTGT-30 and 50-GTAGTGATGTCCAGGAAGTAGGT-30 for iNOS. qPCR reaction was performed with Luminaris Color HiGreen qPCR Master Mix (Thermo Sci- entific., Inc, Rockford, IL) using CFX96 Real Time System (Bio-Rad, Hercules, CA, USA). The following PCR cycling conditions were used for both GADPH and β- actin primer: 120 sec- onds of uracil-DNA glycosylase (UDG) pretreatment at 50˚C, followed by 180 seconds of ini- tial denaturation at 95˚C, followed by 50 cycles of 30 seconds at 95˚C, 30 seconds at 60˚C. Whereas, for iNOS, the following cycling conditions were used: 120 seconds of UDG pretreat- ment at 50˚C, followed by 180 seconds of initial denaturation at 94˚C, followed by 55 cycles of 30 seconds at 94˚C, 30 seconds at 62˚C. Estimation of the lenticular NFκB expression and activation After washing, tissue sections were incubated with horseradish per- oxidase (HRP) conjugated secondary antibody (Ready-to-Use Goat anti rabbit/mouse anti- body, Dako, Glostrup, Denmark) for another 30 minutes. The immunostaining was performed using 3,3’-diaminobenzidine (DAB)/ metal and peroxide mixture for 10 minutes. Tissue sections were then counterstained with hematoxylin for 2 minutes, dehydrated and mounted. All washes in this procedure were done three times for 3 minutes each with TBS mixed with 10% Tween 20 (TBST), whereas antibodies were diluted in blocking buffer. For negative controls, tissue sections were incubated with TBST. For quantification of NFκB and pIκBα activities, extraction of lenticular nuclear and cyto- plasmic fraction was done using NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific, Rockford, IL). Two lenses were pooled together as one sample. Lenses were cut into small pieces and washed with ice-cold PBS followed by centrifugation at 890 g for 5 minutes at 4˚C. The supernatant was discarded and the remaining lens tissue pellet was homogenized with cytoplasmic extraction reagent I (CER I) at a ratio of 1 mg lens tissue: 10 μl CER I using a tissue grinder on ice. Then, tissue suspension was vortexed at highest setting for 15 seconds and incubated on ice for 10 minutes. Cytoplasmic extraction reagent II (CER II) was added to the tissue suspension at the ratio of 1 mg lens tissue: 0.55 μl CER II and vortexed for 5 seconds. After 1 minute incubation on ice, the suspension was vortexed again at highest setting for 5 seconds and then, centrifuged at 11900 g for 5 minutes at 4˚C. The supernatant which contained cytoplasmic extract was used to measure pIκB-α level. The remaining tissue pellet was resuspended with the provided nuclear extraction reagent (NER) at the ratio of 1 mg lens tissue: 5 μl NER. Tissue suspension was vortexed at highest setting for 15 seconds and incubated on ice for 10 minutes and this cycle of vortex and ice incubation was continued for a total of 40 minutes. Then, tissue suspension was centrifuged at 11900 g for 10 minutes at 4˚C. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 5 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats The supernatant which contained nuclear extract was collected for measurement of NFκB. Estimation of iNOS expression Cycle threshold (Ct) values were measured and calcu- lated by the CFX software (version Bio-Rad CFX Manager 2.0). Estimation of the lenticular NFκB expression and activation Both the pIκB-α and NFκB estimations were done using commercially available sandwich- ELISA kits (Elabscience, Wuhan, China) containing wells pre-coated with antibodies specific to pIκB-α and NFκB, respectively, as per manufacture’s instruction. Estimation of lens ATP contents Lens ATP level was determined using a commercially available luminescence ATP assay kit (BioVision, CA, USA). Each lens was homogenized in the supplied reaction buffer in a ratio of 1 mg of tissue: 10 μl of reaction buffer. The homogenate was centrifuged at 890 g for 30 sec- onds at 4˚C and supernatant was used for ATP measurement. 10 μl of standards or samples were added with 90 μl of reaction mix containing reaction buffer and enzyme mix into desig- nated white-walled well plate. Luminescence was read using Victor X5 plate reader (Perkin Elmer, Waltham, MA). Quantification of lens oxidative and nitrosative stress Lens oxidative stress was measured by estimation of malondialdeyhde (MDA), GSH contents, SOD and catalase (CAT) activities in lens tissue. For all oxidative stress parameters, commer- cially available ELISA kits (Cayman Chemicals, Ann Arbor, MI) were used and all estimations were done in duplicate. TBARS Assay kit indirectly measures MDA, a byproduct of lipid per- oxidation. MDA reacts with thiobarbituric acid (TBA) under high temperature and acidic medium to produce coloured complex. For this assay, lenses were homogenized with RIPA lysis buffer containing protease inhibitor in a ratio of 1 mg lens weight: 10 μl RIPA buffer. Samples were then centrifuged at 890 g at 4˚C for 10 minutes and supernatant was used for PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 6 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats analysis. Measurement of SOD activity was based on utilization of tetrazolium salt for detec- tion of superoxide radicals generated by xanthine oxidase and hypoxanthine. Estimation of lens CAT activity was based on the CAT reaction with methanol in the presence of the optimal H2O2 concentration. The production of formaldehyde was measured by adding 4-amino- 3-hydra¬zino-5-mercapto-1, 2, 4-triazole, a chromagen (purpald). Purpald forms a cyclic derivative with aldehyde, which upon oxidation turns from colorless to purple for which absorbance was read at 540 nm. Measurement of lens GSH levels was based on enzymatic recy- cling method [33, 34]. For nitrosative stress, lens nitrotyrosine (3-NT) levels, which indirectly provides estimation of ONOO-, were estimated using a commercially available sandwich-ELISA kit (Elabscience, Wuhan, China) containing wells pre-coated with antibody specific to 3-NT as per manufactur- er’s instructions. Estimation of lens Na+ K+ ATPase activity Na+ K+ ATPase activity was determined by measuring the difference of inorganic phosphorus liberated in the presence or absence of ouabain as described by Khan et al. [35]. Briefly, lenses were homogenized in 50 mM Tris-HCl, pH 7.4 and 300 mM sucrose on ice, followed by centri- fugation at 890 g at 4˚C for 5 minutes. ATPase activity was determined in two reaction solu- tions, solution A and B. In solution A, tissue homogenate was added to 20 mM KCl, 100 mM NaCl, 5mM MgCl2 and 100 mM Tris-HCl followed by incubation at room temperature for 5 minutes. Reaction was started by adding 2.5 mM of ATP disodium salt and reaction mixture was incubated at 37˚C for 15 minutes. In solution B, tissue homogenate was added to 5 mM MgCl2, 100 mM Tris-HCl and 1 mM ouabain (Na+ K+ ATPase specific inhibitor). The mixture was incubated at room temperature for 5 minutes before starting the reaction by adding 120 mM NaCl and 2.5 mM of ATP disodium salt followed by incubation at 37˚C for 15 minutes. Reaction was terminated in both solution A and B by adding 10% trichloroacetic acid (TCA). Mixture was centrifuged at 890 g for 5 minutes and the supernatant was used for estimating inorganic phosphate. The activity of Na+ K+ ATPase was calculated as the difference of the inorganic phosphorus liberated between solution A and B. To measure inorganic phosphate, supernatant was added with 2.5% ammonium molybdate (AM) reagent (2.5g of AM in 100 ml of 3N sulphuric acid) and developer reagent (0.5 g of 1-amino-2-napthol-4-sulphonic acid (ANSA) in 195ml of 15% of sodium metabisulphite and 5 ml of 20% of sodium sulphite). Mixture was vortexed and incubated for 10 minutes at room temperature. Absorbance was read at 640 nm. The enzyme activity was expressed as nano- moles of phosphorus liberated/min/mg protein. Estimation of Ca2+ ATPase activities SERCA and PMCA activities were determined as described by Nagai et al. [36]. Lens tissue homogenate was prepared as described for Na+ K+ ATPase activity. For SERCA activity, PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 7 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats solution containing 200 mM KCl, 100 mM HEPES, 10 mM MgCl2, 2 mM ethylene glycol tetra- acetic acid (EGTA), 2mM ATP disodium salt and 2.2 mM CaCl2, pH 7.4, with or without 1μM thapsigargin (specific SERCA inhibitor) were added to tissue homogenate. The mixture was incubated for 1 hour at 37˚C. For PMCA activity, solution containing 200 mM KCl, 100 mM HEPES, 10 mM MgCl2, 2 mM EGTA, 2 mM ATP disodium salt and 1μM thapsigargin, pH 7.4, with or without 2.2 mM CaCl2 were added to tissue homogenate. The mixture was incubated for 1 hour at 37˚C. The reaction was terminated by adding 10% TCA. The mixture was centrifuged at 890 g for 10 minutes at 4˚C and the supernatant was used for inorganic phosphate determination as described in Na+ K+ ATPase activity. The activity of SERCA and PMCA was calculated as the difference of the inorganic phosphorus liberated in the presence or absence of thapsigargin or calcium. Pilot study Both STZ- induced diabetic groups (SP and VP) showed similar trend of body weight gain which was significantly lower compared to normal rats throughout the experimental period (Fig 2A). Similar level of hyperglycemia was observed in both the STZ- induced diabetic groups starting from 72 hours post STZ injection until the end of experimental period (Fig 2B). The cataractous changes observed in both STZ-induced diabetic groups also showed simi- lar progression over the entire experimental period (Fig 2C). Estimation of calpain 2 activity and lens protein contents Lens calpain 2 activity was measured using a commercially available sandwich-ELISA kit (Elabscience, Wuhan, China) containing wells pre-coated with antibody specific to calpain 2 as per manufacturer’s instructions. Total protein level was determined using an aliquot of homogenized sample, before centri- fugation. The homogenate was then centrifuged at 890 g for 15 minutes at 4˚C and the super- natant was used for quantification of soluble protein. Lens protein level was determined using Bradford method, which detects change in the colour of Coomassie dye from brown to blue as a result of its binding to proteins in acidic medium. Statistical analysis All numerical values were expressed as mean ± standard deviation (SD) while categorical vari- ables expressed as frequency and percentage/bar chart. The difference of proportion of each cataract stage by intervention groups was tested using cross tabulation and Chi-square test. The difference in the mean weight, blood glucose and biochemical between the three interven- tion groups was analyzed using one-way ANOVA with post-hoc Bonferroni test. P value  0.05 was considered significant. IBM SPSS Statistics 24.0 was used to analyze all the data. Main studies Body weight and blood glucose level. Both DV and DT groups showed significantly smaller weight gain compared to NV group from second week post-STZ injection until the end of experimental period. The blood glucose level in STZ injected rats showed significant increase 72 hours post-STZ injection and remained within the range of 27 to 34 mmol/L throughout the experimental period (p<0.001) in both the diabetic groups (Fig 3). PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 8 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats Fig 2. Pilot study showing A) weight gain (grams), B) blood glucose level (mmol/L) and C) cataract progression among 3 groups of rats. N = 6. ap<0.001 versus NP. ANOVA with Bonferroni post-hoc indicated significant difference between NV against diabetic groups (SP and VP). NP: Normal rats, SP: Diabetic rats; VP: Diabetic rats with vehicle treatment. https://doi.org/10.1371/journal.pone.0174542.g002 Fig 2. Pilot study showing A) weight gain (grams), B) blood glucose level (mmol/L) and C) cataract progression among 3 groups of rats. N = 6. ap<0.001 versus NP. ANOVA with Bonferroni post-hoc indicated significant difference between NV against diabetic groups (SP and VP). NP: Normal rats, SP: Diabetic rats; VP: Diabetic rats with vehicle treatment. https://doi.org/10.1371/journal.pone.0174542.g002 Fig 2. Pilot study showing A) weight gain (grams), B) blood glucose level (mmol/L) and C) cataract progression among 3 groups of rats. N = 6. ap<0.001 versus NP. ANOVA with Bonferroni post-hoc indicated significant difference between NV against diabetic groups (SP and VP). NP: Normal rats, SP: Diabetic rats; VP: Diabetic rats with vehicle treatment. https://doi.org/10.1371/journal.pone.0174542.g002 Anterior segment imaging and cataract grading. Anterior segment imaging showed clear lenses in NV group until the end of experimental period (Fig 4). Onset of cataract in both Anterior segment imaging and cataract grading. Anterior segment imaging showed clear lenses in NV group until the end of experimental period (Fig 4). Onset of cataract in both the DV and DT groups started from 3rd week post-STZ injection and persisted until the end of treatment period. However, there was delayed cataract progression in DT group compared clear lenses in NV group until the end of experimental period (Fig 4). Onset of cataract in both the DV and DT groups started from 3rd week post-STZ injection and persisted until the end of treatment period. However, there was delayed cataract progression in DT group compared Fig 3. Main studies A) Weight gain (grams) and B) blood glucose level (mmol/L) among 3 groups of rats during 8 weeks of experimental period. N = 39. ap<0.05 versus NV, aap<0.001 versus NV. ANOVA with Bonferroni post-hoc indicated significant difference between NV against diabetic groups (DV and DT). NV: Normal rats with vehicle treatment; DV: Diabetic rats with vehicle treatment; DT: Diabetic rats with tocotrienol treatment. Fig 3. A) Weight gain (grams) and B) blood glucose level (mmol/L) among 3 groups of rats during 8 weeks of experimental period. N = 39. ap<0.05 versus NV, aap<0.001 versus NV. ANOVA with Bonferroni post-hoc indicated significant difference between NV against diabetic groups (DV and DT). NV: Normal rats with vehicle treatment; DV: Diabetic rats with vehicle treatment; DT: Diabetic rats with tocotrienol treatment. https://doi.org/10.1371/journal.pone.0174542.g003 https://doi.org/10.1371/journal.pone.0174542.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 9 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats Fig 4. Effect of topically applied tocotrienol on development of cataract in STZ-induced diabetic rats during 8 weeks of experimental period. N = 78, ap<0.001 versus NV; bp<0.001 versus DV. NV: Normal rats with vehicle treatment; DV: Diabetic rats with vehicle treatment; DT: Diabetic rats with tocotrienol treatment. https://doi.org/10.1371/journal.pone.0174542.g004 Fig 4. Effect of topically applied tocotrienol on development of cataract in STZ-induced diabetic rats during 8 weeks of experimental period. N = 78, ap<0.001 versus NV; bp<0.001 versus DV. NV: Normal rats with vehicle treatment; DV: Diabetic rats with vehicle treatment; DT: Diabetic rats with tocotrienol treatment. https://doi.org/10.1371/journal.pone.0174542.g004 https://doi.org/10.1371/journal.pone.0174542.g004 to DV group from 5th week onward until the end of treatment period (p<0.001) (Fig 4) Addi- tionally, we did not observe any local or systemic adverse effects throughout the experimental period. Effect of tocotrienol on lenticular AR activity and polyol accumulation. Lenses in both the DV and DT groups showed significantly higher AR activity and sorbitol level compared to NV group. However, DT group showed 1.16-folds lower AR activity and 1.18-folds lower lens sorbitol level compared to DV group and the differences for both parameters were significant (Table 1). Table 1. Effect of topically applied tocotrienol on lens aldose reductase and sorbitol activities in STZ- induced diabetic rats. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 https://doi.org/10.1371/journal.pone.0174542.t001 Main studies Group Lens Aldose Reductase (ng/mg lens protein) Lens Sorbitol (units/ g lens weight) NV 2.19 ± 0.3 4.54 ± 0.2 DV 3.11 ± 0.3 a 6.23 ± 0.4 a DT 2.69 ± 0.1 a,c 5.28 ± 0.3 a,b N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 0.92 & 1.69 respectively, p<0.001), NV-DT (mean difference: 0.49 & 0.74 respectively, p<0.01) and DV-DT (mean difference: 0.42 & 0.95, p<0.05 & p<0.001, respectively). a p<0.001 versus NV; b p<0.01 versus NV; c p<0.001 versus DV; d p<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol https://doi.org/10.1371/journal.pone.0174542.t001 one.0174542 March 28, 2017 10 / 21 Table 1. Effect of topically applied tocotrienol on lens aldose reductase and sorbitol activities in STZ- induced diabetic rats. Group Lens Aldose Reductase (ng/mg lens protein) Lens Sorbitol (units/ g lens weight) NV 2.19 ± 0.3 4.54 ± 0.2 DV 3.11 ± 0.3 a 6.23 ± 0.4 a DT 2.69 ± 0.1 a,c 5.28 ± 0.3 a,b N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 0.92 & 1.69 respectively, p<0.001), NV-DT (mean difference: 0.49 & 0.74 respectively, p<0.01) and DV-DT (mean difference: 0.42 & 0.95, p<0.05 & p<0.001, respectively). a p<0.001 versus NV; b p<0.01 versus NV; c p<0.001 versus DV; d p<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol https://doi.org/10.1371/journal.pone.0174542.t001 Table 1. Effect of topically applied tocotrienol on lens aldose reductase and sorbitol activities in STZ- induced diabetic rats. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 0.92 & 1.69 respectively, p<0.001), NV-DT (mean difference: 0.49 & 0.74 respectively, p<0.01) and DV-DT (mean difference: 0.42 & 0.95, p<0.05 & p<0.001, respectively). a p<0.001 versus NV; b p<0.01 versus NV; c p<0.001 versus DV; d p<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with p NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol 10 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 Anticataract effect of tocotrienol in streptozotocin-induced rats Effect of tocotrienol on lenticular pIκBα and NFκB activities. Main studies Quantitative measure- ment of pIκBα and NFκB by ELISA showed higher cytoplasmic pIκBα activity in DV group compared to DT and NV groups (p<0.05) whereas the same was comparable between DT and NV groups (Fig 5A). Nuclear NFκB activity was 2.28- and 1.24-folds higher in DV and DT groups, respectively, compared to group NV group. However, DT group showed 1.84-folds lesser NFκB activity compared to DV group (p<0.001) (Fig 5B). Immunohistochemical staining for NFκB p65 was observed particularly in the lens epithe- lial and equatorial region. Positive activity was represented by brown-coloured stain that was mainly visible in the DV group and not in both the NV and DT groups (Fig 6). Effect of tocotrienol on lenticular iNOS gene and protein expression. iNOS mRNA expression in DV group was 19.2-folds higher compared to NV group (p<0.001). DT group also showed 7.7-folds higher iNOS mRNA expression compared to NV group (p<0.05). How- ever, there was 2.5-folds lower expression of iNOS mRNA expression in DT compared DV group (p<0.001) (Fig 7A). iNOS protein expression as estimated by ELISA was higher in DV group by 1.25- and 1.19- folds compared to NV (p<0.001) and DT (p<0.01) groups, respec- tively. The iNOS protein in DT group, however, was comparable to NV group (Fig 7B). Effect of tocotrienol on lenticular oxidative and nitrosative stress. We observed 1.27 folds higher level of lenticular MDA in DV group compared to NV group (p<0.001), whereas, DT group had 1.18 folds lower MDA content compared to DV group (p<0.001). Both catalase and SOD enzyme activities in DT group were comparable to that in NV group. The GSH levels were 3.63 and 1.72 folds lower in both DV and DT groups compared to NV group (p<0.001), respectively. However, DT group showed 1.25 fold higher GSH levels compared to DV group (p<0.01) (Table 2). DV group showed higher lenticular 3-NT activity compared to NV group (p<0.001), however, the same in DT group was comparable with that of NV group (Table 2). Effect of tocotrienol on lenticular ATP and ATPases. Lenticular ATP level in DT group was higher in trend compared to DV group and comparable to NV group. Lenticular Na+ K+ ATPase activity in DT group was restored to normal, whereas the DV group showed lower activity compared to NV and DT groups. Main studies Lenticular PMCA activity in DV group was lower compared to NV group (p<0.01), while the same in DT group was comparable to N group. Lenticular SERCA activity in DT group was 1.25-folds higher compared to DV group (Table 3). Effect of tocotrienol on calpain 2 and lenticular protein levels. The lens calpain 2 activ- ity in DV group showed higher mean value compared to NV and DT groups (1.13- and 1.11-folds respectively; p<0.001) whereas, DT group showed no significant difference from NV group (Fig 8). The soluble to insoluble protein ratio was 1.4-folds lower in DV group com- pared to NV group (p<0.05). However, this ratio was restored to normal in DT group (Table 4). Discussion The current study demonstrated that topical application of the microemulsion formulation of tocotrienol delays the progression of cataract in rats with STZ-induced diabetes despite persis- tent hyperglycemia. The delayed cataractogenesis might be due to reduction of lenticular AR activity, polyol accumulation, NFκB expression and activation, iNOS expression, oxidative- nitrosative stress and calpain 2 activity resulting in improved soluble:insoluble protein ratio and delayed lenticular opacities development. Complications of diabetes such as cataract have been attributed to long standing hypergly- cemia [37] and the mechanisms that relate hyperglycemia with diabetic complications have widely been investigated. Since presence of AR as well as polyols has been detected in PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 11 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats Fig 5. Effect of topically applied tocotrienol on lenticular A) pIκBα and B) NFκB in STZ-induced diabetic rats. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 0.71 & 871.80, p<0.05 & p<0.001 respectively) and DV-DT (mean difference: 0.53 & 709.65, p<0.05 & p< 0.001, respectively). ap<0.001 versus NV; bp<0.05 versus NV; cp<0.001 versus DV; dp<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol. https://doi org/10 1371/journal pone 0174542 g005 Fig 5 Effect of topically applied tocotrienol on lenticular A) pIκBα and B) NFκB in STZ induced Fig 5. Effect of topically applied tocotrienol on lenticular A) pIκBα and B) NFκB in STZ-induced diabetic rats. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 0.71 & 871.80, p<0.05 & p<0.001 respectively) and DV-DT (mean difference: 0.53 & 709.65, p<0.05 & p< 0.001, respectively). ap<0.001 versus NV; bp<0.05 versus NV; cp<0.001 versus DV; dp<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol. Fig 5. Effect of topically applied tocotrienol on lenticular A) pIκBα and B) NFκB in STZ-induced diabetic rats. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 0.71 & 871.80, p<0.05 & p<0.001 respectively) and DV-DT (mean difference: 0.53 & 709.65, p<0.05 & p< 0.001, respectively). ap<0.001 versus NV; bp<0.05 versus NV; cp<0.001 versus DV; dp<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol. https://doi.org/10.1371/journal.pone.0174542.g005 Fig 6. Discussion Tissue sections of rat lenses after immunostaining with NFκB p65 antibody in STZ-induced diabetic and normal rats. A- NV group showing normal epithelium with absence of brown stain for NFκB p65, B- DV group showing dense brown-stained lens epithelium, C- DT group showing minimal brown stain for NFκB p65 compared to DV group. Red arrows indicate lens epithelial layer. Scale bar represents 100μm. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol. Fig 6. Tissue sections of rat lenses after immunostaining with NFκB p65 antibody in STZ-induced diabetic and normal rats. A- NV group showing normal epithelium with absence of brown stain for NFκB p65, B- DV group showing dense brown-stained lens epithelium, C- DT group showing minimal brown stain for NFκB p65 compared to DV group. Red arrows indicate lens epithelial layer. Scale bar represents 100μm. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol. https://doi.org/10.1371/journal.pone.0174542.g006 https://doi.org/10.1371/journal.pone.0174542.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 12 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats Fig 7. Effect of topically applied tocotrienol on lenticular A) iNOS gene expression and B) iNOS activity in STZ-induced diabetic rats. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 18.39 & 237.15 respectively, p<0.001) and DV-DT (mean difference: 11.56 & 188.39, p<0.001 & p<0.01, respectively). ap<0.001 versus NV; bp<0.05 versus NV; cp<0.001 versus DV, dp<0.01 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol. https://doi.org/10.1371/journal.pone.0174542.g007 Fig 7. Effect of topically applied tocotrienol on lenticular A) iNOS gene expression and B) iNOS activity in STZ-induced diabetic rats. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 18.39 & 237.15 respectively, p<0.001) and DV-DT (mean difference: 11.56 & 188.39, p<0.001 & p<0.01, respectively). ap<0.001 versus NV; bp<0.05 versus NV; cp<0.001 versus DV, dp<0.01 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated wit vehicle; DT: Diabetic rats treated with tocotrienol. https://doi.org/10.1371/journal.pone.0174542.g007 Fig 7. Effect of topically applied tocotrienol on lenticular A) iNOS gene expression and B) iNOS Fig 7. Effect of topically applied tocotrienol on lenticular A) iNOS gene expression and B) iNOS activity in STZ-induced diabetic rats. Discussion N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 18.39 & 237.15 respectively, p<0.001) and DV-DT (mean difference: 11.56 & 188.39, p<0.001 & p<0.01, respectively). ap<0.001 versus NV; bp<0.05 versus NV; cp<0.001 versus DV, dp<0.01 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol. https://doi.org/10.1371/journal.pone.0174542.g007 Table 2. Effect of topically applied tocotrienol on lenticular MDA, GSH, catalase, SOD and 3-NT in STZ-induced diabetic rats. Group MDA (μM/g lens weight) Catalase activity (μmol/g lens protein) SOD activity (units/mg lens protein) GSH level (μmol/g lens weight) 3-NT activity (ng/mg lens protein) NV 903.29 ± 46.0 65.82 ± 11.4 9.27 ± 0.3 5.49 ± 0.3 8.62 ± 0.2 DV 1143.04 ± 89.6 a 136.09 ± 24.4 a 7.17 ± 0.8 a 2.55 ± 0.1 a 9.75 ± 0.5 a DT 967.26 ± 33.2 b 65.44 ± 16.3 b 9.74 ± 0.6 b 3.19 ± 0.3 a,c 8.62 ± 0.5 b Table 2. Effect of topically applied tocotrienol on lenticular MDA, GSH, catalase, SOD and 3-NT in STZ-induced diabetic rats. Group MDA (μM/g lens weight) Catalase activity (μmol/g lens protein) SOD activity (units/mg lens protein) GSH level (μmol/g lens weight) 3-NT activity (ng/mg lens protein) NV 903.29 ± 46.0 65.82 ± 11.4 9.27 ± 0.3 5.49 ± 0.3 8.62 ± 0.2 DV 1143.04 ± 89.6 a 136.09 ± 24.4 a 7.17 ± 0.8 a 2.55 ± 0.1 a 9.75 ± 0.5 a DT 967.26 ± 33.2 b 65.44 ± 16.3 b 9.74 ± 0.6 b 3.19 ± 0.3 a,c 8.62 ± 0.5 b N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 239.75, 70.27, 2.11, 2.95 & 1.13 respectively, p<0.001) and DV-DT (mean difference: 175.78, 70.65, 2.57, 0.641 & 1.13; p<0.001, p<0.001, p<0.001, p<0.01 & p<0.001 respectively). a p<0.001 versus NV; b p<0 001 versus DV; Table 2. Effect of topically applied tocotrienol on lenticular MDA, GSH, catalase, SOD and 3-NT in STZ-induced diabetic rats. https://doi.org/10.1371/journal.pone.0174542.t002 The role of AR in cataractogenesis is fur- ther supported by other studies that observed rodents having low or no AR activity are resis- tant to development of diabetic cataract [40, 41]. Furthermore, Lee et al. [42] demonstrated that cataract development in type 2 diabetes mellitus patient may be influenced by polymor- phism in AR gene. In the current study, reduced AR activity and polyol accumulation may be the key mechanism in the anticataract effect of tocotrienol. It is noteworthy that lenticular tocotrienol’s effects seen in this study was not secondary to reduction in blood glucose levels as orally administered tocotrienol has been reported to reduce blood glucose level in STZ- induced diabetic rat [43]. Both tocotrienol- and vehicle-treated diabetic group showed similar persistent hyperglycemia and trends in terms of weight gain throughout the study, as demon- strated by earlier studies [44–46]. cataractous lenses [6, 38], polyol pathway seems to play a significant role. Polyol pathway involves two enzymes of which AR is the first one that converts glucose to sorbitol and the sec- ond enzyme, sorbitol dehydrogenase, converts sorbitol to fructose. However, the rate of con- version of sorbitol to fructose is considerably slow and sorbitol does not diffuse out of the cell, hence resulting in its intracellular accumulation [39]. The role of AR in cataractogenesis is fur- ther supported by other studies that observed rodents having low or no AR activity are resis- tant to development of diabetic cataract [40, 41]. Furthermore, Lee et al. [42] demonstrated that cataract development in type 2 diabetes mellitus patient may be influenced by polymor- phism in AR gene. In the current study, reduced AR activity and polyol accumulation may be the key mechanism in the anticataract effect of tocotrienol. It is noteworthy that lenticular tocotrienol’s effects seen in this study was not secondary to reduction in blood glucose levels as orally administered tocotrienol has been reported to reduce blood glucose level in STZ- induced diabetic rat [43]. Both tocotrienol- and vehicle-treated diabetic group showed similar persistent hyperglycemia and trends in terms of weight gain throughout the study, as demon- strated by earlier studies [44–46]. Fig 8. Effect of topically applied tocotrienol on lens calpain 2 activity. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference in lens calpain between NV-DV (mean difference: 45.25, p<0.001) and DV-DT (mean difference: 38.68; p<0.001). ap<0.001 versus NV; bp<0.001 versus DV. Discussion Group MDA (μM/g lens weight) Catalase activity (μmol/g lens protein) SOD activity (units/mg lens protein) GSH level (μmol/g lens weight) 3-NT activity (ng/mg lens protein) NV 903.29 ± 46.0 65.82 ± 11.4 9.27 ± 0.3 5.49 ± 0.3 8.62 ± 0.2 DV 1143.04 ± 89.6 a 136.09 ± 24.4 a 7.17 ± 0.8 a 2.55 ± 0.1 a 9.75 ± 0.5 a DT 967.26 ± 33.2 b 65.44 ± 16.3 b 9.74 ± 0.6 b 3.19 ± 0.3 a,c 8.62 ± 0.5 b N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 239.75, 70.27, 2.11, 2.95 & 1.13 respectively, p<0.001) and DV-DT (mean difference: 175.78, 70.65, 2.57, 0.641 & 1.13; p<0.001, p<0.001, p<0.001, p<0.01 & p<0.001 respectively). a p<0.001 versus NV; b p<0.001 versus DV; c p<0.01 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol https://doi.org/10.1371/journal.pone.0174542.t002 Table 2. Effect of topically applied tocotrienol on lenticular MDA, GSH, catalase, SOD and 3-NT in STZ-in topically applied tocotrienol on lenticular MDA, GSH, catalase, SOD and 3-NT in STZ-induced diabetic rats. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 13 / 21 https://doi.org/10.1371/journal.pone.0174542.t003 Anticataract effect of tocotrienol in streptozotocin-induced rats Table 3. Effect of topically applied tocotrienol on lenticular ATP level and ATPases activity in STZ-induced diabetic rats. Groups ATP (pmol/g lens weight) Na+ K+ ATPase activity (nmol Pi liberated/mg protein/ min) PMCA activity (μmol Pi liberated/ mg protein/ min) SERCA activity (μmol Pi liberated/ mg protein/ min) NV 901.11 ± 79.2 2.73 ± 0.5 3.17 ± 0.3 1.66 ± 0.6 DV 804.21 ± 35.1 1.92 ± 0.7a 2.60 ± 0.3a 1.33 ± 0.2 DT 867.36 ± 51.3 2.63 ± 0.3 3.35 ± 0.3b 1.67 ± 0.3 N = 6, ANOVA with Bonferroni post-hoc indicated significant difference in Na+ K+ ATPase activity between NV-DV (mean difference: 0.81, p<0.05) and PMCA activity between NV-DV (mean difference: 0.57, p<0.05) and DV-DT (mean difference: 0.75; p<0.01). a p<0 05 versus NV; trienol on lenticular ATP level and ATPases activity in STZ-induced diabetic rats. Table 3. Effect of topically applied tocotrienol on lenticular ATP level and ATPases activity in STZ-induced diabetic rats. Groups ATP (pmol/g lens weight) Na+ K+ ATPase activity (nmol Pi liberated/mg protein/ min) PMCA activity (μmol Pi liberated/ mg protein/ min) SERCA activity (μmol Pi liberated/ mg protein/ min) NV 901.11 ± 79.2 2.73 ± 0.5 3.17 ± 0.3 1.66 ± 0.6 DV 804.21 ± 35.1 1.92 ± 0.7a 2.60 ± 0.3a 1.33 ± 0.2 DT 867.36 ± 51.3 2.63 ± 0.3 3.35 ± 0.3b 1.67 ± 0.3 t of topically applied tocotrienol on lenticular ATP level and ATPases activity in STZ-induced diabetic rats. Table 3. Effect of topically applied tocotrienol on lenticular ATP level and ATPases activity in STZ-induced d Table 3. Effect of topically applied tocotrienol on lenticular ATP level and ATPa Table 3. Effect of topically applied tocotrieno N = 6, ANOVA with Bonferroni post-hoc indicated significant difference in Na+ K+ ATPase activity between NV-DV (mean difference: 0.81, p<0.05) and PMCA activity between NV-DV (mean difference: 0.57, p<0.05) and DV-DT (mean difference: 0.75; p<0.01). a 0 05 NV https://doi.org/10.1371/journal.pone.0174542.t003 cataractous lenses [6, 38], polyol pathway seems to play a significant role. Polyol pathway involves two enzymes of which AR is the first one that converts glucose to sorbitol and the sec- ond enzyme, sorbitol dehydrogenase, converts sorbitol to fructose. However, the rate of con- version of sorbitol to fructose is considerably slow and sorbitol does not diffuse out of the cell, hence resulting in its intracellular accumulation [39]. a p<0.05 versus NV; b p<0 05 versus DV DV-DT (mean difference: 30.72, 52.13 & 0.63 respectively, p<0.05). a p<0.05 versus NV; b p<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol https://doi.org/10.1371/journal.pone.0174542.t004 Increased AR activity is positively correlated with oxidative stress primarily by causing NADPH depletion. In this regards, Lou et al. [47] demonstrated restoration of GSH level to near normal with administration of AR inhibitor. Polyphenols including tocopherol, the other analogue of vitamin E, have been shown to possess AR inhibitory activity [48–50], however, the same has not been demonstrated for tocotrienol. Direct aldose reductase inhibition assay may provide evidence if tocotrienol directly inhibits AR. Nevertheless, the reduced oxidative stress in response to treatment with tocotrienol in our study may be attributed to reduce polyol pathway activity as been observed reduced AR activity and polyol accumulation in tocotrie- nol-treated rats. Tocotrienol, however, may also scavenge ROS by donating phenolic hydrogen to free radicals [51]. Reduced lenticular oxidative stress in tocotrienol treated group in this study was in accordance with that observed in galactose-fed rats in our previous study [31]. ROS have been shown to act as secondary signaling messengers that activate many phos- phosignaling pathways including NFκB [52]. Exposure of human lens epithelial cells (HLECs) to ultraviolet radiations was shown to exacerbate ROS production which plays an essential role in the activation of NFκB [53]. Furthermore, this activation of NFκB in HLECs involves phos- phorylation of IκB-α [54]. NFκB exists in cytoplasm complexed with IκB-α and phosphoryla- tion of IκB-α releases NFκB which translocates to nucleus and modulates transcription of several genes including iNOS in the lens [55]. Lower nuclear NFκB and cytoplasmic phosphor- ylated IκB-α levels in the tocotrienol treated group compared to vehicle treated group indi- cated reduced NFκB activation. This is in accordance with other studies which showed ability of tocotrienol to inhibit NFκB activation in vitro [56–59] as well as in vivo [60,61]. It has been suggested that inhibition of NFκB activation by tocotrienol may be attributed to decreased IκB phosphorylation due to suppression of proteasomic activity [60–63]. Furthermore, Wang et al. [58] and Jiang et al. [59] demonstrated the ability of tocotrienol to modulate sphingolipid metabolism and that was shown to cause higher expression and activity of NFκB negative reg- ulator in cytokine-induced RAW 264.7 macrophages. NV: Normal rats with vehicle treatment; DV: Diabetic rats with vehicle treatment; DT: Diabetic rats with tocotrienol treatment. https://doi.org/10.1371/journal.pone.0174542.g008 Fig 8. Effect of topically applied tocotrienol on lens calpain 2 activity. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference in lens calpain between NV-DV (mean difference: 45.25, p<0.001) and DV-DT (mean difference: 38.68; p<0.001). ap<0.001 versus NV; bp<0.001 versus DV. NV: Normal rats with vehicle treatment; DV: Diabetic rats with vehicle treatment; DT: Diabetic rats with tocotrienol treatment Fig 8. Effect of topically applied tocotrienol on lens calpain 2 activity. N = 6, ANOVA with Bonferroni post-hoc indicated significant difference in lens calpain between NV-DV (mean difference: 45.25, p<0.001) and DV-DT (mean difference: 38.68; p<0.001). ap<0.001 versus NV; bp<0.001 versus DV. NV: Normal rats with vehicle treatment; DV: Diabetic rats with vehicle treatment; DT: Diabetic rats with tocotrienol treatment. https://doi.org/10.1371/journal.pone.0174542.g008 PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 14 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats Table 4. Effect of topically applied tocotrienol on lenticular proteins in STZ-induced diabetic rats. Groups Total protein (mg/g lens weight) Soluble protein (mg/g lens weight) Insoluble protein (mg/g lens weight) Soluble:Insoluble protein (ratio) NV 473.78 ± 22.3 309.54 ± 21.5 164.24 ± 28.3 1.93 ± 0.3 DV 490.24 ± 46.9 280.89 ± 11.2a 209.35 ± 39.0a 1.39 ± 0.3a DT 468.83 ± 31.4 311.61 ± 9.8b 157.21 ± 23.5b 2.02 ± 0.3b N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 28.64, 45.11 & 0.54 respectively, p<0.05) and DV-DT (mean difference: 30.72, 52.13 & 0.63 respectively, p<0.05). a p<0.05 versus NV; b p<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol Table 4. Effect of topically applied tocotrienol on lenticular proteins in STZ-induced diabetic rats. Groups Total protein (mg/g lens weight) Soluble protein (mg/g lens weight) Insoluble protein (mg/g lens weight) Soluble:Insoluble protein (ratio) NV 473.78 ± 22.3 309.54 ± 21.5 164.24 ± 28.3 1.93 ± 0.3 DV 490.24 ± 46.9 280.89 ± 11.2a 209.35 ± 39.0a 1.39 ± 0.3a DT 468.83 ± 31.4 311.61 ± 9.8b 157.21 ± 23.5b 2.02 ± 0.3b N = 6 ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 28 64 45 11 & 0 54 respectively p<0 05) and . Effect of topically applied tocotrienol on lenticular proteins in STZ-induced diabetic rats. Table 4. Effect of topically applied tocotrienol on lenticular proteins in STZ-ind N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 28.64, 45.11 & 0.54 respect DV-DT (mean difference: 30.72, 52.13 & 0.63 respectively, p<0.05). a p<0.05 versus NV; b p<0.05 versus DV. NV: Normal rats treated with vehicle; DV: Diabetic rats treated with vehicle; DT: Diabetic rats treated with tocotrienol oc indicated significant difference between NV-DV (mean difference: 28.64, 45.11 & 0.54 respectively, p<0.05) and 3 & 0.63 respectively, p<0.05). N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 28.64, 45.11 & 0.54 respectively, p<0.05) and DV-DT (mean difference: 30.72, 52.13 & 0.63 respectively, p<0.05). a 0 05 NV N = 6, ANOVA with Bonferroni post-hoc indicated significant difference between NV-DV (mean difference: 28.64, 45.11 & 0.54 respectively, p<0.05) and DV-DT (mean difference: 30.72, 52.13 & 0.63 respectively, p<0.05). https://doi.org/10.1371/journal.pone.0174542.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 The NFκB inhibitory effect was previ- ously demonstrated for γ- and δ-tocotrienol [64] and the annatto tocotrienol used in this study consist of both γ and δ isomer [65]. Activation and migration of NFκB to the nucleus in the presence of hyperglycemia leads to its increased binding to the iNOS promoter and a consequent increase in iNOS expression [66]. Increased iNOS expression is positively correlated with cataractogenesis and Inomata et al. [55] detected increased expressions of iNOS mRNA and iNOS protein in cataractous lenses. Since we observed reduced iNOS expression in the lenses of animals treated with toco- trienol, it is likely that reduced NFκB expression secondary to reduced ROS generation was the contributory factor. We demonstrated that topical tocotrienol not only affects iNOS PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 15 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats protein expression, but also reduced its gene expression. Furthermore, this present study also showed reduced nitrotyrosine levels in tocotrienol treated rats also indicate reduced NO pro- duction due to reduced iNOS expression. Additionally, Inomata et al. [55] also showed the induction of iNOS protein occurs before the elevation in calcium content and increase in cal- pain-mediated proteolysis, both of which are closely related to the development of lens opacification. This present study also observed that induction of diabetes causes depletion of lens ATP levels. Mitton et al. [67] have shown earlier that in STZ-induced diabetic rats lenticular ATP/ ADP ratio begin to decrease one week post-induction, even before the onset of cataractous changes. ATP loss may be due to oxidative stress [68] and mitochondrial damage [69] that leads to reduced expression of cytochrome c oxidase, an enzyme important in mitochondrial respiratory pathway for ATP synthesis [70]. We observed normalization of lens ATP contents following tocotrienol treatment which was in accordance with similar observation made by Schloesser et al [71] using brain of aged mice and Nowak et al. [72] using renal proximal tubu- lar cells. Thus, the preservation of lenticular ATP following treatment with tocotrienol in the current study may be attributed to its potent antioxidant effects. Furthermore, effect of toco- trienol on maintaining ATP levels may also be due to its ability to preserve mitochondrial function in the presence of oxidative stress [72] and inhibition of cytochrome c release [73]. As the consequence of preservation of lens ATP levels, lens Na+ K+ ATPase and Ca2+ ATPase activity in tocotrienol treated groups were restored. However, loss of ATPase func- tions may also be a consequence of oxidative stress. Wang et al. [74] have shown that Na+ K+ ATPase activity is highly sensitive to changes in ATP and ROS levels. Huang et al. [75] showed modification of Na+ K+ ATPase is due to oxidative damage. Similarly Ca2+ ATPase activity is affected by oxidative stress [76]. Borchman et al. [77] showed that Ca2+ ATPase activity could be inhibited through oxidation in membrane-enriched sample from rabbit cortical and epithe- lial lens fibers treated with hydrogen peroxide. Moreover, oxidative damage to the lipid mem- branes may cause the uncoupling of membrane bound ATPases, thus reducing their activity [78, 79]. It is likely that tocotrienol associated restoration of ATPase functions involves reduc- tion of lens oxidative stress besides correction of lens ATP levels. Additionally, vitamin E and tocotrienol have also been shown to normalize the oxidative damage-induced reduction in Na+ K+ ATPase activity in carbofuran-treated rat [80] and tert-butylhydroperoxide-treated human erythrocytes [81]. Na+ K+ ATPase has long been recognized for its role in regulating electrolyte concentra- tions in the lens, which is vital to lens transparency. Dysfunction of Na+ K+ ATPase leads to elevated lens Na+ level which promotes lens opacification [24]. Similarly, maintenance of cal- cium homeostasis is imperative for the clarity of the lens. Ca2+ ATPase is essential for the removal of cytosolic calcium, either across the plasma membrane or through intracellular organelles such as the endoplasmic reticulum [82]. The Ca2+ ATPase activity in the mem- branes of cataractous lenses was shown to be 50% lesser compared to normal lenses [83]. Hence, in the current study, restoration of Na+ K+ ATPase and Ca2+ ATPase activity in response to treatment with tocotrienol seems to play a key role in maintaining the lenticular ionic homeostasis. This was also evidenced by reduced calpain 2 activity and normalized solu- ble: insoluble protein ratio in tocotrienol treated diabetic rats in this study. Since the activity of calpain is calcium-dependent, it is evident that increased lenticular Ca2+ subsequent to Ca2+ ATPase dysfunction promotes lens opacification by causing proteolysis of soluble lens proteins into insoluble ones [76]. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 In conclusion, current study demonstrated that topical application of tocotrienol prevents the progression of cataract in STZ-induced diabetic rats in the presence of persistent hypergly- cemia. This delayed cataractogenesis could be attributed to reduced AR activation and reduced PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 16 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats polyol accumulation leading to reduced lenticular oxidative stress. Tocotrienol also reduces lens nitrosative stress due to reduced iNOS expression as a consequence of reduced NFκB acti- vation. Additionally, tocotrienol preserves lens ATP and restores Na+ K+ ATPase and Ca2+ ATPase activity and consequently reduces calpain activity, resulting in restoration of soluble: insoluble protein ratio and delayed cataractogenesis. Further studies to determine the extent of ocular penetration of tocotrienol, and not only preventive but therapeutic anticataract effects of tocotrienol would determine the potential of tocotrienol as an anticataract agent. Acknowledgments We acknowledge the administrative and facility support by Research Management Institute (RMI), Institute of Medical Molecular Biotechnology (IMMB) and Laboratory Animal Care Unit (LACU), Universiti Teknologi MARA, Malaysia. We also would like to thank our hard- working optometrists, Norhafni Razali and Iskandar Supardi for their help in clinical grading. This work was funded by Ministry of Higher Education, Government of Malaysia, under the grant no 600-RMI/RAGS 5/3 (39/2014). S3 Table. Cataract staging data for all experimental groups. (PDF) S3 Table. Cataract staging data for all experimental groups. (PDF) S4 Table. Blood parameters data for all experimental groups. (PDF) S4 Table. Blood parameters data for all experimental groups. (PDF) Supporting information S1 Table. Blood sugar data for all experimental groups. (PDF) S1 Table. Blood sugar data for all experimental groups. (PDF) S1 Table. Blood sugar data for all experimental groups. (PDF) S2 Table. Weight data for all experimental groups. (PDF) S2 Table. Weight data for all experimental groups. (PDF) S3 Table. Cataract staging data for all experimental groups. (PDF) References 1. Mafauzy M, Hussein Z, Chan SP. The status of diabetes control in Malaysia: results of DiabCare 2008. Med. J. Malaysia. 2011; 66(3): 175–181. PMID: 22111435 2. Li L, Wan XH, Zhao GH. Meta-analysis of the risk of cataract in type 2 diabetes. BMC Ophthalmol. 2014; 14(1): 1. 3. Prokofyeva E, Wegener A, Zrenner E. Cataract prevalence and prevention in Europe: a literature review. Acta Ophthalmol. 2013: 91(5): 395–405. https://doi.org/10.1111/j.1755-3768.2012.02444.x PMID: 22715900 4. Kanthan GL, Mitchell P, Burlutsky G, Wang JJ. 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Bar-Shai M, Reznick AZ. Reactive nitrogen species induce nuclear factor-κB-mediated protein degrada- tion in skeletal muscle cells. Free Radic Biol Med. 2006; 40(12): 2112–2125. https://doi.org/10.1016/j. freeradbiomed.2006.02.009 PMID: 16785025 16. Conceptualization: NAAN RA SHSAK SV MT II MII NMI. Data curation: NAAN RA SHSAK SV II AMD NMI. Formal analysis: NAAN RA SHSAK SV II AMD NMI. Funding acquisition: NAAN RA SHSAK SV II MII NMI. Investigation: NAAN. Methodology: NAAN RA SHSAK II NMI. Project administration: NAAN RA NMI. Resources: NAAN RA SHSAK SV MT II MII NMI. Supervision: SHSAK SV II RA AMD NMI. Validation: NAAN RA SHSAK SV II AMD NMI. Visualization: NAAN RA. Writing – original draft: NAAN RA SHSAK SV II. Writing – review & editing: NAAN RA SHSAK SV MT II MII AMD NMI. Conceptualization: NAAN RA SHSAK SV MT II MII NMI. Data curation: NAAN RA SHSAK SV II AMD NMI. Formal analysis: NAAN RA SHSAK SV II AMD NMI. Funding acquisition: NAAN RA SHSAK SV II MII NMI. Investigation: NAAN. Methodology: NAAN RA SHSAK II NMI. Project administration: NAAN RA NMI. Resources: NAAN RA SHSAK SV MT II MII NMI. Supervision: SHSAK SV II RA AMD NMI. Validation: NAAN RA SHSAK SV II AMD NMI. Visualization: NAAN RA. Writing – original draft: NAAN RA SHSAK SV II. Writing – review & editing: NAAN RA SHSAK SV MT II MII AMD NMI. PLOS ONE | https://doi.org/10.1371/journal.pone.0174542 March 28, 2017 17 / 21 Anticataract effect of tocotrienol in streptozotocin-induced rats References Calcium-activated proteolysis in the lens nucleus during selenite cataractogen- esis. Invest Ophthalmol Vis Sci. 1984; 25(11): 1275–1283. PMID: 6386740 29. Yoshida H, Murachi T, Tsukahara I. Age-related changes of calpain II and α-crystallin in the lens of hereditary cataract (Nakano) mouse. Curr Eye Res. 1985; 4(9): 983–988. PMID: 2998702 30. Horwitz J. Alpha-crystallin. Exp Eye Res. 2003; 76(2): 145–153. PMID: 12565801 31. 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Resting heart rate and the risk of incident type 2 diabetes mellitus among non-diabetic and prediabetic Iranian adults: Tehran lipid and glucose study

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RESEARCH Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background  Resting heart rate (RHR) has been found to be a potential risk factor for developing type 2 diabetes mellitus (T2DM), with a highly significant heterogeneity among previous studies. Therefore, we examined the association of RHR and risk of incident T2DM among non-diabetic and prediabetic adults. Methods  The study population included 2431 men and 2910 women aged ≥ 20 years without T2DM at baseline (2001–2005). Participants were followed for incident T2DM by about 3-year intervals up to April 2018. The multivariable Cox proportional models were applied to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs). The models were adjusted for age, body mass index, waist circumference, educational level, physical activity, smoking, hypertension, family history of diabetes, triglycerides/ high-density lipoprotein cholesterol ratio, and fasting plasma glucose. Results  During a median follow-up of 12.2 years, 313 men and 375 women developed T2DM. Interestingly, a significant sex-difference was found (all P-values for sex interaction < 0.025). Among men, compared to the first quintile (< 68 bpm: beats per minute), those who had RHR of over 84 bpm were at higher T2DM risk with a HR (95%CI) of 1.69 (1.16–2.47). Furthermore, considering RHR as a continuous variable, an increase of 10 bpm caused 17% significantly higher risk among men with a HR of 1.17 (1.05–1.30). However, among women, there was no significant association between incident T2DM and RHR. Moreover, among prediabetic participants at baseline, the association of RHR and risk of T2DM progression was generally similar to the general population, which means higher RHR increased the risk of T2DM development only among men with a HR of 1.26 (1.09–1.46) for 10 bpm increase. Conclusions  Among men, being either non-diabetic or prediabetic at baseline, higher RHR can be associated with incident T2DM; however, women didn’t show a significant association. Further studies are needed to determine the added value of RHR as a potential modifiable risk factor in screening and risk prediction of incident T2DM. Keywords  Heart rate, Diabetes Mellitus Type 2, Prediabetic state, Iran Keywords  Heart rate, Diabetes Mellitus Type 2, Prediabetic state, Iran *Correspondence: Farzad Hadaegh [email protected] Full list of author information is available at the end of the article Full list of author information is available at the end of the article © The Author(s) 2023. BMC Public Health BMC Public Health Moazzeni et al. BMC Public Health (2023) 23:2112 https://doi.org/10.1186/s12889-023-17022-7 Resting heart rate and the risk of incident type 2 diabetes mellitus among non-diabetic and prediabetic Iranian adults: Tehran lipid and glucose study Seyyed Saeed Moazzeni1, Kimia Karimi Toudeshki2, Fatemeh Ghorbanpouryami2, Mitra Hasheminia1, Fereidoun Azizi3, Mehdi Pishgahi4 and Farzad Hadaegh1* Abstract Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background (2011–2014), and phase VI (2015–2018). Further details about TLGS rational and design were described before [12]. Type 2 diabetes mellitus (T2DM) caused approximately 1.5 million deaths and was the 8th leading cause of dis­ ability-adjusted life years (DALYs) in 2019 globally [1]. In 2021, the Middle East and North Africa region (MENA) had the highest standardized prevalence of T2DM glob­ ally (18.1%), with an increasing trend [2]. National data from Iran demonstrated that 15.0% and 25.4% of Ira­ nian adults had diabetes and prediabetes, respectively [3]. Moreover, we previously found an age standardized incidence rate of 9.94 per 1000 person-years for T2DM among adults residents of Tehran [4, 5]. A prediabetes tsunami was also reported among residents of Tehran, with > 4% of the population developed prediabetes annu­ ally [6]. From a total of 3891 men and 5036 women aged > 20 years, 443 men and 623 women were excluded due to having T2DM at baseline. Furthermore, we excluded 213 men and 173 women with prevalent cardiovascular disease (CVD) or cancer at baseline. Another exclusion reason was using antihypertensive or vasodilator medica­ tions at baseline (255 men and 612 women), leading to 2980 men and 3628 women. Then we excluded 318 men and 442 women, due to baseline missing data on RHR, fasting plasma glucose (FPG), 2-h post-challenge plasma glucose (2  h PG), and related covariates (considering overlap features). Finally, after further exclusion of 231 men and 276 women because of no follow up data, 2431 men and 2910 women were eligible to enter the analysis. Besides well-known T2DM risk factors, including obe­ sity, physical inactivity, dietary factors, and genetic sus­ ceptibility [7], resting heart rate (RHR) has been shown to be significantly associated with incident T2DM and prediabetes [8–11]. This association has been suggested to be primarily attributed to the insulin resistance (IR) mediated by sympathetic/parasympathetic system [8]. Two previous meta-analyses showed that an increase of 10 beats per minute (bpm) was accompanied with approximately 20% higher T2DM risk; a similar signifi­ cant higher risk was also reported for high RHR catego­ ries versus the lowest categories [8, 10]. However, results from these two meta-analyses had a highly significant heterogeneity (all I2 were about 90%) [8, 10]; the relation­ ship between RHR and incident T2DM was more promi­ nent among Asian populations, compared to Western ones [8]. Study design and populationh The Tehran Lipid and Glucose Study (TLGS), conducting since 1999, is a prospective population-based study about epidemiological features of non-communicable diseases (NCDs). Data from more than 15,000  Tehranian resi­ dents of district 13 has been collected during the recruit­ ment phases. Then it was planned to follow participants by about 3-year intervals [12]. The TLGS aims to make prevention of NCDs through a healthier lifestyle [12]. For the current study, the second phase of TLGS (Octo­ ber 20, 2001 to September 22, 2005) was considered as enrollment. Data gathering for follow-up was carried out in phase III (2005–2008), phase IV (2008–2011), phase V Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. BMC Public Health (2023) 23:2112 (2023) 23:2112 Page 2 of 9 Page 2 of 9 Moazzeni et al. BMC Public Health Clinical and laboratory measurement Using standard questionnaires, data on age, educational level, smoking habits, physical activity, as well as medi­ cal records (history of major illnesses, medication usage, family history of cardiovascular disease and diabetes) obtained. Weight and height were measured while par­ ticipants were wearing light clothes and in a stand­ ing position. Body mass index (BMI) was calculated as weight [kg]/ (height [m])2. We also measured waist cir­ cumference (WC) at the level of the umbilicus with light clothing. Additionally, after 15 min of rest, blood pres­ sure (BP) was assessed twice in a seated position, using manual sphygmomanometer. Through radial palpation, RHR was assessed twice and counted over 60-s periods. The mean of two numbers was considered as pulse rate. h All study participants were asked to fast on the day of the visit for at least 12 h before blood sampling. From all individuals, blood samples were taken with a standard method and in a sitting position. To assess 2 h PG, 82.5 g glucose monohydrate solution (equivalent to 75 g anhy­ drous glucose) was orally taken by individuals who had not a history of taking any glucose-lowering medications. Further details on standard methods for the measure­ ment of 2  h PG, FPG, high-density lipoprotein choles­ terol (HDL-C), and triglycerides (TG) were expressed elsewhere [12]. Therefore, the current study has the aim of investiga­ tion of sex-specific relationship between T2DM devel­ opment and RHR (assessed through palpation) among non-diabetic and prediabetic Iranian adults, using data from the oldest cohort in the MENA region. Background As far as we know, there is no previous study that examined the corresponding relationship in the MENA region. Statistical analysis Baseline demographic and clinical characteristics across the quintiles of RHR are displayed as mean ± standard deviation (SD), median (interquartile range: IQR), and number (%) for normally distributed continuous, highly skewed, and categorical variables, respectively. Chi- square and ANOVA tests were employed to compare baseline characteristics among different groups. During a median follow-up of 12.2 years (IQR: 11.0- 13.3), 313 men and 375 women developed T2DM. Sex- specific multivariable HRs for the association of resting heart rate with incident T2DM is reported in Table  3. Among men, compared to the first quintile (< 68 bpm), those who had RHR of over 78 bpm were at higher age- adjusted risk. After adjustment for age, BMI, WC, edu­ cational level, low physical activity, current smoking, prevalent hypertension, family history of diabetes, and TG/HDL-C in model 3, this higher risk remained sig­ nificant for 5th quintile with the HR of 1.55 (95% CI: 1.06–2.26). Moreover, even after further adjustment for baseline FPG, men with RHR of ≥ 84 bpm (5th quintile) had 69% significantly higher risk with the HR of 1.69 (1.16–2.47). Among men, trend of HRs across quin­ tiles was also significant in all models (all P-values were < 0.05). Furthermore, considering RHR as a continuous variable, an increase of 10 bpm caused 17% significantly higher risk in model 4 [HR: 1.17 (1.05–1.30)]. Among women, on the other hand, there was no significant asso­ ciation between incident T2DM and RHR (both as a cat­ egorical or continuous variable). f Time-to-event was considered as the time of event occurring or censoring, whichever came first. Partici­ pants were censored if they died during the follow-up, left the district, or did not develop T2DM until the end of phase VI (April 2018). Survival time for the censored individuals was the interval between the first and the last observation dates. The event date for the cases of inci­ dent T2DM was considered as the mid-time between the date of follow-up visit at which T2DM was detected for the first time, and the most recent follow-up visit preced­ ing the diagnosis. The Cox proportional hazard models were employed to assess the association of RHR with incident T2DM by calculating the sex-specific hazard ratios (HRs) with 95% confidence intervals (CIs). Four models were used in this analysis: Model 1 adjusted for age. Definition of terms In this study, the level of education was sorted into 3 levels of ≤ 6, 6 to 12, and over 12 years of formal edu­ cation. Subjects were also classified in two groups of current smokers versus previous/non-smokers. Hypertension was considered systolic blood pressure (SBP) ≥ 140 mmHg, or diastolic blood pressure (DBP) ≥ 90 mmHg, or taking anti-hypertensive drugs [13]. Page 3 of 9 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. BMC Public Health (2023) 23:2112 Moreover, using the Modifiable Activity Questionnaire (MAQ), individuals who had less than 1500 min per week of metabolic equivalent tasks were considered as the low physical activity group [12, 14]. assumptions were appropriate. Statistical analyses were employed by the STATA version 14 (Stata Corp LP, Col­ lege Station, Texas) statistical software. P-values < 0.05 were considered statistically significant. T2DM was considered as using glucose lowering drugs (GLDs), or FPG of ≥ 7 mmol/L, or 2 h PG ≥ 11.1 mmol/L. Prediabetes was also considered as 7 mmol/L > FPG ≥ 5.6 mmol/L, or 11.1 mmol/L > 2 h PG ≥ 7.7 mmol/L [15]. Resultsh The mean age of the male and female participants was 41.5 and 39.1 years, respectively. Baseline characteris­ tics of the male and female participants across quantiles of RHR are presented in Tables  1 and 2, respectively. For continuous variables, there were significant differ­ ences in cardiometabolic profile across RHR quantiles among men except for age and HDL-C; however, gener­ ally, there was no significant corresponding difference among women. Moreover, generally, in the higher RHR quantiles, the prevalence of low physical activity was increased in both sexes. Homeostasis Model Assessment of Insulin Resis­ tance (HOMA-IR) was determined using the formula: FPG(mmol/L)*fasting insulin(Mu/ml)/22.5 [16]. Statistical analysis Model 2 adjusted for traditional T2DM risk factors including age, WC, BMI, educational level, current smoking, low physical activ­ ity, family history of diabetes, and hypertension. Model 3: Model 2 + further adjusted for TG/HDL-C ratio; Model 4: Model 3 + further adjusted for FPG. For categorization of RHR, the main exposure of this study, we considered 5 quintiles for each gender separately (the lowest quin­ tile as reference). Considering RHR as a continuous vari­ able, the effect sizes were also calculated for an increase of 10 bpm. As a sensitivity analysis, we also adjusted our models with HOMA-IR for the subgroup of 1394 men and 1849 women with available date for baseline fasting insulin. Due to a significant interaction between sex and RHR for T2DM development (all P-values were < 0.025 through all models), all analyses were done separately for each gender.h As a sensitivity analysis, after adjustment for HOMA- IR, an increase of 10  bpm had HRs of 1.09 (0.94–1.27, P-value: 0.24) and 1.01 (0.90–1.14, P-value: 0.83) among a subgroup of 1394 men and 1849 women, respectively. As a secondary analysis, we evaluated the relation of RHR with incident T2DM among prediabetic partici­ pants (Table 4). Generally, among male and female par­ ticipants, the relationship was similar to non-diabetic ones; although there was no significant association among women with prediabetes at baseline, an increase of 10 bpm was associated with 26% significantly higher risk of T2DM development among prediabetic men in model 4 [HR: 1.26 (1.09–1.46)]. Moreover, compared to the prediabetic men with RHR of < 68 bpm, RHR of The Cox models’ proportionality was measured using the Schoenfeld residual test. All proportionality Moazzeni et al. BMC Public Health (2023) 23:2112 Page 4 of 9 Moazzeni et al. Statistical analysis BMC Public Health Page 4 of 9 Table 1  Baseline characteristics according to the resting heart rate quantiles among men: Tehran Lipid and Glucose Study Q1 Q2 Q3 Q4 Q5 P-value* Number of participants 374 590 355 520 592 Resting heart rate range < 68 68–74 74–78 78–84 ≥ 84 Continuous variables, Mean ± SD Age (year) 42.84 ± 14.52 41.46 ± 13.50 42.37 ± 13.99 41.00 ± 14.28 40.53 ± 38.00 0.087 BMI (kg/m2) 25.67 ± 3.90 25.91 ± 3.91 26.28 ± 3.88 26.19 ± 4.37 26.54 ± 4.43 0.013 Waist circumference (cm) 91.25 ± 10.30 91.82 ± 10.55 93.14 ± 10.28 92.68 ± 11.39 93.38 ± 11.46 0.014 SBP (mmHg) 113.61 ± 12.56 115.17 ± 14.10 115.10 ± 15.38 116.05 ± 14.06 119.74 ± 15.92 < 0.001 DBP (mmHg) 72.12 ± 9.68 74.13 ± 9.66 73.87 ± 9.84 74.83 ± 10.00 77.50 ± 10.68 < 0.001 FPG (mmol/L) 5.01 ± 0.50 4.99 ± 0.47 5.03 ± 0.49 5.08 ± 0.51 5.01 ± 0.50 0.019 HDL-C (mmol/L) 0.94 ± 0.26 0.92 ± 0.21 0.91 ± 0.22 0.90 ± 0.23 0.92 ± 0.22 0.127 TG (mmol/L) 1.66 ± 0.94 1.81 ± 1.11 1.87 ± 0.98 2.06 ± 1.46 1.90 ± 1.08 < 0.001 Categorical variables, n (%) Education level, years 0.281   - ≤ 6 77 (20.6%) 99 (16.8%) 84 (23.7%) 98 (18.8%) 103 (17.4%)   - 6–12 218 (58.3%) 365 (61.9%) 198 (55.8%) 301 (57.9%) 362 (61.1%)   - > 12 79 (21.1%) 126 (21.3%) 73 (20.5%) 121 (23.3%) 127 (21.5%) Current smoker, yes 129 (34.5%) 161 (27.3%) 96 (27.0%) 125 (24.0%) 120 (20.3%) < 0.001 Low physical activity, yes 208 (55.6%) 344 (58.3%) 229 (64.5%) 309 (59.4%) 405 (68.4%) < 0.001 Family history of DM, yes 102 (27.3%) 174 (29.5%) 89 (25.1%) 142 (27.3%) 183 (30.9%) 0.324 SD: Standard deviation; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; FPG: fasting plasma glucose; HDL-C: high density lipoprotein cholesterol; TG: triglycerides; DM: diabetes mellitus. Values are shown as Mean ± SD and number (%), for continuous and categorical variables, respectively; for TG values are shown as Median (interquartile range). * The comparison p-value between groups was calculated using ANOVA test for continues variables and chi-square test for categorical variables. p p g y Values are shown as Mean ± SD and number (%), for continuous and categorical variables, respectively; for TG values are shown as Median (interquartile range). n ± SD and number (%), for continuous and categorical variables, respectively; for TG values are shown as Median (interquartile range). e between groups was calculated using ANOVA test for continues variables and chi-square test for categorical variables. are shown as Mean ± SD and number (%), for continuous and categorical variables, respectively; for TG values are shown as Median (inte ( ), g , p y; ( q g ) * The comparison p-value between groups was calculated using ANOVA test for continues variables and chi-square test for categorical variables. hown as Mean ± SD and number (%), for continuous and categorical variables, respectively; for TG values are shown as Median (interqua arison p-value between groups was calculated using ANOVA test for continues variables and chi-square test for categorical variables. ues are shown as Mean ± SD and number (%), for continuous and categorical variables, respectively; for TG values are shown as Median he comparison p-value between groups was calculated using ANOVA test for continues variables and chi-square test for categorical va Statistical analysis * The comparison p-value between groups was calculated using ANOVA test for continues variables and chi-square test for categorical variables. Table 2  Baseline characteristics according to the resting heart rate quantiles among Women: Tehran Lipid and Glucose Study Q1 Q2 Q3 Q4 Q5 P-value* Number of participants 550 339 702 598 721 Resting heart rate range < 76 76–80 80–86 86–92 ≥ 92 Continuous variables, Mean ± SD Age (year) 41.17 ± 12.20 40.32 ± 11.67 38.69 ± 12.41 38.04 ± 12.69 38.13 ± 12.18 < 0.001 BMI (kg/m2) 28.03 ± 4.54 27.92 ± 4.50 27.33 ± 4.82 27.79 ± 5.09 27.65 ± 5.12 0.087 Waist circumference (cm) 88.40 ± 11.79 87.97 ± 11.81 86.88 ± 12.59 87.76 ± 13.15 87.64 ± 12.69 0.299 SBP (mmHg) 109.57 ± 15.39 109.04 ± 13.58 110.24 ± 14.57 110.47 ± 14.11 110.84 ± 15.80 0.332 DBP (mmHg) 72.00 ± 9.80 71.68 ± 8.96 72.86 ± 9.28 73.16 ± 9.23 72.66 ± 9.88 0.090 FPG (mmol/L) 4.95 ± 0.52 4.93 ± 0.49 4.90 ± 0.50 4.89 ± 0.47 4.90 ± 0.50 0.203 HDL-C (mmol/L) 1.08 ± 0.27 1.10 ± 0.29 1.07 ± 0.29 1.10 ± 0.28 1.09 ± 0.27 0.332 TG (mmol/L) 1.46 ± 1.27 1.55 ± 1.01 1.54 ± 0.90 1.54 ± 0.92 1.57 ± 0.95 0.270 Categorical variables, n (%) Education level, years 0.754   - ≤ 6 159 (28.9%) 90 (26.5%) 175 (24.9%) 147 (24.6%) 188 (26.1%)   - 6–12 309 (56.2%) 206 (60.8%) 418 (59.5%) 360 (60.2%) 425 (58.9%)   - > 12 82 (14.9%) 43 (12.7%) 109 (15.6%) 91 (15.2%) 108 (15.0%) Current smoker, yes 25 (4.5%) 9 (2.7%) 21 (3.0%) 12 (2.0%) 15 (2.1%) 0.062 Low physical activity, yes 319 (58.0%) 194 (57.2%) 438 (62.4%) 405 (67.7%) 473 (65.6%) 0.001 Family history of DM, yes 171 (31.1%) 101 (29.8%) 194 (27.6%) 180 (30.1%) 226 (31.3%) 0.590 SD: Standard deviation; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; FPG: fasting plasma glucose; HDL-C: high density lipoprotein cholesterol; TG: triglycerides; DM: diabetes mellitus. Values are shown as Mean ± SD and number (%), for continuous and categorical variables, respectively; for TG values are shown as Median (interquartile range). * The comparison p-value between groups was calculated using ANOVA test for continues variables and chi-square test for categorical variables. Page 5 of 9 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. Discussion In this prospective population-based cohort study, dur­ ing over a decade of follow up, we found a significant interaction between sex and RHR for the T2DM risk. After adjustment for traditional T2DM risk factors, TG/ HDL-C ratio, and baseline FPG, an increase of 10 bpm was associated with about 17% higher risk of T2DM development among men. Moreover, compared to RHR of < 68 bpm, men with RHR of ≥ 84 bpm had about 70% increased risk of incident T2DM; however, this higher risk attenuated to an insignificant level after adjustment for HOMA-IR. Among women, on the other hand, there is no association between RHR and T2DM development. Furthermore, the relationship between RHR and T2DM risk among prediabetic men and women was also similar to non-diabetic ones. Although several studies have been published about the association of RHR and T2DM, their findings are not completely comparable to ours, due to differences in study design, study setting, outcome assessment, level of adjustment, and other methodological aspects. Similar to our findings for men, in a prospective cohort study of 31,156 male health professionals, highest versus lowest categories of RHR had an about 70% increased risk of T2DM development; moreover, an increase of 10 bpm showed 19% higher risk [10]. In a meta-analysis of the mentioned study [10] and another 13 prospective cohort studies, a positive association between RHR and T2DM risk was found; the summary relative risk (RR) per 10 bpm increase was 1.17 (95% CI, 1.09–1.26); more­ over, the summary RR for highest versus lowest RHR was reported to be 1.44 (1.20–1.74) in the meta-analysis [10]. Similarly, two other meta-analyses [8, 11] showed higher risk of T2DM for increased range of RHR. Findings of recently published cohort studies from Asian countries were also similar [17–19]. In contrast to a meta-analysis study [8] and some other previous studies that did not find a significant interac­ tion between RHR and sex on the T2DM risk [11, 18, 30], this interaction was significant in the current study, even after adjustment for age, BMI,  WC, educational level, low physical activity, current smoking, prevalent hypertension, family history of diabetes, TG/HDL-C ratio, and FPG. Despite a significant association between RHR and T2DM among men, our female participants did not show any significant difference in T2DM risk across RHR quantiles. Statistical analysis BMC Public Health Table 3  Multivariable hazard ratios (HR) and 95% confidence intervals (CI) for the association of resting heart rate with incident type 2 diabetes mellitus: Tehran Lipid and Glucose Study Quantile Range (bpm) E/N Model 1 Model 2 Model 3 Model 4  h (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Men First Quantile < 68 40/374 Reference Reference Reference Reference Second Quantile 68–74 64/590 1.11 (0.75–1.65) 0.598 1.08 (0.73–1.61) 0.696 1.08 (0.72–1.60) 0.719 1.15 (0.77–1.72) 0.489 Third Quantile 74–78 40/355 1.12 (0.72–1.74) 0.608 1.08 (0.69–1.67) 0.748 1.05 (0.68–1.63) 0.831 1.21 (0.78–1.89) 0.389 Fourth Quantile 78–84 76/520 1.56 (1.06–2.29) 0.023 1.49 (1.01–2.19) 0.045 1.43 (0.97–2.11) 0.072 1.33 (0.90–1.96) 0.159 Fifth Quantile ≥ 84 93/592 1.76 (1.21–2.54) 0.003 1.57 (1.07–2.30) 0.020 1.55 (1.06–2.26) 0.025 1.69 (1.16–2.47) 0.007 P-value for trend 0.004 0.032 0.048 0.049 Increase of 10 bpm 313/2431 1.21 (1.09–1.34) < 0.001 1.17 (1.05–1.30) 0.005 1.16 (1.04–1.30) 0.007 1.17 (1.05–1.30) 0.005 Women First Quantile < 76 80/550 Reference Reference Reference Reference Second Quantile 76–80 46/339 0.98 (0.68–1.41) 0.931 0.98 (0.68–1.41) 0.900 0.92 (0.64–1.32) 0.648 1.00 (0.70–1.44) 0.993 Third Quantile 80–86 84/702 0.90 (0.66–1.22) 0.502 0.90 (0.66–1.23) 0.514 0.88 (0.65–1.20) 0.418 0.97 (0.71–1.32) 0.838 Fourth Quantile 86–92 70/598 0.93 (0.67–1.28) 0.645 0.83 (0.60–1.15) 0.264 0.80 (0.58–1.11) 0.185 0.88 (0.64–1.21) 0.430 Fifth Quantile ≥ 92 95/721 1.07 (0.79–1.44) 0.674 0.96 (0.71–1.30) 0.806 0.92 (0.68–1.25) 0.608 0.97 (0.72–1.31) 0.845 P-value for trend 0.822 0.813 0.758 0.941 Increase of 10 bpm 375/2910 1.02 (0.93–1.12) 0.658 0.98 (0.89–1.08) 0.656 0.97 (0.88–1.07) 0.532 0.98 (0.89–1.08) 0.661 E: event; N: number; bpm: beat per minute. Model 1: Adjusted for age. Model 2: adjusted for age, body mass index, waist circumference, educational level, low physical activity, current smoking, prevalent hypertension, and family history of diabetes. Model 3: Model 2 + further adjusted for Triglycerides/ High-density lipoprotein cholesterol ratio (TG/HDL-C). p p Model 1: Adjusted for age. Model 2: adjusted for age, body mass index, waist circumference, educational level, low physical activity, current smoking, prevalent hypertension, and family history of diabetes. Model 3: Model 2+further adjusted for Triglycerides/ High-density lipoprotein cholesterol ratio (TG/HDL-C). Moazzeni et al. BMC Public Health (2023) 23:2112 Page 6 of 9 (2023) 23:2112 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. Statistical analysis BMC Public Health Several different mechanisms were introduced pre­ viously for the explanation of the association between increased RHR and risk of T2DM development that mostly attributed to insulin resistance (IR) induction by autonomic system [10]. Since RHR is an indicator of autonomic activity [21], higher RHR indicates a change in the sympathetic-parasympathetic balance in favor of the sympathetic. It causes glucose metabolism dysregulation through: (1) reduced insulin secretion, (2) reduced skel­ etal muscle glucose uptake by vasoconstriction, and (3) elevated IR in the skeletal muscle cells, by the stimulation of renin-angiotensin-aldosterone system [8, 10, 22, 23]. It should be noted that based on a meta-analysis, it was sug­ gested that baseline glucose and/or IR accounts for part, though not all of the RHR and incident T2DM relation [8]. Recently, it was reported that only 27.5% of the RHR effect on incident T2DM was explained by the indirect effect of IR [24]. Importantly, chronic sympathetic over- activity was linked to high blood pressure, obesity, and the metabolic syndrome that all of them are accompa­ nied by T2DM development, on the basis of high inflam­ matory state [25]. In our models, even after adjustment for obesity, hypertension, TG/HDL-C ratio and baseline FPG, the association remained significant among men; however, after adjustment for HOMA-IR, this higher risk did not remain significant. Moreover, lower RHR is a potential marker of better cardiorespiratory fitness [26], which can provide protection against cardio-metabolic diseases including T2DM [27]; however, the low-physi­ cal-activity, which was adjusted in our models, could not completely evaluate the cardiorespiratory fitness. Finally, evidence of genetic causal correlations between RHR and T2DM/cardiometabolic traits was also found [28]. Nev­ ertheless, further investigations are needed yet to clarify this complex relationship [29]. > 80  bpm showed significant increased risk of incident T2DM in model 4. Discussion Based on a prospective cohort study among Inner Mongolian, a similar significant interac­ tion was also found for sex; however, higher quartile of RHR caused higher T2DM risk in both genders, although it is more prominent among men [19]. Similarly, some other studies also reported this higher impact of RHR on T2DM risk among men [31, 32].ff Furthermore, in the current study, we evaluated the relationship of RHR with risk of progression from pre­ diabetes to T2DM; similar to non-diabetic participants, increased RHR caused significantly higher risk of T2DM development only among men. Similarly, in another lon­ gitudinal study, it was shown that higher RHR at baseline was associated with a modestly increased incidence rate of T2DM among American overweight adults with pre­ diabetes [20]. Additionally, from a prospective study from China, the researchers have found that fasting RHR was associated with higher risk of progression from impaired fasting glucose to diabetes [11]; however, their findings were significant not only for men but also for women, which was different from our findings [11]. Several physiological differences may explain differ­ ent findings in males and females. Firstly, sex steroid Page 7 of 9 Page 7 of 9 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. Data Availability Th d d Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. As strengths, this is the first prospective study inves­ tigating the impact of RHR on incident T2DM in the MENA region, with a high burden of T2DM. Another strength of our study is adjusting sex-specific models for several potential confounders. We also acknowledge sev­ eral limitations. First, in the current study, RHR was mea­ sured by radial pulse counting over 60-s periods that was less accurate than using electrocardiogram which mea­ sures the heart rate directly; this issue may become more important in older age when atherosclerosis is more involved. Second, the present study only included Tehra­ nian citizens; hence, the results may be unable to be gen­ eralizable to the other ethnicities or rural populations. Ethics approval and consent to participate This study was approved by the Institutional Review Board (IRB) of the Research Institute for Endocrine Sciences (RIES), Shahid Beheshti University of Medical Sciences, and all participants provided written informed consent. All methods were done in accordance with the relevant guidelines and regulations. Funding g No funding from any source was obtained for this study. Received: 26 March 2023 / Accepted: 19 October 2023 Received: 26 March 2023 / Accepted: 19 October 2023 Received: 26 March 2023 / Accepted: 19 October 2023 Abbreviations T2DM Type 2 diabetes mellitus DALYs Disability-adjusted life years MENA Middle East and North Africa RHR Resting heart rate IR Insulin resistance bpm Beats per minute TLGS Tehran Lipid and Glucose Study NCDs Non-communicable diseases CVD Cardiovascular disease FPG Fasting plasma glucose 2h PG 2-h post-challenge plasma glucose BMI Body mass index BP Blood pressure HDL-C High-density lipoprotein cholesterol TG Triglyceride SBP Systolic blood pressure Author contributions Study conception and design: S.S.M and F.H; analysis and interpretation of data: M.H, S.S.M, and F.H; drafting of the manuscript: S.S.M, K.K.T, F.G and F.H; critical revision: S.S.M, F.A, M.P, and F.H. All authors read and approved the final manuscript. Discussion BMC Public Health (2023) 23:2112 DBP Diastolic blood pressure MAQ Modifiable Activity Questionnaire GLDs Glucose lowering drugs HOMA-IR Homeostasis Model Assessment of Insulin Resistance SD Standard deviation IQR Interquartile range HRs Hazard ratios CIs Confidence intervals RRs Relative risk DBP Diastolic blood pressure MAQ Modifiable Activity Questionnaire GLDs Glucose lowering drugs HOMA-IR Homeostasis Model Assessment of Insulin Resistance SD Standard deviation IQR Interquartile range HRs Hazard ratios CIs Confidence intervals RRs Relative risk hormones play an important role in protecting women against T2DM development through enhancing insulin sensitivity by activating estrogen receptor α in insulin sensitive tissues such as skeletal muscles, adipose tissue, and hepatocytes [33, 34]. Moreover, higher mitochon­ drial activity in different tissues such as adipose tissue and skeletal muscle in female gender caused further protection against T2DM development [34]. Secondly, considering the autonomic nervous system, which regu­ lates RHR, vagal and parasympathetic activity in female heart is more prominent than male [35]. Oxytocin also increases vagal activity and decreases RHR in women [36]. Furthermore, the association between RHR and elevated levels of all the inflammatory markers is fur­ ther prominent in men than in women [37]. Therefore, in women, RHR may not be as accurate as among men for indication of high inflammatory state and sympathetic- parasympathetic imbalance. Acknowledgements The authors would like to express their appreciation to the TLGS participants and staff for their kind cooperation. Competing interests h h d l The authors declare no competing interests. Conclusion 1Prevention of Metabolic Disorders Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran To sum up, among non-diabetic and prediabetic men, higher RHR was significantly associated with higher risk of incident T2DM. For women, on the other hand, there was no significant relationship. Further studies are needed to determine the added value of RHR as a poten­ tial modifiable risk factor in screening and prediction of incident T2DM. 2Medical student, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Medical student, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 4Department of Cardiology, Shohada-e-Tajrish Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran Discussion BMC Public Health Table 4  Multivariable hazard ratios (HR) and 95% confidence intervals (CI) for the association of resting heart rate with incident type 2 diabetes mellitus among subjects with pre- diabetes at baseline: Tehran Lipid and Glucose Study Quantile Range (bpm) E/N Model 1 Model 2 Model 3 Model 4  h (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Men First Quantile < 68 20/73 Reference Reference Reference Reference Second Quantile 68–75 34/95 1.53 (0.88–2.66) 0.131 1.50 (0.86–2.61) 0.155 1.49 (0.86–2.61) 0.157 1.49 (0.85–2.61) 0.160 Third Quantile 75–80 40/118 1.39 (0.81–2.38) 0.231 1.52 (0.89–2.61) 0.129 1.52 (0.88–2.61) 0.133 1.56 (0.91–2.69) 0.107 Fourth Quantile 80–84 34/78 1.93 (1.11–3.35) 0.020 1.93 (1.10–3.39) 0.021 1.92 (1.10–3.38) 0.023 1.79 (1.01–3.14) 0.045 Fifth Quantile ≥ 84 53/119 2.09 (1.25–3.50) 0.005 2.12 (1.26–3.59) 0.005 2.12 (1.25–3.58) 0.005 2.22 (1.31–3.76) 0.003 P-value for trend 0.039 0.055 0.057 0.046 Increase of 10 bpm 181/483 1.25 (1.08–1.44) 0.002 1.25 (1.08–1.44) 0.003 1.25 (1.08–1.44) 0.003 1.26 (1.09–1.46) 0.002 Women First Quantile < 74 30/76 Reference Reference Reference Reference Second Quantile 74–80 36/90 0.91 (0.56–1.48) 0.706 0.89 (0.55–1.46) 0.652 0.88 (0.54–1.43) 0.600 0.93 (0.57–1.52) 0.772 Third Quantile 80–89 59/147 1.04 (0.67–1.61) 0.872 0.99 (0.64–1.54) 0.963 0.97 (0.62–1.51) 0.882 1.09 (0.70–1.70) 0.699 Fourth Quantile 89–94 50/102 1.29 (0.82–2.02) 0.277 1.19 (0.76–1.88) 0.451 1.14 (0.72–1.80) 0.584 1.24 (0.78–1.96) 0.367 Fifth Quantile ≥ 94 45/112 1.03 (0.65–1.64) 0.899 0.88 (0.55–1.41) 0.598 0.88 (0.55–1.42) 0.609 0.95 (0.59–1.52) 0.824 P-value for trend 0.584 0.619 0.741 0.654 Increase of 10 bpm 220/527 1.03 (0.91–1.16) 0.480 0.99 (0.88–1.12) 0.884 0.99 (0.88–1.12) 0.870 1.01 (0.90–1.14) 0.859 E: event; N: number; bpm: beat per minute. Model 1: Adjusted for age. Model 2: adjusted for age, body mass index, waist circumference, educational level, low physical activity, current smoking, prevalent hypertension, and family history of diabetes. Model 3: Model 2 + further adjusted for Triglycerides/ High-density lipoprotein cholesterol ratio (TG/HDL-C). Model 4: Model 3 + further adjusted for fasting plasma glucose. Page 8 of 9 Page 8 of 9 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. References Diabet Medicine: J Br Diabet Association. 2017;34(1):69–78. 25. Esser N, Legrand-Poels S, Piette J, Scheen AJ, Paquot N. Inflammation as a link between obesity, metabolic syndrome and type 2 Diabetes. Diabetes Res Clin Pract. 2014;105(2):141–50. 7. Bellou V, Belbasis L, Tzoulaki I, Evangelou E. Risk factors for type 2 Diabetes Mellitus: an exposure-wide umbrella review of meta-analyses. PLoS ONE. 2018;13(3):e0194127. 7. Bellou V, Belbasis L, Tzoulaki I, Evangelou E. Risk factors for type 2 Diabetes Mellitus: an exposure-wide umbrella review of meta-analyses. PLoS ONE. 2018;13(3):e0194127. 26. Sandvik L, Erikssen J, Thaulow E, Erikssen G, Mundal R, Rodahl K. Physical fitness as a predictor of mortality among healthy, middle-aged Norwegian men. N Engl J Med. 1993;328(8):533–7. 8. Aune D, B ÓH, Vatten LJ. Resting heart rate and the risk of type 2 Diabetes: a systematic review and dose–response meta-analysis of cohort studies. Nutr Metab Cardiovasc Dis. 2015;25(6):526–34. 8. Aune D, B ÓH, Vatten LJ. Resting heart rate and the risk of type 2 Diabetes: a systematic review and dose–response meta-analysis of cohort studies. Nutr Metab Cardiovasc Dis. 2015;25(6):526–34. 27. Blair SN, Kohl HW, Paffenbarger RS, Clark DG, Cooper KH, Gibbons LW. Physi­ cal fitness and all-cause mortality: a prospective study of healthy men and women. JAMA. 1989;262(17):2395–401. 9. Zhang SY, Wu JH, Zhou JW, Liang Z, Qiu QY, Xu T, Zhang MZ, Zhong CK, Jiang W, Zhang YH. Overweight, resting heart rate and prediabetes/diabetes: a population-based prospective cohort study among inner mongolians in China. Sci Rep. 2016;6(1):23939. 28. Guo Y, Chung W, Zhu Z, Shan Z, Li J, Liu S, Liang L. Genome-wide Assessment for resting Heart Rate and Shared Genetics with Cardiometabolic traits and Type 2 Diabetes. J Am Coll Cardiol. 2019;74(17):2162–74. 10. Lee DH, de Rezende LFM, Hu FB, Jeon JY, Giovannucci EL. Resting heart rate and risk of type 2 Diabetes: a prospective cohort study and meta-analysis. Diabetes Metab Res Rev. 2019;35(2):e3095. 29. Munroe PB, Ramírez J, van Duijvenboden S. Resting heart rate and type 2 Diabetes: a complex relationship in need of Greater understanding. J Am Coll Cardiol. 2019;74(17):2175–7. 30. Liu D, Qin P, Liu Y, Sun X, Li H, Wu X, Zhang Y, Han M, Qie R, Huang S. Sex- specific association of resting heart rate with type 2 Diabetes Mellitus. J Diabetes Complicat. 2020;34(12):107754. 11. References 1. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet (London, England). 2020, 396(10258):1204–1222. 2. Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, Stein C, Basit A, Chan JCN, Mbanya JC, et al. IDF Diabetes Atlas: Global, regional and country-level Diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res Clin Pract. 2022;183:109119. 3. Khamseh ME, Sepanlou SG, Hashemi-Madani N, Joukar F, Mehrparvar AH, Faramarzi E, Okati-Aliabad H, Rahimi Z, Rezaianzadeh A, Homayounfar R, et al. Nationwide Prevalence of Diabetes and Prediabetes and Associated Risk factors among Iranian adults: analysis of data from PERSIAN Cohort Study. Page 9 of 9 Page 9 of 9 Moazzeni et al. BMC Public Health (2023) 23:2112 Moazzeni et al. BMC Public Health Moazzeni et al. BMC Public Health (2023) 23:2112 (2023) 23:2112 Diabetes Therapy: Research Treatment and Education of Diabetes and Related Disorders. 2021;12(11):2921–38. 21. Grassi G, Vailati S, Bertinieri G, Seravalle G, Stella ML, Dell’Oro R, Mancia G. Heart rate as marker of sympathetic activity. J Hypertens. 1998;16(11):1635–9. Diabetes Therapy: Research Treatment and Education of Diabetes and Related Disorders. 2021;12(11):2921–38. 22. Julius S, Gudbrandsson T, Jamerson K, Andersson O. The interconnection between sympathetics, microcirculation, and insulin resistance in Hyperten­ sion. Blood Press. 1992;1(1):9–19. 4. Derakhshan A, Sardarinia M, Khalili D, Momenan AA, Azizi F, Hadaegh F. Sex specific incidence rates of type 2 Diabetes and its risk factors over 9 years of follow-up: Tehran lipid and glucose study. PLoS ONE. 2014;9(7):e102563–3. 23. Perin PC, Maule S, Quadri R. Sympathetic nervous system, Diabetes, and Hypertension. Clin Exp Hypertens. 2001;23(1–2):45–55. 5. Moazzeni SS, Ghafelehbashi H, Hasheminia M, Parizadeh D, Ghanbarian A, Azizi F, Hadaegh F. Sex-specific prevalence of coronary Heart Disease among Tehranian adult population across different glycemic status: Tehran lipid and glucose study, 2008–2011. BMC Public Health. 2020;20(1):1510. 24. Saito I, Maruyama K, Kato T, Takata Y, Tomooka K, Kawamura R, Osawa H, Tan­ igawa T. Role of insulin resistance in the association between resting heart rate and type 2 Diabetes: a prospective study. J Diabetes Complications. 2022;36(11):108319. f y glucose study, 2008–2011. BMC Public Health. 2020;20(1):1510. 6. Hadaegh F, Derakhshan A, Zafari N, Khalili D, Mirbolouk M, Saadat N, Azizi F. Pre-diabetes tsunami: incidence rates and risk factors of pre-diabetes and its different phenotypes over 9 years of follow-up. References Wang L, Cui L, Wang Y, Vaidya A, Chen S, Zhang C, Zhu Y, Li D, Hu FB, Wu S, et al. Resting heart rate and the risk of developing impaired fasting glucose and Diabetes: the Kailuan prospective study. Int J Epidemiol. 2015;44(2):689–99. 31. Nagaya T, Yoshida H, Takahashi H, Kawai M. Resting heart rate and blood pressure, Independent of each other, proportionally raise the risk for type-2 Diabetes Mellitus. Int J Epidemiol. 2010;39(1):215–22. 12. Azizi F, Ghanbarian A, Momenan AA, Hadaegh F, Mirmiran P, Hedayati M, Mehrabi Y, Zahedi-Asl S. Prevention of non-communicable Disease in a popu­ lation in nutrition transition: Tehran lipid and glucose study phase II. Trials. 2009;10(1):1–15. 32. Grantham N, Magliano D, Tanamas SK, Söderberg S, Schlaich M, Shaw J. Higher heart rate increases risk of Diabetes among men: the Australian Diabetes obesity and lifestyle (AusDiab) Study. Diabet Med. 2013;30(4):421–7. 13. Unger T, Borghi C, Charchar F, Khan NA, Poulter NR, Prabhakaran D, Ramirez A, Schlaich M, Stergiou GS, Tomaszewski M. 2020 International Society of Hypertension global Hypertension practice guidelines. Hypertension. 2020;75(6):1334–57. 33. Tramunt B, Smati S, Grandgeorge N, Lenfant F, Arnal J-F, Montagner A, Gourdy P. Sex differences in metabolic regulation and Diabetes susceptibility. Diabe­ tologia. 2020;63(3):453–61. 14. Momenan AA, Delshad M, Sarbazi N, Rezaei_Ghaleh N, Ghanbarian A, Azizi F. Reliability and validity of the modifiable activity questionnaire (MAQ) in an Iranian urban adult population. Arch Iran Med. 2012;15(5):279–82. 34. Goossens GH, Jocken JW, Blaak EE. Sexual dimorphism in cardiometabolic health: the role of adipose tissue, muscle and liver. Nat Reviews Endocrinol. 2021;17(1):47–66.f p p 15. Association AD. Diagnosis and classification of Diabetes Mellitus. Diabetes Care. 2010;33(Supplement1):62–S69. 35. Koenig J, Thayer JF. Sex differences in healthy human heart rate variability: a meta-analysis. Neurosci Biobehavioral Reviews. 2016;64:288–310. 16. Khalili D, Khayamzadeh M, Kohansal K, Ahanchi NS, Hasheminia M, Hadaegh F, Tohidi M, Azizi F, Habibi-Moeini AS. Are HOMA-IR and HOMA-B good predictors for Diabetes and pre-diabetes subtypes? BMC Endocr Disorders. 2023;23(1):1–9. 36. Higa KT, Mori E, Viana FF, Morris M, Michelini LC. Baroreflex control of heart rate by oxytocin in the solitary-vagal complex. Am J Physiology-Regulatory Integr Comp Physiol. 2002;282(2):R537–45. 17. Long T, Wang J, Han X, Wang F, Hu H, Yu C, Yuan J, Yao P, Wei S, Wang Y, et al. Association between resting heart rate and incident Diabetes risk: a mende­ lian randomization study. Acta Diabetol. 2019;56(9):1037–44. 37. References Whelton SP, Narla V, Blaha MJ, Nasir K, Blumenthal RS, Jenny NS, Al-Mallah MH, Michos ED. Association between resting heart rate and inflamma­ tory biomarkers (high-sensitivity C-reactive protein, interleukin-6, and fibrinogen)(from the multi-ethnic study of Atherosclerosis). Am J Cardiol. 2014;113(4):644–9. 18. Wang W, Wang J, Lv J, Yu C, Shao C, Tang Y, Guo Y, Bian Z, Du H, Yang L. Association of heart rate and Diabetes among 0.5 million adults in the China Kadoorie biobank: results from observational and mendelian randomization analyses. Nutr Metabolism Cardiovasc Dis. 2021;31(8):2328–37. Publisher’s Note 19. Wang T, Zhang W, Zhang M, Zhang Y, Zhang S. Higher heart rates increase risk of Diabetes and cardiovascular events: a prospective cohort study among inner mongolians. Diabetes Metab. 2020;46(1):20–6. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 20. Carnethon MR, Prineas RJ, Temprosa M, Zhang ZM, Uwaifo G, Molitch ME. The association among autonomic nervous system function, incident Diabetes, and intervention arm in the Diabetes Prevention Program. Diabetes Care. 2006;29(4):914–9.

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Description of decorative finishes

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Panel No. 1—Tiffany Glazed Effect for Living Room. Stencil Novelty Company, 13 East 14th Street, New York City. Molding around edge—Orchid Dulamel. Baseboard and Crown Molding—Orchid Utilac. Top Molding— 1 part Old Rose and 8 parts White Sani- Flat. Two bands above baseboard and below crown molding painted with Weatherproof Aluminum Paint. Stencil Novelty Company, 13 East 14th Street, New York City. Molding around edge—Orchid Dulamel. Baseboard and Crown Molding—Orchid Utilac. Top Molding— 1 part Old Rose and 8 parts White Sani- Flat. Ground Color—Light Buff Sani-Flat glazed with Cobalt Blue and Turkey Red Pure Oil Colors. Panel—Muresco #5, Light Green. Panel—Muresco #5, Light Green. Inner Molding—Equal parts of Light Gray and White Sani-Flat. Molding around edge. Baseboard and Top Molding— Ivory Utilac. Upper Wall—Light Blue Dulamel. Upper Wall—Light Blue Dulamel. Panel— 1 part Light Gray and 4 parts White Sani-Flat. Stile—Equal parts Light Gray and White Sani-Flat. Panel— 1 part Light Gray and 4 parts White Sani-Flat. Upper Wall—Light Blue Dulamel. Top Molding—Light Blue Dulamel. Decoration of Sea Gulls, soaring at various heights White Sani-Flat, with Black (Utilac) bill, feet and tip of wings. Stencil #923, price $1.20, purchased from the Stencil Novelty Company, I 3 East 1 4th Street, New York City. Wainscoting—Marked off to imitate tile, painted with Peach Dulamel, with alternate blocks in Light Blue Dulamel. Baseboard—Light Blue Utilac. Four feet from floor a darker Blue stripe divides wainscot- ing from the upper wall, and just above this stripe is a Conventional Wave design border in Weatherproof Aluminum Paint. Stencil #922—30c, from Stencil Novelty Company, 1 3 East 1 4th Street, New York City. Stile—Equal parts Light Gray and White Sani-Flat. Top Molding—Light Blue Dulamel. Decoration of Sea Gulls, soaring at various heights White Sani-Flat, with Black (Utilac) bill, feet and tip of wings. Stencil #923, price $1.20, purchased from the Stencil Novelty Company, I 3 East 1 4th Street, New York City. Baseboard and Top Molding—Light Gray Utilac. Panel No. 3—Modernistic Effect for Bedroom. Upper Wall—Orchid Dulamel. Upper Wall—Orchid Dulamel. Wainscoting—Marked off to imitate tile, painted with Peach Dulamel, with alternate blocks in Light Blue Dulamel. Wainscoting—Marked off to imitate tile, painted with Peach Dulamel, with alternate blocks in Light Blue Dulamel. Baseboard—Light Blue Utilac. Four feet from floor a darker Blue stripe divides wainscot- Divided horizontally in three equal sections. CATALOGUE LfBRARV EFEREMCT R 5 1935 Description of Decorative Finishes As Exhibited At Booth F41 The Better Housing Exposition March 25th to 31st, inclusive Port Authority Commerce Building New York City Beniamin Moore& Co. A 511 Canal St. New^forkCity Paints,Varnishes and Muresco As Exhibited At Booth F41 The Better Housing Exposition March 25th to 31st, inclusive Port Authority Commerce Building New York City Beniamin Moore& Co. A 511 Canal St. New^forkCity Paints,Varnishes and Muresco The Better Housing Exposition March 25th to 31st, inclusive Port Authority Commerce Building New York City Panel No. 5 Cont’d. Panel No. 3 ConCd. Panel No. 1—Tiffany Glazed Effect for Living Room. Ground Color—Light Buff Sani-Flat glazed with Cobalt Blue and Turkey Red Pure Oil Colors. Panel No. 1—Tiffany Glazed Effect for Living Room. Panel No. 6~A Modern Sea Gull Effect for Bathroom. Panel—Muresco #5, Light Green. REFERHNCF CATALOGUE LI8RAR' APR 5 1935 Description of Decorative Finishes As Exhibited At Booth F41 The Better Housing Exposition March 25th to 31st, inclusive Port Authority Commerce Building New York City Benjamin Moore & Co. Paints.Varnishes and Muresco NewYorkCity Panel No. 7 Red, White and Blue Kitchen. Description of Entire Panel, including edge of molding—Ivory Interior Gloss. Decorative Finishes As Exhibited At Booth F41 In connection with Benjamin Moore & Co.’s Exhibit, Miss Betty Moore, interior decorator and well known radio personality, is scheduled to present a daily talk at 4:00 P.M. in the auditorium. Stencil four feet from the floor of a Tea Set Parade done in Bright Red Impervo Enamel. Stencil #950—35c, purchased from the Stencil Novelty Company, 13 East 14th Street, New York City. Top of Molding—Light Blue Utilac. Top of Molding—Light Blue Utilac. Betty Moore will gladly assist you personally with your decorating problem. She will send you color cards, and sug- gest color schemes for interior or exterior painting, if you will write to Baseboard—Royal Blue Utilac. Baseboard—Royal Blue Utilac. Divided horizontally in three equal sections. Imitation Tile Wainscoting—Orchid Utilac marked out in White. The first third, next to baseboard—equal parts of Old Rose and White Sani-Flat. Just above wainscoting are stencilled Tulips and Daffodils. Just above wainscoting are stencilled Tulips and Daffodils. The next third— 1 part Old Rose and 4 parts White Sani- Flat. Four feet from floor a darker Blue stripe divides wainscot- ing from the upper wall, and just above this stripe is a Conventional Wave design border in Weatherproof Aluminum Paint. Stencil #922—30c, from Stencil Novelty Company, 1 3 East 1 4th Street, New York City. Tulips—#372—20c—Old Rose and Lavender Sani- Flat with Light Green Utilac for foliage. Tulips—#372—20c—Old Rose and Lavender Sani- Flat with Light Green Utilac for foliage. Tulips—#372—20c—Old Rose and Lavender Sani- Flat with Light Green Utilac for foliage. The last third— 1 part Old Rose and 8 parts White Sani- Flat. Daffodils—# 1 1 8—20c—^Yellow Utilac with Light Green Utilac for foliage. Daffodils—# 1 1 8—20c—^Yellow Utilac with Light Green Utilac for foliage. Baseboard—Old Rose Sani-Flat (Standard Shade). Baseboard—Old Rose Sani-Flat (Standard Shade). Molding around edge—Weatherproof Aluminum Paint. Panel No. 8—Sunshine Kitchen. The Better Housing Exposition March 25th to 31st, inclusive Port Authority Commerce Building New York City BETTY MOOBE BETTY MOOBE 511 Carnal Street New York City BETTY MOOBE 511 Carnal Street New York City Upper Wall—Light Daflodil Yellow Utilac (!4 pint Yellow to 1 gallon White). Wainscoting—A tone deeper in Yellow Utilac (J4 P*nt Yellow to I quart White). BETTY MOOKE New York City Consult Your Benjamin Moore Dealer Employ A Reliable Painter and Use Paiat Consult Your Benjamin Moore Dealer Employ A Reliable Painter and Use Paiat Stencil four feet from the floor of a Tea Set Parade done in Black Impervo Enamel. Stencil #950—35c, purchased from the Stencil Novelty Company, 13 East 14th Street, New York City. In connection with Benjamin Moore & Co.^s Exhibit^ Miss Betty Moore, interior decorator and well known radio personality, is scheduled to present a daily talk at 4:00 P.M. in the auditorium. Betty Moore will gladly assist you personally with your decorating problem. She will send you color cards, and sug- gest color schemes for interior or exterior painting, if you will write to 511 Carnal Street 511 Carnal Street Consult Your Benjamin Moore Dealer Employ A Reliable Painter and Use Paint

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Cervical cancer screening of underserved women in the United States: results from the National Breast and Cervical Cancer Early Detection Program, 1997–2012

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Cancer Causes Control (2015) 26:671–686 DOI 10.1007/s10552-015-0524-5 ORIGINAL PAPER Cervical cancer screening of underserved women in the United States: results from the National Breast and Cervical Cancer Early Detection Program, 1997–2012 Florence K. L. Tangka • David H. Howard • Janet Royalty • Lucinda P. Dalzell • Jacqueline Miller • Brett J. O’Hara • Susan A. Sabatino • Kristy Joseph • Kristy Kenney • Gery P. Guy Jr. • Ingrid J. Hall Florence K. L. Tangka • David H. Howard • Janet Royalty • Lucinda P. Dalzell • Jacqueline Miller • Brett J. O’Hara • Susan A. Sabatino • Kristy Joseph • Kristy Kenney • Gery P. Guy Jr. • Ingrid J. Hall Received: 11 September 2014 / Accepted: 8 January 2015 / Published online: 18 March 2015  The Author(s) 2015. This article is published with open access at Springerlink.com uninsured women using data from the US Census Bureau. We adjusted our estimates for hysterectomy status using the National Health Interview Survey and the Behavioral Risk Factor Surveillance System. We used data from the NBCCEDP to describe the number of women receiving NBCCEDP-funded screening and calculated the proportion of eligible women who received screening through the NBCCEDP at the national level (by age group, race/eth- nicity) and at the state level by age group. We used the Medical Expenditure Panel Survey to estimate the pro- portion of NBCCEDP-eligible women who were screened outside the NBCCEDP and the proportion that are not screened. Abstract Objective The National Breast and Cervical Cancer Early Detection Program (NBCCEDP) provides breast and cer- vical cancer screens to low-income, uninsured, and un- derinsured women. We describe the number and proportion of women eligible for cervical cancer screening services and the proportion of eligible women screened over the period 1997–2012. Methods Low-income, uninsured, and underinsured women aged 18–64 years who have not had a hysterectomy are eligible for cervical cancer screening through the NBCCEDP. We estimated the number of low-income, Results We estimate that in 2010–2012, 705,970 women aged 18–64 years, 6.5 % (705,970 of 9.8 mil- lion) of the eligible population, received NBCCEDP- funded Pap tests. We estimate that 60.2 % of eligible women aged 18–64 years were screened outside the NBCCEDP and 33.3 % were not screened. The NBCCEDP provided 623,603 screens to women aged 40–64 years, an estimated 16.5 % of the eligible population, and 83,660 screens to women aged 18–39 years, representing an estimated 1.2 % of the eligible population. The estimated proportions of eligible women screened in each state ranged from 1.5 to 32.7 % and 5 % to 73.2 % among the 18–64 and 40–64 years age groups, respectively. Changes in the proportion of eligible women screened over the study period were nonsignificant. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the US Census Bureau. F. K. L. Tangka (&)  J. Royalty  J. Miller  S. A. Sabatino  K. Kenney  G. P. Guy Jr.  I. J. Hall Division of Cancer Prevention and Control, Centers for Disease Control and Prevention (CDC), 4770 Buford Highway NE, Mailstop F-76, Atlanta, GA 30341-3717, USA il FT k @ d D. H. Howard Department of Health Policy and Management, Emory University, 1518 Clifton Road NE, Atlanta, GA 30322, USA L. P. Dalzell  B. J. O’Hara Social, Economics and Household Statistics Division, US Census Bureau, Washington, DC 20233-8510, USA Conclusions Although the program provided cervical screening to over 700,000 women between 2010 and 2012, it served a small percent of those eligible. The proportion of women screened varied substantially across age groups, racial/ethnic groups, and states. Many low-income, unin- sured women are not being screened. K. Joseph Division of Global Health Protection, Center for Global Health, CDC, 4770 Buford Highway NE, Mailstop E-93, Atlanta, GA 30329, USA Introduction The incidence of and mortality from cervical cancer in the USA have declined over time as a result of progress in primary prevention, early-stage disease detection, and treatment [1]. However, there are still opportunities to improve the care of women with cervical cancer. In 2010, 11,918 women were diagnosed with cervical cancer, and 3,939 women died from the disease [2]. y Our previous report [10] was the first to describe the extent to which the NBCCEDP helped meet the cervical cancer screening needs of the underserved population in the USA. Previously, we estimated that the NBCCEDP screened 8.7 % of eligible women aged 18–64 years for cervical cancer over the period 2004–2006. The proportion of women screened varied by age group, race/ethnicity, and across states. We estimated that the NBCCEDP screened 22.6 % of eligible women aged 40–64 years, 2.3 % of eligible women aged 18–39 years, 7.3 % of eligible His- panic women, 6.5 % of eligible non-Hispanic black women, and 9.7 % of eligible non-Hispanic white women [10]. The purpose of this study is to update the 2004–2006 analysis by Tangka et al. [10], using data from 2010 to 2012 and to describe trends in the number of women eligible and proportion of eligible women screened be- tween 1997 and 2012. We also estimated the proportion of women screened by state and by race/ethnicity, the pro- portion of eligible women who received non-NBCCEDP- funded Pap tests, and the proportion of eligible women who are not screened. This report describes the extent of the nation’s only organized screening program provision of cervical cancer screening services to underserved women in the USA over time. Information from this study is cru- cial for understanding the reach of the NBCCEDP, iden- tifying populations that could benefit from better access to screening services, and targeting specific interventions for hard-to-reach women. The United States Preventive Services Task Force (USPSTF) recommends screening for cervical cancer in women aged 21–65 every 3 years with Papanicolaou smear testing (Pap test) or in women aged 30–65 every 5 years with a combination of Pap test and human papillomavirus testing [3]. The Healthy People 2020 objective for cervical cancer screening is to screen 93 % of women aged 21–65 years by the year 2020 [4]. Current screening proportions fall short of this target and, in fact, have exhibited a small but statistically significant decline from 2000–2010. K. Joseph K. Joseph Division of Global Health Protection, Center for Global Health, CDC, 4770 Buford Highway NE, Mailstop E-93, Atlanta, GA 30329, USA 12 3 Cancer Causes Control (2015) 26:671–686 672 Keywords Cervical cancer  Papanicolaou test utilization  Screening proportions  Medically underserved  National Breast and Cervical Cancer Early Detection Program for program management, data collection, quality assur- ance and improvement, partnership development, profes- sional education, public education, outreach, and evaluation. The NBCCEDP provides cervical cancer screening services to low-income, uninsured women aged 21–64 years. Estimates of NBCCEDP reach for breast cancer screening are reported elsewhere in this monograph [7]. Treatment for women diagnosed with cervical cancer through the NBCCEDP is covered by state Medicaid funding through the Breast and Cervical Cancer Treatment Act of 2000 (Public Law 106-354), the Native American Breast and Cervical Cancer Treatment Technical Amend- ment Act of 2001 (Public Law 107-121), and outside sources. A detailed description of the NBCCEDP and its history are available elsewhere [6, 8, 9]. Introduction As of 2010, only 83 % of women in this 21–65 years age group were up-to-date with screening. Screening proportions for some groups of women are even lower, including Asian women (75 %), and women lacking a usual source of care (65 %) or health in- surance (64 %) (CDC 2012b) [5]. To reduce disparities in cervical cancer screening pro- portions, the US Congress established the National Breast and Cervical Cancer Early Detection Program (NBCCEDP) in 1991 to help low-income, underinsured, and uninsured women gain access to screening and diag- nostic exams for breast and cervical cancer [6]. The NBCCEDP is implemented through cooperative agree- ments between the Centers for Disease Control and Prevention (CDC) and 67 grantees representing health departments in all 50 states, the District of Columbia (DC), 5 US territories, and 11 American Indian and Alaska Na- tive (AIAN) tribes or tribal organizations. The grantees typically establish subcontracts with healthcare providers across the state to deliver screening services. The local healthcare providers are diverse and include local health departments, Federally Qualified Health Centers, commu- nity health centers, Indian Health Service clinics, private clinics, hospitals, and other healthcare systems. Methods Eligibility for the NBCCEDP cervical cancer screening services Eligibility for the NBCCEDP cervical cancer screening services Eligibility for the NBCCEDP cervical cancer screening services In 2012, the NBCCEDP provided $158 million to these 67 grantees. Per congressional mandate, at least 60 % of federal funds received by the grantees must be spent on provision of clinical services. The remaining funds are used We used data from the Current Population Survey Annual Social and Economic Supplement (CPS ASEC) for calen- dar years 1997–2012 to estimate the number of eligible 123 123 673 Cancer Causes Control (2015) 26:671–686 women for 3-year time intervals by age group, state, and race/ethnicity. The CPS is a monthly survey conducted by the US Census Bureau for the Bureau of Labor Statistics that collects information on employment; as well as de- mographic information including age, family size, sex, race, and Hispanic origin. The US Census Bureau applies a probability sample to draw about 100,000 addresses (78,000 households) participating in the CPS for the CPS ASEC. Interviewed households are asked a set of supple- mentary questions about their health insurance coverage and income during the previous year [11]. Respondents were considered uninsured if they were not covered by any type of private or government health insurance for the entire previous year. The methods used to collect and re- port CPS ASEC are described elsewhere [11–14]. sample of the civilian non-institutionalized US population. Conducted annually by CDC’s National Center for Health Statistics (NCHS), the NHIS collects health information during in-person interviews. Each year one or more sup- plements are included in the NHIS that focus on specific health topics. The 2005 NHIS supplement on cancer con- trol was able to provide estimates of the national proportion of US women who have had a hysterectomy by socioeco- nomic group [16]. A full description of the NHIS is available online [17]. We used the BRFSS to estimate the proportion of women who have had a hysterectomy at the state level. BRFSS is a state-based telephone survey of the civilian, non-institutionalized adult population and collects infor- mation on health practices and risk behaviors. A full de- scription of the BRFSS is available online [18]. Because of the small proportion of women without a cervix in the three age groups in each state, we used the percentages of women who had a hysterectomy for each age category, irrespective of income and insurance status, to make the adjustment at the state level. Eligibility for the NBCCEDP cervical cancer screening services In this article, ‘‘eligible women,’’ refers to the women eligible for NBCCEDP- funded Pap test, ‘‘women screened’’ refers to women who received Pap tests within a given 3-year period, and ‘‘screened by the NBCCEDP’’ means screened by provi- ders who received CDC funding from grantees of the NBCCEDP. The NBCCEDP provides cervical cancer screening services to uninsured or underinsured low-income women aged 21–64 years (18–64 years before the USPSTF cervi- cal cancer recommendation of 2012 [3]) with a cervix (those who have not had a hysterectomy with removal of the cervix). Underinsured women are those with limited coverage or a high deductible or co-payment for cervical cancer screening. Women with incomes 250 % of the federal poverty level (FPL) are classified as ‘‘low income’’ [6]. NBCCEDP grantees have the flexibility to establish their own eligibility criteria within federal guidelines. As of 2012, 31 grantees set income eligibility criteria at 250 % of FPL and 20 set income eligibility criteria at lower poverty levels (17 at 200 % FPL, two at 225 % FPL, and one at 185 % FPL). Alaska and Hawaii have higher poverty level thresholds that are not reflected in the US Census Bureau’s definition of poverty. Estimates were adjusted to approximate the FPL in those states. The number of women screened by NBCCEDP The number of women screened by NBCCEDP Information on the number of women screened by the NBCCEDP was obtained from the NBCCEDP grantees. CDC administers and collects a standardized set of data re- ported by grantees on all screening services funded in part or in full by the NBCCEDP, known as minimum data elements (MDEs). Service-level records are reported and include a unique patient identifier to track women receiving services over time. The MDEs include data on demographic char- acteristics, service dates, tests performed, test results, and outcomes. Demographic data are self-reported. The MDEs provide information on the number of women who received NBCCEDP-funded Pap tests during the study period. For the purposes of our study, we counted women screened based on their state of residency within the 50 states and DC. We classified women by age and race/ethnicity groups. More information on the components and structure of NBCCEDP and methods for collecting and reporting NBCCEDP data have been described elsewhere [6, 19, 20]. The CDC prioritizes screening of women aged 40–64 years who have not been screened in the past 5 years for cervical cancer [15]. In 2012, about half of all NBCCEDP grantees restricted eligibility to women aged 40 or older. Although the majority of women 65 years and older are covered by Medicare and are not eligible for the NBCCEDP, some NBCCEDP grantees screen women age 65 years and older who are either ineligible or cannot af- ford the premium to enroll in Medicare part B. Our analysis includes only women aged 18–64 years. Women without a cervix are not eligible for cervical cancer screening through the NBCCEDP. The CPS ASEC does not record whether respondents have had a hysterectomy, and so we adjusted estimates of the number of eligible women downward to account for the proportion of women who have had a hysterectomy. Following the previous report [10], groups were categorized for aged 18–64, 18–39, and 40–64 years. Based on Census Bureau convention, we categorized women who reported that they were of Hispanic origin as Hispanic regardless of race. We categorized the remaining We used the National Health Interview Survey (NHIS) to estimate the proportion of women in the USA who have had a hysterectomy. Eligible women screened outside the NBCCEDP We used the 2009–2011 Medical Expenditure Panel Survey (MEPS) to estimate the proportion of women receiving Pap tests in the eligible population based on the USPSTF-rec- ommended 3-year screening interval. MEPS is a nationally representative survey of the civilian and non-institutional- ized population, and is administered by the Agency for Healthcare Research and Quality. MEPS collects infor- mation including individuals’ demographics, and their health and insurance status. Using the 2011 MEPS, we calculated the proportion of women aged 18–64 years who were uninsured for the entire year, lived in households with incomes B250 % of the FPL, and reported having received a Pap test in the past 3 years. We applied sample weights to produce nationally representative estimates. We calculated the proportion of women screened outside the NBCCEDP by subtracting the proportion screened by the NBCCEDP from the proportion of the eligible population screened that we estimated using MEPS. Estimates of the number of women eligible and the proportion of eligible women screened are based on ran- dom surveys and are thus subject to sampling error. The technique for computing confidence intervals for the esti- mates of the eligible women has been described previously [22]. Consistent with Census Bureau convention [14], we report 90 % confidence intervals for estimates of the eligible population and the proportion of the eligible population screened. We used t tests to assess the sig- nificance of differences in the proportion of women screened. Apparent differences in the trends between the various race/ethnicity groups were not tested for statistical significance. The number of women screened by NBCCEDP is an exact count, and so we do not report inferential statistics for screening totals. Number and percent eligible Number and percent eligible Table 1 reports the estimated number of eligible women and the number and proportion of eligible women screened by race/ethnicity. Between 2010 and 2012, approximately 98 million women aged 18–64 years resided in the USA. We estimate that of those women, approximately 10.9 million or 11.1 % were eligible for a NBCCEDP-funded Pap test. We estimate that more women aged 18–39 years were eligible than women aged 40–64 years (7.1, 3.8 million). We estimate that although fewer Hispanic than non-Hispanic women aged 18–64 years were eligible (3.8, 7.1 million), the percentage of all Hispanic women who were eligible was significantly larger than that of non- Screening proportion The number of women screened by NBCCEDP The NHIS is the principal source of information on the health of a nationally representative 123 12 3 Cancer Causes Control (2015) 26:671–686 674 age groups for all US and eligible women, we estimated the proportion of all US women and eligible women screened through the NBCCEDP. We examined the distribution of NBCCEDP cervical cancer screening by age groups, by race/ethnicity at the national level, and by age groups at the state level. State designation is based on the woman’s residence rather than the grantee providing the service. For states with tribal organizations, the state percentages in- clude the screening data from AIAN grantees. We report the number of women eligible and the proportion of women in the population who are eligible in each state. In compliance with the NBCCEDP data use agreement, grantee- and state-specific reports of the number and pro- portion of eligible women screened are de-identified. We excluded two states from the analysis of variation in the proportion of women screened at the state level because they use different NBCCEDP implementation and eligi- bility criteria. women, who were non-Hispanic, into one of the following racial groups: non-Hispanic white, non-Hispanic black, non-Hispanic AIAN, and non-Hispanic Asian and Native Hawaiian and other Pacific Islander (ANHOPI). The non- Hispanic ANHOPI race category includes those who re- ported any combination of Asian, Native Hawaiian, or other Pacific Islander, and no other race or ethnicity. Re- porting of race and Hispanic origin is optional in the NBCCEDP. Eligible population estimates were not avail- able for 1.9 % of screened women with unknown or mul- tiple race/ethnicity information. These women were excluded from the screening proportion calculations. 2010–2012 National results 2010–2012 National results Number and percent eligible ¼ Unduplicated women screened within a 3year interval 3-year average size of the eligible population ¼ Unduplicated women screened within a 3year interval ¼ Unduplicated women screened within a 3year interval ¼ Unduplicated women screened within a 3year interval 3-year average size of the eligible population 123 Results We calculated the proportion of eligible women screened for 15 successive periods from 1997–2012 based on 3-year screening intervals (e.g., 1997–1999, 1998–2000, and 2010– 2012). We calculated the numerator as the number of unduplicated women screened by the NBCCEDP within the 3-year interval. We chose the 3-year period to be consistent with the cervical cancer screening interval recommended by USPSTF for cervical cancer screening at the time [21]. We calculated the denominator (size of the eligible population) as an average of the 3 years within the time period. 3-year average size of the eligible population The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System Table 1 National Breast and Cervical Cancer Early Detection Program (NBCCEDP) eligibility and screening for Cervical Cancer, by age group, race and ethnicity; 2010–2012 Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System AIAN American Indian/Alaska Native, ANHOPI Asian, Native Hawaiian, and Pacific Islander. Details may not sum to totals because of rounding a The US population represents the Current Population Survey sample universe of the resident civilian non-institutionalized population of the USA AIAN American Indian/Alaska Native, ANHOPI Asian, Native Hawaiian, and Pacific Islander. Details may not sum to totals because of rounding a The US population represents the Current Population Survey sample universe of the resident civilian non-institutionalized population of the USA b Women eligible for NBCCEDP-funded Pap tests include those 18–64 years of age who have a cervix, are uninsured, and have low income (based on eligibility used in each state) aggregated to the nation. The number of eligible women could be underestimated because it excludes those who have health insurance but whose insurance does not cover cervical cancer screening and those who are uninsured for \1 year. See ‘‘Methods’’ section for details b Women eligible for NBCCEDP-funded Pap tests include those 18–64 years of age who have a cervix, are uninsured, and have low income (based on eligibility used in each state) aggregated to the nation. The number of eligible women could be underestimated because it excludes those who have health insurance but whose insurance does not cover cervical cancer screening and those who are uninsured for \1 year. See ‘‘Methods’’ section for details c Number of eligible US women in a given age subgroups may not sum to totals across race and ethnicity categories. Hysterectomy adjustment factors were held constant for increased reliability across age groups within a given race or Hispanic-origin category and not across age groups for all races c Number of eligible US women in a given age subgroups may not sum to totals across race and ethnicity categories. 3-year average size of the eligible population Based on the number of women screened and estimates of the number of women in the 18–64, 18–39, and 40–64 123 Cancer Causes Control (2015) 26:671–686 675 Table 1 National Breast and Cervical Cancer Early Detection Program (NBCCEDP) eligibility and screening for Cervical Cancer, by age group, race and ethnicity; 2010–2012 Race/ethnicity US populationa Women eligible for NBCCEDP screeningb Eligible women screened for cervical cancer via NBCCEDP Number (in thousands) Number (in thousands)c (90 % CI) %d (90 % CI) Number %e (90 % CI) 18–64 Totalf 98,212 10,887 (10,714–11,061) 11.1 (10.9–11.3) 705,970 6.5 (6.4–6.6) Non-Hispanic 82,709 7,063 (6,921–7,206) 8.5 (8.3–8.7) 503,263 7.1 (7.0–7.3) White 62,901 4,516 (4,401–4,631) 7.2 (7.0–7.4) 321,892 7.1 (6.9–7.3) Black 12,884 1,826 (1,754–1,898) 14.2 (13.6–14.8) 99,743 5.5 (5.2–5.7) AIAN 713 153 (128–178) 21.5 (18.0–25.0) 34,718 22.7 (18.9–26.4) ANHOPI 6,211 568 (520–616) 9.1 (8.3–9.9) 42,339 7.5 (6.8–8.1) Multiracial – – – – – 4,571 – – Hispanic 15,503 3,824 (3,717–3,931) 24.7 (24.0–25.4) 193,763 5.1 (4.9–5.2) Unknown – – – – – 8,944 – – 18–39 Total 45,432 7,107 (6,971–7,244) 15.6 (15.3–15.9) 83,660 1.2 (1.2–1.2) Non-Hispanic 36,453 3,970 (3,870–4,070) 10.9 (10.6–11.2) 50,713 1.3 (1.2–1.3) White 26,445 2,489 (2,410–2,568) 9.4 (9.1–9.7) 26,705 1.1 (1.0–1.1) Black 6,379 1,066 (1,014–1,118) 16.7 (15.9–17.5) 4,939 0.5 (0.4–0.5) AIAN 370 98 (77–119) 26.5 (20.8–32.2) 15,362 15.7 (12.3–19.0) ANHOPI 3,259 319 (285–352) 9.8 (8.8–10.8) 2,406 0.8 (0.7–0.8) Multiracial – – – – – 1,301 – – Hispanic 8,978 2,477 (2,395–2,559) 27.6 (26.7–28.5) 31,837 1.3 (1.2–1.3) Unknown – – – – – 1,110 – – 40–64 Total 52,780 3,780 (3,683–3,877) 7.2 (7.0–7.4) 623,603 16.5 (16.1–16.9) Non-Hispanic 46,256 3,093 (3,002–3,185) 6.7 (6.5–6.9) 453,246 14.7 (14.2–15.1) White 36,456 2,027 (1,953–2,101) 5.6 (5.4–5.8) 295,613 14.6 (14.1–15.1) Black 6,506 760 (719–800) 11.7 (11.1–12.3) 94,849 12.5 (11.8–13.2) AIAN 343 55 (45–65) 16.1 (13.2–19.0) 19,505 35.3 (29.0–41.7) ANHOPI 2,952 250 (225–275) 8.5 (7.7–9.3) 39,992 16.0 (14.4–17.6) Multiracial – – – – – 3,287 – – Hispanic 6,524 1,347 (1,293–1,400) 20.6 (19.8–21.4) 162,508 12.1 (11.6–12.5) Unknown – – – – – – 7,849 – Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. Percent screened from outside the NBCCEDP Figure 1 depicts the estimated proportion of eligible women screened by the NBCCEDP, the proportion screened outside the NBCCEDP, and the proportion not screened. Comparing NBCCEDP with MEPS screening data for the NBCCEDP-eligible population, we estimated that approximately 60.2 % [90 % confidence intervals (CI): 57.5–62.7] of NBCCEDP-eligible women aged 18–64 years received a Pap test outside the NBCCEDP and the remaining 33.3 % (90 % CI: 30.8–36.0) did not receive a Pap test from any provider. We estimated that 44.4 % (90 % CI: 40.2–47.0) of women in the 40–64 years age group were screened outside the NBCCEDP and 39.1 % (90 % CI: 35.7–42.5) were not screened. 3-year average size of the eligible population Hysterectomy adjustment factors were held constant for increased reliability across age groups within a given race or Hispanic-origin category and not across age groups for all races d Percent of all US women in a given age, racial, and ethnic group who were eligible for NBCCEDP-funded Pap tests e Percent of all US women in a given age, racial, and ethnic group who are eligible and who were provided with NBCCEDP-funded Pap tests f Number of women screened at aged 18–39 and 40–64 years do not sum to the numbers for 18–64 years because age groups are not mutually exclusive over the 3-year period f Number of women screened at aged 18–39 and 40–64 years do not sum to the numbers for 18–64 years because age groups are not mutually exclusive over the 3-year period 12 3 676 Cancer Causes Control (2015) 26:671–686 screened ranging from 12.5 % among black women to 35.3 % among AIAN women. Hispanic women (24.7 %, 8.5 %). We estimate that within the non-Hispanic eligible population, white women con- stituted the largest group (4.5 million). However, the per- centage of all white women eligible for the NBCCEDP was smaller than that of black women (7.2 %, 14.2 %). Number and percent screened by the NBCCEDP In 2012, 705,970 women aged 18–64 years received at least one NBCCEDP-funded Pap test within the prior 3 years (Table 1). Eighty-eight percent (88.3 %) were in the 40–64 years age group. We estimate that the NBCCEDP provided cervical cancer screening to ap- proximately 0.7 % of USA women aged 18–64 years, 0.2 % of USA women aged 18–39 years, and 1.2 % of USA women aged 40–64 years. We estimate that 1.2 % of Hispanic women and 0.6 % of non-Hispanic women aged 18–64 years received a Pap test through the NBCCEDP. Among non-Hispanic women, we estimate that 0.5 % of white women and 0.8 % of black women were screened through the NBCCEDP. 2010–2012 State-level results Table 2 and ‘‘Appendix’’ show the estimated number and percentage of women who were eligible for NBCCEDP in each state for the 18–64, 40–64, and 18–39 years age groups. Across states, the number of eligible women ranges from about 11,000 in Vermont to about 1.5 million in California for the 18–64 years age group. The percentage of eligible women ranged from 2.5 to 17.9 % for women aged 18–64 years, and from 1.5 to 10.8 % for women aged 40–64 years. We estimate that 6.5 % of eligible women were screened one or more times between 2010 and 2012 (Table 1). The proportion of eligible women who were screened varied by age group and race/ethnicity. We esti- mate that the proportion of eligible women aged 40–64 years screened (16.5 %) was higher than the pro- portion of women aged 18–39 years screened (1.2 %). Among women in the 18–64 years and 40–64 years age groups, the estimated proportion of non-Hispanic women screened (7.1 % among women aged 18–64 years and 14.7 % among women aged 40–64 years) was higher than the proportion of Hispanic women screened (5.1 and 12.1 %, respectively). Among non-Hispanics in the 18–64 years age group, the estimated proportion of eligible women screened ranged from 5.5 % among black women to 22.7 % among AIAN women. Screening patterns are similar among non-Hispanic women in the 40–64 years age group, with an estimated proportion of eligible women Figure 2 depicts the estimated proportion of eligible women screened by state. The proportion of eligible women who were screened from 2010 to 2012 through the NBCCEDP varied across states. The estimated proportions of women screened by state state-level screening propor- tion ranged from 1.5 to 32.7 %, 0.001 to 22.4 %, and 5.0 % to 73.2 % in the 18–64, 18–39, and 40–64 years age groups, respectively. The median estimated proportion of women aged 18–64 and 40–64 years screened was 10.4 and 31.5 %, respectively. The 25th and 75th percentiles for Not screened 33.3% Other providers 60.2% 6.5% 18-64 Not screened 39.1% Other providers 44.4% 16.5% 40-64 NBCCEDP Fig. 1 Percentage of low- income uninsured women screened for cervical cancer in the United States, 2010–2012. Source: Authors’ tabulations of modified data from Medical Expenditure Panel Survey 2011, US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements and from NBCCEDP October 2013 data 123 Fig. 2010–2012 State-level results 1 Percentage of low- income uninsured women screened for cervical cancer in the United States, 2010–2012. 2010–2012 State-level results Source: Authors’ tabulations of modified data from Medical Expenditure Panel Survey 2011, US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements and from NBCCEDP October 2013 data 40-64 18-64 NBCCEDP Other providers 60.2% Not screened 33.3% 123 Cancer Causes Control (2015) 26:671–686 677 Table 2 Estimated number of women eligible for cervical cancer screening in NBCCEDP, by state: 3-year averages for 2010–2012 Poverty criterionc 18–64 40–64 US populationa Eligible women* US populationa Eligible womenb Numberd Numberd (90 % CI)d % of totale (90 % CI)d Numberd Numberd (90 % CI)d % of totale (90 % CI)d USA 98,212 10,887 (10,714–11,061) 11.1 (10.9–11.3) 52,780 3,780 (3,683–3,877) 7.2 (7.0–7.4) Alabama 200 1,535 146 (124–167) 9.5 (8.1–10.9) 870 44 (35–54) 5.1 (4.0–6.2) Alaska 250 221 31 (27–34) 13.8 (12.0–15.6) 116 11 (9–13) 9.2 (7.6–10.8) Arizona 250 2,011 267 (234–299) 13.3 (11.7–14.9) 1,095 103 (86–119) 9.4 (7.9–10.9) Arkansas 200 895 122 (107–137) 13.6 (11.9–15.3) 469 38 (31–45) 8.0 (6.5–9.5) California 200 11,975 1,480 (1,410–1,550) 12.4 (11.8–13.0) 6,141 563 (521–605) 9.2 (8.5–9.9) Colorado 250 1,614 155 (137–173) 9.6 (8.5–10.7) 842 48 (41–54) 5.6 (4.8–6.4) Connecticut 200 1,143 53 (43–62) 4.6 (3.8–5.4) 666 20 (15–24) 2.9 (2.2–3.6) Delaware 250 287 20 (17–23) 7.1 (6.0–8.2) 159 6 (5–8) 4.1 (3.1–5.1) District of Columbia 250 228 15 (12–17) 6.4 (5.3–7.5) 89 4 (3–5) 4.8 (3.6–6.0) Florida 200 5,958 798 (744–852) 13.4 (12.5–14.3) 3,407 294 (265–324) 8.6 (7.7–9.5) Georgia 200 3,190 427 (390–464) 13.4 (12.2–14.6) 1,706 135 (117–153) 7.9 (6.9–8.9) Hawaii 250 417 26 (22–31) 6.3 (5.2–7.4) 225 12 (10–15) 5.4 (4.3–6.5) Idaho 200 464 66 (58–75) 14.3 (12.4–16.2) 240 16 (12–19) 6.6 (5.2–8.0) Illinois 250 4,034 446 (405–487) 11.1 (10.1–12.1) 2,138 166 (146–187) 7.8 (6.8–8.8) Indiana 200 2,008 194 (166–222) 9.7 (8.3–11.1) 1,056 66 (50–82) 6.2 (4.7–7.7) Iowa 250 950 78 (68–88) 8.2 (7.2–9.2) 523 27 (23–31) 5.2 (4.4–6.0) Kansas 225 854 82 (71–94) 9.7 (8.4–11.0) 434 20 (16–25) 4.7 (3.7–5.7) Kentucky 250 1,387 180 (159–200) 13.0 (11.5–14.5) 753 57 (48–66) 7.5 (6.3–8.7) Louisiana 250 1,416 236 (208–264) 16.7 (14.7–18.7) 751 78 (65–91) 10.4 (8.6–12.2) Maine 250 432 28 (23–32) 6.4 (5.3–7.5) 258 13 (10–16) 5.1 (4.0–6.2) Maryland 250 1,927 160 (141–178) 8.3 (7.3–9.3) 1,061 58 (49–68) 5.5 (4.6–6.4) Massachusetts 250 2,143 54 (39–69) 2.5 (1.8–3.2) 1,177 18 (11–25) 1.5 (0.9–2.1) Michigan 250 3,084 283 (254–312) 9.2 (8.3–10.1) 1,723 112 (96–129) 6.5 (5.6–7.4) Minnesota 250 1,643 100 (86–114) 6.1 (5.3–6.9) 866 28 (22–34) 3.2 (2.5–3.9) Mississippi 250 907 121 (106–136) 13.3 (11.7–14.9) 486 34 (27–41) 7.0 (5.6–8.4) Missouri 200 1,853 184 (160–209) 10.0 (8.7–11.3) 1,029 59 (47–72) 5.8 (4.6–7.0) Montana 200 300 41 (34–47) 13.5 (11.4–15.6) 161 13 (10–16) 8.2 (6.4–10.0) Nebraska 225 564 50 (43–58) 8.9 (7.6–10.2) 299 14 (11–17) 4.7 (3.8–5.6) Nevada 250 848 143 (129–157) 16.9 (15.3–18.5) 441 46 (39–53) 10.5 (8.9–12.1) New Hampshire 250 427 32 (28–36) 7.5 (6.5–8.5) 255 13 (11–16) 5.3 (4.4–6.2) New Jersey 250 2,779 297 (260–334) 10.7 (9.4–12.0) 1,548 124 (106–142) 8.0 (6.8–9.2) New Mexico 250 634 114 (100–127) 17.9 (15.8–20.0) 351 38 (31–45) 10.8 (8.9–12.7) 678 Cancer Causes Control (2015) 26:671–686 Table 2 continued Poverty criterionc 18–64 40–64 US populationa Eligible women* US populationa Eligible womenb Numberd Numberd (90 % CI)d % of totale (90 % CI)d Numberd Numberd (90 % CI)d % of totale (90 % CI)d New York 250 6,312 569 (521–616) 9.0 (8.2–9.8) 3,347 207 (183–232) 6.2 (5.5–6.9) North Carolina 250 3,039 400 (362–439) 13.2 (11.9–14.5) 1,652 135 (118–153) 8.2 (7.1–9.3) North Dakota 200 213 15 (12–17) 6.8 (5.6–8.0) 111 5 (4–6) 4.5 (3.5–5.5) Ohio 200 3,566 310 (277–343) 8.7 (7.8–9.6) 1,964 123 (104–143) 6.3 (5.3–7.3) Oklahoma 185 1,145 125 (109–141) 10.9 (9.5–12.3) 606 38 (30–46) 6.3 (4.9–7.7) Oregon 250 1,243 150 (132–168) 12.0 (10.6–13.4) 681 46 (37–55) 6.7 (5.4–8.0) Pennsylvania 250 4,043 341 (305–377) 8.4 (7.5–9.3) 2,276 134 (116–152) 5.9 (5.1–6.7) Rhode Island 250 340 29 (24–33) 8.4 (7.1–9.7) 188 12 (10–14) 6.3 (5.2–7.4) South Carolina 200 1,503 172 (152–193) 11.5 (10.1–12.9) 812 55 (45–65) 6.8 (5.6–8.0) South Dakota 200 247 24 (21–28) 9.8 (8.4–11.2) 127 6 (5–8) 5.0 (3.8–6.2) Tennessee 250 2,030 201 (176–226) 9.9 (8.7–11.1) 1,109 81 (67–94) 7.3 (6.1–8.5) Texas 200 8,068 1,371 (1,307–1,436) 17.0 (16.2–17.8) 4,066 396 (365–426) 9.7 (8.9–10.5) Utah 250 826 87 (73–101) 10.5 (8.8–12.2) 353 20 (15–25) 5.7 (4.2–7.2) Vermont 250 204 11 (9–13) 5.5 (4.5–6.5) 122 3 (2–5) 2.9 (2.1–3.7) Virginia 200 2,606 217 (192–242) 8.3 (7.3–9.3) 1,424 83 (70–95) 5.8 (4.9–6.7) Washington 250 2,175 242 (214–270) 11.1 (9.8–12.4) 1,175 83 (68–97) 7.0 (5.8–8.2) West Virginia 200 595 61 (52–69) 10.2 (8.7–11.7) 355 23 (19–27) 6.5 (5.3–7.7) Wisconsin 250 1,755 116 (96–137) 6.6 (5.4–7.8) 981 43 (32–54) 4.4 (3.3–5.5) Wyoming 250 175 19 (16–22) 10.9 (9.4–12.4) 96 6 (5–8) 6.6 (5.3–7.9) Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements. 2010–2012 State-level results The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System Details may not sum to totals because of rounding a The US population represents the Current Population Survey sample universe which consists of the resident civilian non-institutionalized population of the USA b Women eligible for NBCCEDP-funded Pap tests include those 18–64 years of age who have a cervix, are uninsured, and have low income (based on eligibility used in each state) aggregated to the nation. The number of eligible women could be underestimated because it excludes those who have health insurance but whose insurance does not cover cervical cancer screening and those who are uninsured for \1 year. See ‘‘Methods’’ section for details c 30 states and District of Columbia set income eligibility at 250 % of poverty, 18 states at 200 % of poverty, 2 states at 225 %, and 1 state at 185 % of poverty. The estimated number of women for the USA is based on the eligibility criteria used in each state d Number in thousands e Eligible women as percentage of all women in a given age in that state 1 679 Cancer Causes Control (2015) 26:671–686 0 10 20 30 40 50 60 70 80 Percent Screened US States and District of Columbia Women 40-64 Women 18-64 Women 18-39 Women 40-64, US percentage is 16.5% Women 18-64, US percentage is 6.5% Women 18-39, US percentage is 1.2% Fig. 2 Percent of NBCCEDP-eligible women screened for cervical cancer screening by state and DC compared to national average, 2010–2012. Source: Authors’ tabulations of modified data from the US Factor Surveillance System. Notes: The symbols show the percentage of eligible women screened by each state and District of Columbia. Two states that use different eligibility/implementation criteria are not 0 10 20 30 40 50 60 70 80 Percent Screened US States and District of Columbia Women 40-64 Women 18-64 Women 18-39 Women 40-64, US percentage is 16.5% Women 18-64, US percentage is 6.5% Women 18-39, US percentage is 1.2% Percent Screened US States and District of Columbia Fig. 2 Percent of NBCCEDP-eligible women screened for cervical cancer screening by state and DC compared to national average, 2010–2012. Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. 2010–2012 State-level results The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System. Notes: The symbols show the percentage of eligible women screened by each state and District of Columbia. Two states that use different eligibility/implementation criteria are not included. Data points for each age group are sorted by percentage of eligible women screened. The proportion of NBCCEDP-eligible women screened by the NBCCEDP across the US is 1.2 % aged 18–39, 6.5 % aged 18–64, and 16.5 aged 40–64 Factor Surveillance System. Notes: The symbols show the percentage of eligible women screened by each state and District of Columbia. Two states that use different eligibility/implementation criteria are not included. Data points for each age group are sorted by percentage of eligible women screened. The proportion of NBCCEDP-eligible women screened by the NBCCEDP across the US is 1.2 % aged 18–39, 6.5 % aged 18–64, and 16.5 aged 40–64 the 18–64 years age group are 5.2 and 14.0 %, respec- tively. The equivalent figures for women in the 40–64 years age group are 14.1 and 39.5 %. Increases in the number of women eligible outpaced the number of women screened, leading to a statistically sig- nificant decline in the proportion of women screened be- tween 1999–2001 and 2010–2012. The estimated proportion of eligible women age 18–64 years who were screened fell from 7.6 % in 1999–2001 to 6.5 % in 2010–2012. The estimated proportion of eligible women aged 18–39 years who were screened decreased slightly from 1.4 % in 1999–2001 to 1.2 % in 2010–2012. The estimated proportion of eligible women aged 40–64 years who were screened decreased from 22.3 % in 1999–2001 to 16.5 % in 2011–2012. Trends, 1997–2012 national results Trends, 1997–2012 national results Tables 3 and Fig. 3 report the number of eligible women and the number and proportion of women screened by age group and period. The estimated number of eligible women aged 18–64 years increased by 3.5 million from 7.4 million in 1999–2001 to 10.9 million in 2010–2012. Over the same period, the estimated number of eligible women aged 18–39 years increased by 1.9 million from 5.2 million to 7.1 million. Meanwhile, the estimated number of eligible women aged 40–64 years increased by 1.6 million from 2.2 million to 3.8 million. Figure 4 shows trends in the estimated proportion of eligible women aged 18–64 years screened by race/eth- nicity. Estimates of changes in the proportion of women screened over the study period for black [from 4.6 % (90 % CI: 4.2–5.0 %) to 5.5 % (90 % CI: 5.3–5.7 %)], ANHOPI [from 5.6 % (90 % CI: 4.5–6.7 %) to 7.5 % (90 % CI: 6.9–8.1 %)], and Hispanic [from 4.2 % (90 % CI: 3.9–4.5 %) to 5.1 % (90 % CI: 5.0–5.2 %)] were sig- nificant at the 1 % level. The estimate of the change in the proportion of white women screened over the study period The number of women aged 18–64 years screened in- creased by 142,000 (from 564,000 to 706,000), with women aged 40–64 years accounting for most of the in- crease (132,000). Trends in the number eligible and num- ber screened were similar among racial and ethnic groups (data not shown). Trends, 1997–2012 national results 12 3 Cancer Causes Control (2015) 26:671–686 680 Table 3 NBCCEDP trends in the number of women eligible and the number and percent of women screened for cervical cancer Year Women eligible for NBCCEDP screening Eligible women screened for cervical cancer via NBCCEDP Number (in thousands) (90 % CI) Number (in thousands) Percent of eligible screened (90 % CI) 18–64 1997–1999 7,681 (7,373–7,989) 487 6.3 (6.1–6.6) 1998–2000 7,458 (7,178–7,738) 520 7.0 (6.7–7.2) 1999–2001 7,417 (7,172–7,661) 564 7.6 (7.4–7.9) 2000–2002 7,570 (7,350–7,789) 605 8.0 (7.8–8.2) 2001–2003 8,039 (7,813–8,264) 643 8.0 (7.8–8.2) 2002–2004 8,341 (8,130–8,552) 688 8.2 (8.0–8.5) 2003–2005 8,594 (8,414–8,774) 722 8.4 (8.2–8.6) 2004–2006 8,730 (8,579–8,882) 760 8.7 (8.5–8.9) 2005–2007 8,787 (8,639–8,934) 764 8.7 (8.6–8.8) 2006–2008 8,945 (8,796–9,094) 774 8.7 (8.5–8.8) 2007–2009 9,415 (9,257–9,573) 781 8.3 (8.2–8.4) 2008–2010 10,196 (10,023–10,369) 772 7.6 (7.4–7.7) 2009–2011 10,768 (10,588–10,947) 746 6.9 (6.8–7.0) 2010–2012 10,887 (10,714–11,061) 706 6.5 (6.4–6.6) Change 3,207 219 0.14 18–39 1997–1999 5,416 (5,133–5,698) 68 1.3 (1.2–1.3) 1998–2000 5,258 (5,001–5,514) 65 1.2 (1.2–1.3) 1999–2001 5,206 (4,983–5,429) 75 1.4 (1.4–1.5) 2000–2002 5,264 (5,066–5,463) 90 1.7 (1.7–1.8) 2001–2003 5,552 (5,348–5,755) 107 1.9 (1.8–2.0) 2002–2004 5,740 (5,557–5,924) 123 2.1 (2.1–2.2) 2003–2005 5,899 (5,746–6,051) 130 2.2 (2.2–2.3) 2004–2006 5,967 (5,845–6,088) 136 2.3 (2.2–2.3) 2005–2007 5,978 (5,858–6,099) 134 2.3 (2.2–2.3) 2006–2008 6,033 (5,916–6,149) 134 2.2 (2.2–2.3) 2007–2009 6,301 (6,180–6,423) 125 2.0 (2.0–2.0) 2008–2010 6,731 (6,604–6,858) 111 1.7 (1.6–1.7) 2009–2011 7,064 (6,930–7,198) 92 1.3 (1.3–1.3) 2010–2012 7,107 (6,971–7,244) 84 1.2 (1.2–1.2) Change 1,692 15 -0.1 40–64 1997–1999 2,265 (2,103–2,428) 421 18.6 (17.3–19.9) 1998–2000 2,200 (2,052–2,348) 457 20.8 (19.4–22.2) 1999–2001 2,211 (2,081–2,341) 492 22.3 (21.0–23.6) 2000–2002 2,305 (2,188–2,423) 518 22.5 (21.3–23.6) 2001–2003 2,487 (2,365–2,609) 539 21.7 (20.6–22.7) 2002–2004 2,600 (2,487–2,714) 568 21.9 (20.9–22.8) 2003–2005 2,695 (2,598–2,792) 595 22.1 (21.3–22.9) 2004–2006 2,764 (2,682–2,845) 626 22.7 (22.0–23.3) 2005–2007 2,808 (2,727–2,889) 633 22.5 (21.9–23.2) 2006–2008 2,912 (2,830–2,994) 643 22.1 (21.5–22.7) 2007–2009 3,114 (3,027–3,201) 658 21.1 (20.5–21.7) 2008–2010 3,465 (3,370–3,561) 663 19.1 (18.6–19.7) 2009–2011 3,703 (3,603–3,804) 656 17.7 (17.2–18.2) 12 Cancer Causes Control (2015) 26:671–686 681 Table 3 continued Year Women eligible for NBCCEDP screening Eligible women screened for cervical cancer via NBCCEDP Number (in thousands) (90 % CI) Number (in thousands) Percent of eligible screened (90 % CI) 2010–2012 3,780 (3,683–3,877) 624 16.5 (16.1–16.9) Change 1,515 203 -2.1 Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. Discussion We estimate that the NBCCEDP screened approximately 7 % of the eligible population during the period 2010–2012. More than half of the eligible women screened were racial/ethnic minorities, consistent with the NBCCEDP’s goal of reducing disparities. We estimate that 60.2 % of the eligible women aged 18–64 years were screened outside the NBCCEDP, leaving approximately 33.3 % of the eligible women not screened within a 3-year period. Previously, we reported that from 2004 to 2006, 9 % of the eligible population aged 18–64 received a Pap test from the NBCCEDP, while 56.2 % were screened by other providers and 34.8 % were not screened [10]. The Government Accountability Office (GAO) conducted a separate study to examine, among other things, the NBCCEDP’s screening of eligible women [15]. GAO used the MEPS as the source of data to esti- mate the number of women eligible for the NBCCEDP, from 2004 to 2006. GAO findings were similar to our previous findings (2004–2006). GAO estimated that from 2004 to 2006, 9 % of eligible women were screened by the NBCCEDP, 59 % by other providers, and 33 % were not screened [15]. The decline in the proportion of eligible women screened is largely due to increases in the number of women eligible. Declines in the proportion screened occurred across all race/ethnicity groups. The data in this paper provide vital information for planning, monitoring, and evaluating the only nationally organized screening program in the USA. Variation in screening rates across states could be ex- plained by differences in CDC funding levels, eligibility criteria, availability of other resources, clinical costs, grantee infrastructure for management and service deliv- ery, recruitment strategies, and the number of eligible women [10, 15]. As to be expected, eligibility rates tend to be higher in states with lower average incomes. Local characteristics such as the average cost of service delivery, size of the state population, percentage of eligible women, and the percentage of the population that resides in an urban area affect screening proportions [31]. Grantees re- ceive varying levels of funding from the CDC, state gov- ernment, and other sources. It is likely that this may influence the number of women served. Similar to previous findings, the number and percent- ages of eligible women screened varied widely by age, race/ethnicity [10, 15], and state of residence [10]. Trends, 1997–2012 national results The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System Details may not sum to totals because of rounding 2010–2012 3,780 (3,683–3,877) 624 16.5 (16.1–16.9) Change 1,515 203 -2.1 Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System Details may not sum to totals because of rounding [from 7.5 % (90 % CI: 7.1–7.9 %) to 7.1 % (90 % CI: 6.9–7.3 %)] was not significant. [from 7.5 % (90 % CI: 7.1–7.9 %) to 7.1 % (90 % CI: 6.9–7.3 %)] was not significant. Some grantees prioritize serving women aged 40–64 years to focus outreach efforts to women for both breast and cervical cancer screenings. Some grantees prioritize serving women aged 40–64 years to focus outreach efforts to women for both breast and cervical cancer screenings. The percentage of the eligible women screened by the NBCCEDP is small. A large number of federally funded community health centers, hospitals, family planning clinics, and voluntary associations provide cervical cancer screening services to underserved women outside of the NBCCEDP. Although most eligible women receive cervi- cal screening services from other providers, 33.3 % of eligible women aged 18–64 years were not screened. This figure is consistent with other recent estimates of screening among uninsured women [5]. Possible reasons why more eligible women do not receive cervical cancer screening include fear of painful procedures, fear of having cancer, lack of insurance, high deductibles and co-payments, lack of a usual source of care, lack of knowledge about screening or recommended screening intervals, lack of transportation, and lack of nearby providers [10, 15, 23– 28]. Among women using the NBCCEDP services, low education level and foreign-born status were associated with not returning for repeat screening, suggesting that low education and factors associated with foreign-born status are barriers to the use of the NBCCEDP [29]. A large proportion of eligible women do not know about the NBCCEDP [30]. Discussion The NBCCEDP was most successful in meeting the needs of women aged 40–64 years, although the percent of women eligible for services is higher in the 18–39 years age group. 12 3 123 682 Cancer Causes Control (2015) 26:671–686 0 2000 4000 6000 8000 10000 12000 Number (in thousands) Number of women eligible by age groupa 18-64 18-39 40-64 18-64 18-39 40-64 0 100 200 300 400 500 600 700 800 900 Number (in thousands) Number of women screened by age groupb 18-39 40-64 18-64 0 5 10 15 20 25 97-99 98-00 99-01 00-02 01-03 02-04 03-05 04-06 05-07 06-08 07-09 08-10 09-11 10-12 % Three year period Percent of eligible women screened by age groupc 18-64 18-39 40-64 18-64 18-39 40-64 682 Cancer Causes Control (2015) 26 0 2000 4000 6000 8000 10000 12000 Number (in thousands) Number of women eligible by age groupa 18-64 18-39 40-64 18-64 18-39 40-64 0 100 200 300 400 500 600 700 800 900 Number (in thousands) Number of women screened by age groupb 18-39 40-64 18-64 0 5 10 15 20 25 97-99 98-00 99-01 00-02 01-03 02-04 03-05 04-06 05-07 06-08 07-09 08-10 09-11 10-12 % Three year period Percent of eligible women screened by age groupc 18-64 18-39 40-64 18-64 18-39 40-64 0 2000 4000 6000 8000 10000 12000 Number (in thousands) Number of women eligible by age groupa 18-64 18-39 40-64 18-64 18-39 40-64 0 100 200 300 400 500 600 700 800 900 Number (in thousands) Number of women screened by age groupb 18-39 40-64 18-64 Number of women eligible by age groupa Number of women eligible by age groupa Number (in thousands) Number of women screened by age groupb Number (in thousands) 0 18-39 0 5 10 15 20 25 97-99 98-00 99-01 00-02 01-03 02-04 03-05 04-06 05-07 06-08 07-09 08-10 09-11 10-12 % Three year period Percent of eligible women screened by age groupc 18-64 18-39 40-64 18-64 18-39 40-64 Percent of eligible women screened by age groupc Percent of eligible women screened by age groupc Three year period Three year period Fig. 3 Trends in NBCCEDP-eligible population and reach for cervical cancer screening by age group. aWomen eligible for NBCCEDP-funded Pap tests include those 18–64 years of age who have a cervix, are uninsured, and have low income (based on eligibility used in each state) aggregated to the nation. Discussion 4 NBCCEDP trends in the percent of eligible women screened for cervical cancer, aged 18–64, by race and ethnicity. aWomen eligible for NBCCEDP-funded Pap tests include those 18–64 years of age who have a cervix, are uninsured, and have low income (based on eligibility criteria used in each state) aggregated to the nation. The number of eligible women could be underestimated because it excludes those who have health insurance, but whose insurance does not cover cervical cancer screening and those who are uninsured for \1 year. See ‘‘Methods’’ section for details. abPercent of all US women aged 18–64 who were eligible for NBCCEDP-funded Pap tests and who were provided with NBCCEDP-funded Pap tests. Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System. Notes: AIAN American Indian or Alaska Native; ANHOPI Asian American, Native Hawaiian, or Pacific Islander. Data are presented in two graphs because of differences in scale. Highest points are marked to point out scale Cancer Causes Control (2015) 26:671–686 683 Cancer Causes Control (2015) 26:671–686 683 9.8 0 1 2 3 4 5 6 7 8 9 10 Percent screened via NBCCEDPa White Asian All races Hispanic Black Hispanic Black All races White ANHOPI 42.8 0 5 10 15 20 25 30 35 40 45 Percent screened via NBCCEDPa Th i d AIAN All races Percent screened via NBCCEDPa Percent screened via Three year period Three year period Fig. 4 NBCCEDP trends in the percent of eligible women screened for cervical cancer, aged 18–64, by race and ethnicity. aWomen eligible for NBCCEDP-funded Pap tests include those 18–64 years of age who have a cervix, are uninsured, and have low income (based on eligibility criteria used in each state) aggregated to the nation. The number of eligible women could be underestimated because it excludes those who have health insurance, but whose insurance does not cover cervical cancer screening and those who are uninsured for \1 year. See ‘‘Methods’’ section for details. abPercent of all US women aged 18–64 who were eligible for NBCCEDP-funded Pap tests and who were provided with NBCCEDP-funded Pap tests. Discussion The number of eligible women could be underestimated because it excludes those who have health insurance, but whose insurance does not cover cervical cancer screening and those who are uninsured for \1 year. See ‘‘Methods’’ section for details. bPercent of all US women in a given age group who were eligible for NBCCEDP-funded Pap tests. cPercent of all US women in a given age group who are eligible and who were provided with NBCCEDP-funded Pap tests. Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. The modifi- cation of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System NBCCEDP. In 1999, there were 32.3 million people in poverty compared with 46.5 million people in 2012. The poverty rate increased by 3.2 percentage points, from 11.8 % in 1999 to 15.0 % in 2012 [11, 32]. In 2010–2012, both the number of women eligible for the NBCCEDP and the number of women screened through the NBCCEDP increased, in comparison with 1999–2001. However, increases in the number of women eligible out- paced the number of women screened, resulting in a decrease in the proportion of women screened between 1999–2001 and 2010–2012. The decline in the proportion of eligible women screened is largely due to increases in the number of eligible women. Unemployment rates, which increase during and shortly after recession periods (March 2001–November 2001 and December 2007–June 2009), increased the number of people in poverty, hence the number of women eligible for the Our study is subject to a number of limitations. First, our study may underestimate the number of women who are eligible for the NBCCEDP. Women who are un- derinsured (those whose insurance does not cover pre- ventive services or those who have high co-payments) and are eligible for the NBCCEDP are not included in the CPS ASEC uninsured estimates and thus are not included in the denominators of the screening proportions. The CPS 123 123 9.8 0 1 2 3 4 5 6 7 8 9 10 Percent screened via NBCCEDPa White Asian All races Hispanic Black Hispanic Black All races White ANHOPI 42.8 0 5 10 15 20 25 30 35 40 45 Percent screened via NBCCEDPa Three year period AIAN All races Fig. Discussion Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements, and from NBCCEDP October 2013 data. The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System. Notes: AIAN American Indian or Alaska Native; ANHOPI Asian American, Native Hawaiian, or Pacific Islander. Data are presented in two graphs because of differences in scale. Highest points are marked to point out scale ASEC measures the number of women who are insured, but not the number who are underinsured. No general definition of being underinsured is available, and the number of low-income, underinsured women in the population is unknown. This could result in an overesti- mate of the screening proportions. Estimates are also subject to recall bias. The CPS ASEC uses annual retro- spective questions, and respondents may have difficulty recalling information about insurance coverage [33]. Considering only women uninsured for the whole year excludes women who are uninsured for only a part of the year and would be eligible for the NBCCEDP for the period that they were uninsured. Our inability to define the race and ethnicity of some women in the study could result in an underestimate of the screening proportion for any given race or ethnic group. Analyses that further stratify the data (by age group at the state level and by age group for individual race/ethnic groups) were impossible due to small sample sizes. Dalzell et al. [12] describe three US Census Bureau’s data sources for estimating the NBCCEDP-eligible population. Second, we used BRFSS and NHIS data to adjust the estimates of the eligible population derived from the CPS ASEC for the proportion of women who have had a hys- terectomy. Using data from various sources may introduce some errors in the estimates because the questionnaires, data collection methods, and sampling methods are dif- ferent. Also, in these data sources, it is not possible to 12 3 3 3 Cancer Causes Control (2015) 26:671–686 684 implementation on screening uptake. There are many fac- tors other than access to insurance that serve as barriers for underserved women to receive cancer screening [10, 15, 23–26, 28–30]. CDC, through the NBCCEDP, funds grantees to recruit women, address these barriers and im- prove access to screening and diagnostic services. Discussion Since the state grantees reach underserved women, the NBCCEDP provides a unique opportunity to reduce disparities in cervical cancer and increase the proportion of women screened among the underserved population. Although the number of women screened by NBCCEDP has increased since 1997, a large share of NBCCEDP-eligible women did not receive recommended Pap tests. Results of this study indicate there continues to be an unmet need for screening services for underserved populations. distinguish between women who had a partial hysterecto- my instead of a total hysterectomy. Third, because BRFSS was administered by landline telephones only during the year used in this analysis, less affluent groups, such as low-income uninsured women, may be underrepresented because they are less likely to have landline telephone service [34, 35]. In contrast, NHIS is conducted primarily by in-person interview. Last, the unit of analysis for this study is the state and not the grantee, as standardized estimates of eligible populations are not available for NBCCEDP grantees that are tribal organizations and US territories. In 2010, Healthy People 2020 set the objective of in- creasing the proportion of women aged 21–65 years who receive a Pap test within a 3-year period to 93 % [36]. Although cervical cancer screening proportions have in- creased over time, screening proportions among the unin- sured still lag far behind those among women with private or public health insurance [5]. The Affordable Care Act (ACA) should increase access to cervical cancer screening services for many low-income, underserved women by making health insurance more available and by eliminating cost-sharing for cervical cancer screening. Additional studies will be needed to assess the impact of ACA Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. Appendix See Table 4. Appendix See Table 4. Appendix Table 4 Estimated number of women, aged 18–39, eligible for cervical cancer screening in NBCCEDP, by state: 3-year averages for 2010–2012 Poverty criterionc US populationa Eligible womenb Number (in thousands) Number (in thousands) 90 % CI (in thousands) % of totald 90 % CI (%) 18–39 USA 45,432 7,107 (6,971–7,244) 15.6 (15.3–15.9) Alabama 200 665 101 (84–119) 15.2 (12.5–17.9) Alaska 250 105 20 (17–23) 18.9 (15.8–22.0) Arizona 250 916 164 (138–190) 17.9 (15.0–20.8) Arkansas 200 426 84 (72–97) 19.8 (16.9–22.7) California 200 5,834 917 (865–969) 15.7 (14.8–16.6) Colorado 250 772 107 (92–123) 13.9 (11.9–15.9) Connecticut 200 478 33 (26–40) 6.9 (5.4–8.4) Delaware 250 128 14 (11–16) 10.7 (8.8–12.6) District of Columbia 250 139 10 (8–12) 7.4 (5.9–8.9) Florida 200 2,550 503 (461–545) 19.7 (18.0–21.4) Georgia 200 1,484 292 (262–323) 19.7 (17.7–21.7) Hawaii 250 192 14 (11–18) 7.3 (5.5–9.1) Idaho 200 224 51 (43–58) 22.6 (19.2–26.0) Illinois 250 1,896 280 (246–313) 14.8 (13.0–16.6) Indiana 200 952 128 (107–149) 13.5 (11.3–15.7) Iowa 250 427 51 (43–59) 12.0 (10.1–13.9) Kansas 225 420 62 (52–72) 14.8 (12.4–17.2) Kentucky 250 634 123 (106–140) 19.4 (16.7–22.1) Louisiana 250 666 158 (135–181) 23.8 (20.3–27.3) Maine 250 174 15 (11–18) 8.3 (6.3–10.3) Cancer Causes Control (2015) 26:671–686 685 Table 4 continued Poverty criterionc US populationa Eligible womenb Number (in thousands) Number (in thousands) 90 % CI (in thousands) % of totald 90 % CI (%) Maryland 250 866 101 (86–117) 11.7 (9.9–13.5) Massachusetts 250 966 36 (24–48) 3.7 (2.5–4.9) Michigan 250 1,361 171 (148–193) 12.5 (10.9–14.1) Minnesota 250 776 72 (60–84) 9.3 (7.8–10.8) Mississippi 250 420 87 (74–99) 20.6 (17.6–23.6) Missouri 200 825 125 (105–145) 15.2 (12.8–17.6) Montana 200 139 28 (22–33) 19.8 (16.0–23.6) Nebraska 225 265 36 (30–43) 13.7 (11.2–16.2) Nevada 250 407 97 (85–108) 23.8 (21.0–26.6) New Hampshire 250 172 19 (15–22) 10.8 (8.9–12.7) New Jersey 250 1,231 173 (143–203) 14.0 (11.6–16.4) New Mexico 250 283 76 (65–86) 26.8 (23.0–30.6) New York 250 2,965 362 (323–400) 12.2 (10.9–13.5) North Carolina 250 1,387 265 (233–298) 19.1 (16.8–21.4) North Dakota 200 102 10 (7–12) 9.4 (7.2–11.6) Ohio 200 1,602 186 (162–211) 11.6 (10.1–13.1) Oklahoma 185 539 87 (74–100) 16.2 (13.8–18.6) Oregon 250 562 104 (89–118) 18.5 (15.9–21.1) Pennsylvania 250 1,767 207 (177–236) 11.7 (10.0–13.4) Rhode Island 250 152 17 (13–20) 11.1 (8.8–13.4) South Carolina 200 691 117 (100–134) 17.0 (14.5–19.5) South Dakota 200 119 18 (15–21) 14.8 (12.3–17.3) Tennessee 250 921 120 (100–140) 13.0 (10.9–15.1) Texas 200 4,002 976 (922–1,030) 24.4 (23.1–25.7) Utah 250 473 67 (55–79) 14.2 (11.6–16.8) Vermont 250 82 8 (6–9) 9.4 (7.4–11.4) Virginia 200 1,182 135 (114–155) 11.4 (9.7–13.1) Washington 250 1,001 159 (137–182) 15.9 (13.7–18.1) West Virginia 200 240 38 (31–45) 15.7 (12.7–18.7) Wisconsin 250 774 73 (57–89) 9.5 (7.4–11.6) Wyoming 250 79 13 (11–15) 16.0 (13.3–18.7) Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements. References References 18. CDC. Behavioral risk factor surveillance system (cited 2014 April 21). http://www.cdc.gov/brfss/about/about_brfss.htm 1. Pierce Campbell CM, Menezes LJ, Paskett ED, Giuliano AR (2012) Prevention of invasive cervical cancer in the United States: past, present, and future. Cancer Epidemiol Biomarkers Prev 21(9):1402–1408 19. Miller JW, Plescia M, Ekwueme DU (2014) Public health na- tional approach to reducing breast and cervical cancer disparities. Cancer 15(120 Suppl 16):2537–2539 2. U.S. Cancer Statistics Working Group (2013) United States Cancer Statistics: 1999–2010 incidence and mortality web-based report. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Health Promotion, and National Cancer Institute (cited 2014 June 2). www.cdc.gov/uscs 20. Yancy B, DeGroff A, Royalty J, Marroulis S, Mattingly C, Be- nard VB (2014) Using data to effectively manage a national screening program. Cancer 15(120 Suppl 16):2575–2583 21. U.S. Preventive Services Task Force (2003) Recommendations and rationale screening for cervical cancer: recommendations and rationale. Am Fam Physician 67(8):1759–1766 3. U.S. Preventive Services Task Force (2012) Screening for cer- vical cancer: clinical summary of U.S. Preventive Services Task Force recommendation. AHRQ Publication No. 11-05156-EF-3 (cited 2014 June 6). http://www.uspreventiveservicestaskforce. org/uspstf11/cervcancer/cervcancersum.htm 22. Tangka FKL, Dalaker J, Chattopadhyay SK, Gardner JG, Royalty J, Hall IJ, DeGroff A, Blackman DK, Coates RJ (2006) Meeting the mammography screening needs of underserved women: the performance of the National Breast and Cervical Cancer Early Detection Program in 2002–2003. Cancer Causes Control 17:1145–1154 4. U.S. Department of Health and Human Services. Healthy people 2020 (cited 2014 June 6). http://www.healthypeople.gov/2020/ topicsobjectives2020/objectiveslist.aspx?topicId=5 23. Corcoran J, Crowley M (2014) Latinas’ attitudes about cervical cancer prevention: a meta-synthesis. J Cult Divers 21(1):15–21 p j j p p 5. CDC (2012) Cancer screening—United States, 2010. MMWR 61:41–46 24. Fayanju OM, Kraenzle S, Drake BF, Oka M, Goodman MS (2014) Perceived barriers to mammography among underserved women in a Breast Health Center Outreach Program. Am J Surg 208(3):425–434 6. CDC. National Breast and Cervical Cancer Early Detection Program (cited 2014 April 19). http://www.cdc.gov/cancer/ nbccedp/about.htm 7. Howard D, Tangka FKL, Royalty J, Danzell LP, Miller J, O’Hara B, Joseph K, Kenney K, Guy G, Hall IJ (2015) Breast cancer screening of underserved women in the United States: results from the National Breast and Cervical Cancer Early Detection Program, 1998–2012. Cancer Causes Control (forthcoming, this supplement) 25. References Henry KA, McDonald K, Sherman R, Kinney AY, Stroup AM (2014) Association between individual and geographic factors and nonadherence to mammography screening guidelines. J Womens Health 23(8):664–674 26. Fang DM, Baker DL (2013) Barriers and facilitators of cervical cancer screening among women of Hmong origin. J Health Care Poor Underserved 24(2):540–555 8. Lantz P, Mullen J (2015) Overview of the NBCCEDP (forth- coming, this supplement) 27. CDC (2012) Breast cancer screening among adult women—be- havioral Risk Factor Surveillance System, United States, 2010. MMWR 61:46–50 9. Lee NC, Wong FL, Jamison PM et al (2014) Implementation of the National Breast and Cervical Cancer Early Detection Pro- gram: the beginning. Cancer 15(120 Suppl 16):2540–2548 28. Ackerson K, Gretebeck K (2007) Factors influencing cancer screening practices of underserved women. J Am Acad Nurse Pract 19(11):591–601 10. Tangka FKL, O’Hara B, Gardner JG, Turner J, Royalty J et al (2010) Meeting the cervical cancer screening needs of underserved women: the National Breast and Cervical Cancer Early Detection Program, 2004–2006. Cancer Causes Control 21:1081–1090 29. Song L, Fletcher R (1998) Breast cancer rescreening in low- income women. Am J Prev Med 15(2):128–133 11. DeNavas-Walt C, Proctor BD, Smith J (2013) U.S. Census Bureau, current population reports, series P60–245, income, poverty, and health insurance coverage in the United States 2012. U.S. Government Printing Office, Washington 30. Hall IJ, Rim SH, Johnson-Turbes CA, Vanderpool R, Kamalu NN (2012) The African American women and mass media campaign: a CDCbreastcancerscreeningproject.JWomensHealth21:1107–1113 31. Subramanian S, Tangka FKL, Ekwueme DU, Trogdon J, Crouse W, Royalty J (2015) Explaining variation by state in breast and cervical cancer screening proportions in the NBCCEDP (forth- coming, this supplement) 12. Dalzell LP, Tangka FKL, Powers DS, Holmes, O’Hara BJ, Kristy J, Janet R (2015) Data sources to identify low income, uninsured populations: application to public health—National Breast and Cervical Early Detection Program (forthcoming, this supplement) g pp 32. Dalaker J, Proctor BD (2000) U.S. Census Bureau, current population reports, series P60–210, poverty in the United States: 1999. U.S. Government Printing Office, Washington 13. U.S. Census Bureau (2006) Current population survey: design and methodology. Technical paper 66 (cited 2014 April 21). http://www.census.gov/prod/2006pubs/tp-66.pdf 33. Pascale J, Rodean J, Leeman J, Cosenza C, Schoua-Glusberg A (2013) Preparing to measure health coverage in federal surveys post-reform: lessons from Massachusetts. Inquiry 50(2):106–123 14. U.S. Appendix The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System Source: Authors’ tabulations of modified data from the US Census Bureau, Current Population Survey, 2010–2012 Annual Social and Economic Supplements. The modification of the data was the authors’ tabulations of data from 2005 National Health Interview Survey and 2005 Behavioral Risk Factor Surveillance System Details may not sum to totals because of rounding a The US population represents the Current Population Survey sample universe which consists of the resident civilian non-institutionalized population of the USA a The US population represents the Current Population Survey sample universe which consists of the resident civilian non-institutionalized population of the USA b Women eligible for NBCCEDP–funded Pap tests include those 18–39 years of age who have a cervix, are uninsured, and have low income (based on eligibility criteria used in each state) aggregated to the nation. The number of eligible women could be underestimated because it excludes those who have health insurance but whose insurance does not cover cervical cancer screening and those who are uninsured for\1 year. See ‘‘Methods’’ section for details b Women eligible for NBCCEDP–funded Pap tests include those 18–39 years of age who have a cervix, are uninsured, and have low income (based on eligibility criteria used in each state) aggregated to the nation. The number of eligible women could be underestimated because it excludes those who have health insurance but whose insurance does not cover cervical cancer screening and those who are uninsured for\1 year. See ‘‘Methods’’ section for details c 30 states and District of Columbia set income eligibility at 250 % of poverty, 18 states at 200 % of poverty, 2 states at 225 %, and 1 state at 185 % of poverty. The estimated women for the USA are based on the eligibility criteria used in each state c 30 states and District of Columbia set income eligibility at 250 % of poverty, 18 states at 200 % of poverty, 2 states at 225 %, and 1 state at 185 % of poverty. The estimated women for the USA are based on the eligibility criteria used in each state d Eligible women as percentage of all women in a given age in that state 123 123 123 Cancer Causes Control (2015) 26:671–686 686 17. Appendix CDC (2014) About the National Health Interview Survey (cited 2014 April 21). http://www.cdc.gov/nchs/nhis/about_nhis.htm References Census Bureau (2012) Source and accuracy of estimates for income, poverty, and health insurance coverage in the United States (cited 2014 June 8). http://www.census.gov/hhes/www/ p60_245sa.pdf 34. Miller JW, King JB, Ryerson AB, Eheman CR, White MC (2009) Mammography use from 2000 to 2006: state-level trends with corresponding breast cancer incidence rates. Am J Roentgenol 192:352–360 15. United States Government Accountability Office (2009) Report to congressional requesters. MEDICAID. Source of screening affect women’s eligibility for coverage of breast and cervical cancer treatment in some states. GAO-09-384 (cited 2014 June 2). http://www.gao.gov/new.items/d09384.pdf 35. Ochner MH, Salvail FR, Ford ES, Ajani U (2008) Obesity and self-reported general health, Hawaii BRFSS: are polynesians at higher risk? Obesity 16(4):923–926 16. National Center for Health Statistics (2006) Data file documen- tation, National Health Interview Survey, 2005 (machine-read- able data file and documentation). Hyattsville: U.S. Department of Health and Human Services (cited June 6 2014). ftp://cdc.gov/ pub/Health_Statistics/NCHS/Dataset_Documentation/NHIS/2005/ srvydesc.pdf 36. Brown ML, Klabunde CN, Cronin KA, White MC, Richardson LC, McNeel TS (2014) Challenges in meeting healthy people 2020 objectives for cancer-related preventive services, National Health Interview Survey, 2008 and 2010. Prev Chronic Dis 11:130174 12 3

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SERUM FERRITIN LEVEL IN PATIENTS WITH ACUTE HAEMORRAGIC STROKE AND ITS ASSOCIATION WITH OUTCOME

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SERUM FERRITIN LEVEL IN PATIENTS WITH ACUTE HAEMORRAGIC STROKE AND ITS ASSOCIATION WITH OUTCOME Singh Kulbinder1, Sen AK2 and Roy Mainak3 1. Post Graduate Trainee, Department Of Medicine, Jorhat Medical College Hospital, Jorhat, Assam. 2. Professor, Hod, Department Of Medicine, Jorhat Medical College Hospital, Jorhat, Assam. 3. Associate Proffesor , Department Of Biochemistry, Jorhat Medical College Hospital, Jorhat , Assam. …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Received: 28 February 2022 Final Accepted: 30 March 2022 Published: April 2022 Background:Stroke is characterized by acute onset of focal neurological deficit lasting 24 hours or longer. With the increasing incidence of hemorrhagic stroke in Indians, the use of biochemical markers like Ferritin can predict outcome. This study was undertaken to study level of Serum Ferritin levels in patients with acute hemorrhagic stroke and it association with the outcome. Materials and Methods: This was a hospital based cross sectional observational study with 90 patients with acute hemorrhagic stroke admitted into the Department of Medicine, JMCH since 1st July 2020 till June 30th 2021. All the patients subjected to investigations and inclusion and exclusion criteria. Equal number of age and sex matched healthy individuals were included in the study as control Results: In this study the mean age group with Acute Hemorrhagic Stroke was 63.3 +-7.2 years. Of the total cases 57 were males (63.3%) and 33 female (37.7%). Hypertension was the most common risk factor associated present in 67.8% patients which is 53.3. The most common site of bleed was the basal ganglia. The mean serum Ferritin value was 333.44 +92.12ng/ml in patients with acute hemorrhagic stroke .The serum ferritin values correlated with the hematoma volume and higher values were associated with increased mortality . Conclusion: In the study Serum Ferritin values were significantly higher 445.29+-97.70 ng/ml (p value<.001) in patients which deteriorated and showed a positive correlation. ……………………………………………………………… Background:Stroke is characterized by acute onset of focal neurological deficit lasting 24 hours or longer. With the increasing incidence of hemorrhagic stroke in Indians, the use of biochemical markers like Ferritin can predict outcome. This study was undertaken to study level of Serum Ferritin levels in patients with acute hemorrhagic stroke and it association with the outcome. Materials and Methods: This was a hospital based cross sectional observational study with 90 patients with acute hemorrhagic stroke admitted into the Department of Medicine, JMCH since 1st July 2020 till June 30th 2021. All the patients subjected to investigations and inclusion and exclusion criteria. SERUM FERRITIN LEVEL IN PATIENTS WITH ACUTE HAEMORRAGIC STROKE AND ITS ASSOCIATION WITH OUTCOME Equal number of age and sex matched healthy individuals were included in the study as control Results: In this study the mean age group with Acute Hemorrhagic Stroke was 63.3 +-7.2 years. Of the total cases 57 were males (63.3%) and 33 female (37.7%). Hypertension was the most common risk factor associated present in 67.8% patients which is 53.3. The most common site of bleed was the basal ganglia. The mean serum Ferritin value was 333.44 +92.12ng/ml in patients with acute hemorrhagic stroke .The serum ferritin values correlated with the hematoma volume and higher values were associated with increased mortality . Conclusion: In the study Serum Ferritin values were significantly higher 445.29+-97.70 ng/ml (p value<.001) in patients which deteriorated and showed a positive correlation. Copy Right, IJAR, 2022,. All rights reserved. Copy Right, IJAR, 2022,. All rights reserved. Copy Right, IJAR, 2022,. All rights reserved. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 10(04), 761-764 Journal Homepage: - www.journalijar.com Article DOI: 10.21474/IJAR01/14613 DOI URL: http://dx.doi.org/10.21474/IJAR01/14613 Materials And Methods:- A Hospital based observational study conducted in Jorhat Medical College & Hospital, Department of Medicine , Jorhat for a durationof 1 year from 1st July 2020- 31stJune 2021 upon a total number of 90 patients . The patients attending the hospital with the diagnosis of acute hemorrhagic stroke and willing to participate were enrolled in the study Inclusion Criteria: 1. Patients equal to or above 18 years of age with acute hemorrhagic stroke 2. Patients/ their attendants giving informed consent for the study Exclusion criteria: 1. Ischemic stroke 2. Anemia 3. Chronic liver disease 4. Chronic kidney disease 5. Hematological malignancy 1. Ischemic stroke 2. Anemia 3. Chronic liver disease 4. Chronic kidney disease 5. Hematological malignancy 90 Age and Sex matched healthy individuals were included in the study as control group g y y g p Patients diagnosed clinically and radiologically as acute hemorrhagic stroke were included in the study following informed consent either from them or their relatives. All patients were then subjected tonon-contrast CT scan of brain. Important findings such as site, size, intraventricular extensions, midline shifts if any were noted.Clinical examination involved recording of vitals, GCS(Glasgow Coma Scale), MRS (Modified Rankin Scale) on admission, neurological examination. All data were then analyzed using the IBMSPSS 23. Chi square test wasdone to establish the association. A p value of <0.05 considered to be statistically significant. Introduction:- A stroke is a clinically defined syndrome of rapidly developing symptoms or signs of focal loss of cerebral function with no apparent cause other than that of vascular origin, but the loss of function can at times be global (applied to patients in deep coma and to those with subarachnoid hemorrhage). Symptoms last more than 24 h or lead to death .1.Stroke is one of the leading causes of death and disability in India. The estimated adjusted prevalence rate of stroke range, 84-262/100,000 in rural and 334-424/100,000 in urban areas. The incidence rate is 119-145/100,000 based on the recent population based studies. There is also a wide variation in case fatality rates with the highest being 42% .2. Hypertension is considered to be one of the most important risk factor in middle aged and elderly person.3 The increased risk of intracerebral hemorrhage is greater in the set of patients who have stopped taking their 761 Corresponding Author:- Singh Kulbinder Address:- Post Graduate Trainee,Department Of Medicine, Jorhat Medical College Hospital, Jorhat, Assam. 761 Int. J. Adv. Res. 10(04), 761-764 ISSN: 2320-5407 antihypertensive medication, are relatively young or are smokers. ICH accounts for approximately 10-20% of all strokes antihypertensive medication, are relatively young or are smokers. ICH accounts for approximately 10-20% of all strokes Iron has been involved in cerebral injury after the occurrence of intracerebralhemorrhage. Free iron released from the lysis of red blood cells plays a role in injury. Iron toxicity is generally thought to result from the generation of free radicals via the Fenton reaction .4. In that reaction, ferrous iron reacts with hydrogen peroxide to form radical oxygen. The resulting ferric iron can be reduced back to Fe2+ by a variety of reducing agents and thereby regenerate the starting reagents. In this way, the radical reaction cycle can begin again. Some cellular antioxidants like GSH and superoxide dismutase work to limit this damage, but these antioxidants have limited efficacy to combat the amount of oxidative stress during ICH.5 Free iron isconsidered toxic to cells and the body has established an elaborate set of protective mechanisms to bind iron in various tissue compartments. Within tissues the iron is stored complexed to protein as ferritin or hemosiderin. Stored iron in the form of ferritin is not essential for sustaining life or for preventing anemia, but when liberated, it can promote tissue injury by provoking iron mediated Fenton reaction 5 Result:- Result: Table 1:- Serum Ferritin level in stroke patients and in control group . Table 2:- Serum Ferritin and GCS score in patients with Hemorrhagic Stroke. Group Sex N Serum Ferritin ng/ml (Mean ± SD) P value Haemorrhagic Stroke Male 57 327.02± 88.9 <.001 Female 33 300.55± 97.7 Total 90 333.44± 92.12 Control Male 57 126.72±41.06 Female 33 94.53±18.767 Total 90 115.28± 37.65 GCS Serum Ferritin ( Mean ±SD) ng/dl p value 3-8 444.00±87.59 762 Int. J. Adv. Res. 10(04), 761-764 Int. J. Adv. Res. 10(04), 761-764 ISSN: 2320-5407 ISSN: 2320 5407 Int. J. Adv. Res. 10(04), 761 764 Table 3:- Serum Ferritin level in different size of Hemorrhage and across various MRS groups. MRS (Modified Rankin Scale) Serum Ferritin ( ng/dl) Mean± SD Hematoma volume (ml) ( Mean± SD) P value ≤ 2 265.54±75.51 18.48± 11.03 < .001 3-6 445.29±97.70 36.76±18.04 Table 4:- Serum Ferritin values in fatal and Non-Fatal group. In Hospital Mortality Number Serum Ferritin( Mean ± SD) ng/dl P Value Fatal 24 487.08±95.7 <.001 Non-Fatal 66 277.58± 75.2 9-12 272.22± 75.70 <.001 13-15 258.65± 62.25 We observed that the mean age of the patient was 63.3±7.2 years. The mean value of serum Ferritin in the patients with acute hemorrhagic stroke was 333.44± 92.12 ng/dl ng/ml and the value of serum Ferritin in the control group was 115.28± 37.65 ng/dl. The difference is statistically significant (p value < .001). The mean serum ferritin in the male patients was 327.02±88.9 ng/dl and the mean value in male control group is 126.72±41.06 ng/dl . The mean value of serum ferritin in the female cases was300.55 ±97.7 ng/dl and the mean ferritin value in the control group is 94.53±18.767ng/dl. The difference is found to be statistically significant. The mean serum Ferritin in patients with MRS score ≤ 2 is 265.54±75.51 ng/ml, whereas the mean serum Ferritin in patients with MRS score 3-6 is 445.29±97.70 ng/ml. The difference is found to be statistically significant. The mean serum ferritin values in the patients with GCS score between 3- 8 was 444.00±87.59ng/dl as compared to patients having GCS score 9-12 and 13-15 whose respective Ferritin values are 272.22 ±75.70ng/dl and 258.65± 62.25ng/dl, that in patients with MRS score≤ 2, the mean serum Ferritin value is 265.54±75.51ng/ml and the mean volume of hematoma is 18.48± 11.03 ml . Result:- In patients with MRS scores 3-6, the mean serum Ferritin value is 445.29±97.70ng/ml and the mean volume of the hematoma is 36.76±18.04ml. The inpatient mortality of the patients was 26.6% Discussion:- In the present study we found that the mean value of serum Ferritin in the patients with acute hemorrhagic stroke was 333.44± 92.12ng/ml and the value of serum Ferritin in the control group was 115.28± 37.6ng/dl. The findings are consistent with the studies conducted by Pankaj P etal. who found the serum ferritin values in the patients with hemorrhagic stroke which deteriorated were 463.91±181.2 ng/dl.7Similar results were found in the study conducted by Hemant Mahur etal6. In hemorrhagic group the mean serum ferritin level was 355.759 in those deteriorated. They concluded that the patients with stroke with increased serum ferritin concentrations have a higher risk of poorer outcome, hemorrhagic transformation, and cerebral edema than patients with low ferritin values.6Pankaj P etal. conducted a study on the patient of acute stroke where they found out that level of serum ferritin had direct correlation with poorer prognosis in patients of stroke. The mean level of serum ferritin was found to be 463.91ng/dl in deteriorated group of patients.7These findings are consistent with the findings of study conducted by Dr Manish Narayanetal. (2018) where they found the mean serum ferritin in hemorrhagic stroke patients in the to be 463.91ng/ml. The difference was statistically significant, elevated ferritin levels were seen in patients with poor clinical outcome 8 The present study found that the mean value of serum ferritin in the patients which improved , MRS ≤ 2 was 265.5± 75.51 ng/ml whereas the mean value of serum ferritin in the patients which deteriorated, MRS 3-6 was relatively higher that is 445.29±97.70ng/ml. The difference was found to be statistically significant as reflected by p values < .001. Similar results were observed in the study conducted by Hemant Mahur etal. conducted a study on a total of 100 patients of stroke who presented to the hospital . In hemorrhagic group the mean serum ferritin level was 86.838ng/ml in clinically improvement patients and 355.759ng/ml in those deteriorated. They concluded that, the patients with stroke with increased serum ferritin concentrations have a higher risk of poorer outcome, hemorrhagic transformation, and cerebral edema than patients with low ferritin values 6 763 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 10(04), 761-764 Int. J. Adv. Res. 10(04), 761-764 The findings are consistent with the studies conducted by Pankaj P etal. Conclusion:- Hemorrhagic Strokes are common in Males as compared to Females. It is more common in age group of 60 years and above. Mean Serum Ferritin level was higher in the patients with HemorrhagicStroke. The Patients with higher MRS scores and lower GCS scores had higher serum Ferritin levels. The larger size of hemorrhage was associated with higher levels of serum Ferritin. In patients with Hemorrhagicstroke, higher Ferritin level was associated with higher rates of in hospital mortality. Discussion:- who conducted a study on the patient of acute stroke where they found out that level of serum ferritin had direct correlation with poorer prognosis in patients of stroke. The mean level of serum ferritin in the group of clinically improved was 87.01ng/ml was much lesser compared to the group clinically deteriorated or died 458.7 ng/ml among patients of ischemic stroke. Similarly in patients of hemorrhagic stroke it was 96.4ng/ml in improved group compared to 463.91ng/ml in deteriorated. The differences were statistically significant7 Similar results were observed in the study conducted by Rajendran S R etal. c where they concluded that elevated levels of serum ferritin were seen in patients of haemorrhagic stroke and those elevated levels were associated with poor outcome of the patients 10 p gy JD, Sudhan P. Stroke epidemiology and stroke care services in India. J Stroke. 2013;15(3):128-134. C d i i " b li f di l d id i i j " i di i Bibliography:- g p y 1. Warlow CP. Epidemiology of stroke.The Lancet. 1998 Oct 1;352:S1-4. 1. Warlow CP. Epidemiology of stroke.The Lancet. 1998 Oct 1;352:S1-4. 2. Pandian JD, Sudhan P. Stroke epidemiology and stroke care services in India. J Stroke. 2013 , p gy ; ( ) 3. Emerit, J., C. Beaumont, and F. Trivin. "Iron metabolism, free radicals, andoxidative injury." Biomedicine & pharmacotherapy 55.6 (2001): 333-339. 3. Emerit, J., C. Beaumont, and F. Trivin. "Iron metabolism, free radicals, andoxidative injury." Biomedicine & pharmacotherapy 55.6 (2001): 333-339. 4. Floyed RA ,ZaleskaMM,Harman H:Possible involvement of iron andoxygen free radicals in aspects of aging brain ;free radicals in molecularbiology;Raven press N.Y.1984:143-161 4. Floyed RA ,ZaleskaMM,Harman H:Possible involvement of iron andoxygen free radicals in aspects of aging brain ;free radicals in molecularbiology;Raven press N.Y.1984:143-161 5. Fischbach, F. A., et al. "On the structure of hemosiderin and its relationshipto ferritin." Journal of ultrastructure research 37.5 (1971): 495-503. 5. Fischbach, F. A., et al. "On the structure of hemosiderin and its relationshipto ferritin." Journal of ultrastructure research 37.5 (1971): 495-503. 6. Mathur H, Ralot T K, Singh DP ,Ken P, Patel J To establish the role of serum ferritin as a prognostic marker in patients of stroke IP Indian Journal of Neurosciences, 2018;4(2):64-68 7. Pankaj P, Das M, Singh M K. “Association between Level of Serum Ferritin and Outcome of Patients of Stroke”. Journal of Evolution of Medical and Dental Sciences 2015; Vol. 4, Issue 12, February 09; Page: 2023- 2036 8. Narayan M, Singh SK. Study of association between serum ferritin and prognosis of patients in acute ischemic and haemorrhagic stroke. IOSR Journal of Dental and Medical Sciences. 2018;17:46-56. 9. Hegde A, Menon G, Kumar V, Lakshmi Prasad G, Kongwad LI, Nair R, Nayak R. Clinical profile and predictors of outcome in spontaneous intracerebralhemorrhage from a tertiary care center in South India. Stroke research and treatment. 2020 Jan 27; 2020. ; 10. Rajendran SR1,Periswamy T,1 Manjuladevi MT,2 and George N3 etalEvaluation of serumferritin as a prognostic marker in acute hemorrhagic stroke J Neurosci Rural Pract 2020; 11(01): 072-07. 764

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Correspondence: Luke D. Schiferl ([email protected]) Correspondence: Luke D. Schiferl ([email protected]) Correspondence: Luke D. Schiferl ([email protected]) Received: 9 August 2022 – Discussion started: 29 August 2022 Received: 9 August 2022 – Discussion started: 29 August 2022 Received: 9 August 2022 – Discussion started: 29 August 2022 Revised: 17 November 2022 – Accepted: 29 November 2022 – Published: 22 December 2022 Revised: 17 November 2022 – Accepted: 29 November 2022 – Published: 22 December 2022 Abstract. The continued warming of the Arctic could release vast stores of carbon into the atmosphere from high-latitude ecosystems, especially from thawing permafrost. Increasing uptake of carbon dioxide (CO2) by vegetation during longer growing seasons may partially offset such release of car- bon. However, evidence of significant net annual release of carbon from site-level observations and model simulations across tundra ecosystems has been inconclusive. To address this knowledge gap, we combined top-down observations of atmospheric CO2 concentration enhancements from aircraft and a tall tower, which integrate ecosystem exchange over large regions, with bottom-up observed CO2 fluxes from tun- dra environments and found that the Alaska North Slope is not a consistent net source nor net sink of CO2 to the atmo- sphere (ranging from −6 to +6 TgCyr−1 for 2012–2017). Our analysis suggests that significant biogenic CO2 fluxes from unfrozen terrestrial soils, and likely inland waters, dur- ing the early cold season (September–December) are ma- jor factors in determining the net annual carbon balance of the North Slope, implying strong sensitivity to the rapidly warming freeze-up period. At the regional level, we find no evidence of the previously reported large late-cold-season (January–April) CO2 emissions to the atmosphere during the study period. Despite the importance of the cold-season CO2 Research article Research article Biogeosciences, 19, 5953–5972, 2022 https://doi.org/10.5194/bg-19-5953-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Using atmospheric observations to quantify annual biogenic carbon dioxide fluxes on the Alaska North Slope Luke D. Schiferl1,2, Jennifer D. Watts3, Erik J. L. Larson4, Kyle A. Arndt3,5,6, Sébastien C. Biraud7, Eugénie S. Euskirchen8, Jordan P. Goodrich5,9, John M. Henderson10, Aram Kalhori5,11, Kathryn McKain12,13, Marikate E. Mountain10, J. William Munger2, Walter C. Using atmospheric observations to quantify annual biogenic carbon dioxide fluxes on the Alaska North Slope Oechel5,14, Colm Sweeney12, Yonghong Yi15,16, Donatella Zona5,17, and Róisín Commane1,18 1Lamont-Doherty Earth Observatory, Columbia University, Palisades, New York, USA 3Woodwell Climate Research Center, Falmouth, Massachusetts, USA 4Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachu 4Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA 5 5Department of Biology, San Diego State University, San Diego, California, USA 6Earth Systems Research Center, Institute for the Study of Earth, Oceans, and Space, University of New Hampshire, Durham, New Hampshire, USA 7Lawrence Berkeley National Laboratory, Berkeley, California, USA y y y 8Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA y y y 8Institute of Arctic Biology, University of Alaska Fairbanks, Fair 9Ministry for the Environment, Wellington, New Zealand 9Ministry for the Environment, Wellington, New Zealand 10Atmospheric and Environmental Research, Inc., Lexington, Massachusetts, USA 10Atmospheric and Environmental Research, Inc., Lexington, M 11GFZ German Research Centre for Geosciences, Potsdam, Germany Global Monitoring Laboratory, Earth System Research Laboratories, NOAA, Boulder, Colorado, U 12Global Monitoring Laboratory, Earth System Research Laboratories, N itute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado, USA Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Co 13Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado, USA 14Department of Geography University of Exeter Exeter UK Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado, USA 14Department of Geography University of Exeter Exeter UK Department of Geography, University of Exeter, Exeter, UK 14Department of Geography, University of Exeter, Exeter, UK 15Joint Institute for Regional Earth System Science and Engineering, University of California, Los Angeles, California, USA 16College of Surveying and Geo-Informatics, Tongji University, Shanghai, China l Earth System Science and Engineering, University of California, Los Angeles, California, USA Joint Institute for Regional Earth System Science and Engineering, University of California, Los A 15Joint Institute for Regional Earth System Science and Engineering, University of California, 16College of Surveying and Geo-Informatics, Tongji University, Shanghai, China College of Surveying and Geo-Informatics, Tongji University, Shanghai, China 17Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, UK 18Department of Earth and Environmental Sciences, Columbia University, New York, New York, p y 18Department of Earth and Environmental Sciences, Columbia University, New York, New York, USA 1 Introduction The Arctic surface air temperature is warming at twice the rate of the global average (Box et al., 2019; Meredith et al., 2019). Continued thawing of Arctic permafrost has the potential to release vast stores of carbon into the atmosphere, thereby further accelerating warming (Schuur et al., 2015; Hugelius et al., 2014). In the biosphere, the net CO2 flux is the balance between the uptake of CO2 by vegetation through photosynthesis (negative net CO2 flux indicates re- moval from the atmosphere) and the release of CO2 into the atmosphere by plant and microbial respiration (positive net CO2 flux indicates a source to the atmosphere). Arctic grow- ing seasons are short (∼3 months), and the long cold sea- son dominates the seasonal cycle. The transition between the growing and cold seasons is marked by the soil zero- curtain period, when belowground temperatures of the ac- tive layer above frozen permafrost remain near freezing; the active layer is insulated by snow and ice at the surface and warmed by the latent heat release of freezing water (Outcalt et al., 1990). During the zero-curtain period, soil respiration can remain active in deeper soils for weeks to months af- ter the end of the growing season (Zona et al., 2016; Ro- manovsky and Osterkamp, 2000). As the climate warms, the active layer above permafrost deepens, thawed soils become wetter, a larger volume of soil remains unfrozen for a longer period of time, and the duration of the zero-curtain period plays an increasingly important role in determining the net carbon exchange in Arctic ecosystems (Kim et al., 2012; Arndt et al., 2019). Recent work has shown a significant cold-season source of CO2 from Arctic ecosystems, includ- ing more than a 70 % increase in October–December CO2 concentration enhancements in the past 40 years, consistent with an increase in cold-season respiration, which is not well represented in Earth system models (Commane et al., 2017; Natali and Watts et al., 2019). Neglecting these processes could lead to a large underestimation of CO2 emissions, bi- assing current and future climate projections. Currently, observations and models do not agree on the sign of the annual net CO2 flux across the Alaska North Slope region. Site-level measurements and atmospheric observa- tions suggest that this region is a net CO2 source (Commane et al., 2017; Oechel et al., 2014; Euskirchen et al., 2017). L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope This study shows that quantification and characterization of year-round CO2 fluxes from the heterogeneous terrestrial and aquatic ecosystems in the Arctic using both site-level and atmospheric observations are important to accurately project the Earth system response to future warming. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5954 tundra during the growing season is relatively small and may be offset by emissions from respiration that can continue well into the cold season (Watts et al., 2021). In the past, year- round CO2 flux measurements from tundra ecosystems were rare due to difficulties in maintaining instrumentation under remote and extreme cold conditions (Euskirchen et al., 2017; Kittler et al., 2017; Goodrich et al., 2016). Long-term year- round CO2 concentration measurements have been made in the Arctic at a small number of tall towers, which have been situated to sample clean marine air off the ocean (Jeong et al., 2018; Worthy et al., 2009). While aircraft provide greater spatial coverage over land than these towers, they tend to op- erate for short durations, and their temporal coverage is lim- ited by weather and visibility during the cold season (Chang et al., 2014; Commane et al., 2017; Miller et al., 2016). How- ever, the recent increase in the availability of observations of gas fluxes and concentrations within a particular tundra re- gion, the Alaska North Slope (Fig. 1a), is making it possible to better conduct year-round multi-scale assessments of tun- dra ecosystems, with the aim of improving our understanding of CO2 sink/source activity and carbon budgets in these en- vironments. emissions to the annual total, the interannual variability in the net CO2 flux is driven by the variability in growing season fluxes. During the growing season, the regional net CO2 flux is also highly sensitive to the distribution of tundra vegeta- tion types throughout the North Slope. This study shows that quantification and characterization of year-round CO2 fluxes from the heterogeneous terrestrial and aquatic ecosystems in the Arctic using both site-level and atmospheric observations are important to accurately project the Earth system response to future warming. emissions to the annual total, the interannual variability in the net CO2 flux is driven by the variability in growing season fluxes. During the growing season, the regional net CO2 flux is also highly sensitive to the distribution of tundra vegeta- tion types throughout the North Slope. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope erl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5955 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5955 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5955 Figure 1. Alaska North Slope study region, eddy flux site locations, area sampled by aircraft and the tower, and example results from the eddy flux site measurement–model comparison. (a) North Slope region (red box) within Alaska and northwestern Canada; tundra ar- eas are shown in purple, and boreal forest areas are shown in green (Luus et al., 2017). (b) Location of eddy flux measurement sites on the Alaska North Slope used in this analysis. (c) The 10 d WRF-STILT (Weather Research and Forecasting–Stochastic Time-Inverted La- grangian Transport) footprints used to sample CO2 flux models, summed for all aircraft and tall-tower CO2 observations used in this analysis; colors represent values greater than 0 and are saturated at 60 ppm(µmolm−2 s−1)−1, and the maximum value near Utqia˙gvik, Alaska, is 324 ppm(µmolm−2 s−1)−1. (d) Time series of the observed (black dots) and simulated (colored lines) site-level daily mean net CO2 flux for 2014 at the Ivotuk (IVO; left) and Climate Monitoring and Diagnostics Laboratory (CMDL; right) eddy flux measurement sites, where site- level Tundra Vegetation Photosynthesis and Respiration Model (TVPRM) net CO2 flux simulations are driven by North American Regional Reanalysis (NARR) meteorology and the solar-induced chlorophyll fluorescence (SIF) product from the contiguous SIF (CSIF) dataset. Pos- itive net CO2 flux values indicate CO2 fluxes into the atmosphere throughout this study. A comparison for all eight eddy flux sites is provided in Fig. S1 in the Supplement. Figure 1. Alaska North Slope study region, eddy flux site locations, area sampled by aircraft and the tower, and example results from the eddy flux site measurement–model comparison. (a) North Slope region (red box) within Alaska and northwestern Canada; tundra ar- eas are shown in purple, and boreal forest areas are shown in green (Luus et al., 2017). (b) Location of eddy flux measurement sites on the Alaska North Slope used in this analysis. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope (c) The 10 d WRF-STILT (Weather Research and Forecasting–Stochastic Time-Inverted La- grangian Transport) footprints used to sample CO2 flux models, summed for all aircraft and tall-tower CO2 observations used in this analysis; colors represent values greater than 0 and are saturated at 60 ppm(µmolm−2 s−1)−1, and the maximum value near Utqia˙gvik, Alaska, is 324 ppm(µmolm−2 s−1)−1. (d) Time series of the observed (black dots) and simulated (colored lines) site-level daily mean net CO2 flux for 2014 at the Ivotuk (IVO; left) and Climate Monitoring and Diagnostics Laboratory (CMDL; right) eddy flux measurement sites, where site- level Tundra Vegetation Photosynthesis and Respiration Model (TVPRM) net CO2 flux simulations are driven by North American Regional Reanalysis (NARR) meteorology and the solar-induced chlorophyll fluorescence (SIF) product from the contiguous SIF (CSIF) dataset. Pos- itive net CO2 flux values indicate CO2 fluxes into the atmosphere throughout this study. A comparison for all eight eddy flux sites is provided in Fig. S1 in the Supplement. centrations sub-weekly from June to September 2015 over the North Slope (Biraud et al., 2016; Tadi´c et al., 2021). The US National Aeronautics and Space Administration (NASA) Arctic-Boreal Vulnerability Experiment (ABoVE) Arctic Carbon Atmospheric Profiles (Arctic-CAP) airborne campaign flew throughout Alaska and northwestern Canada approximately every month from May to November 2017 (Sweeney and McKain, 2019; Sweeney et al., 2022). Car- bon dioxide concentration observations from the NASA Car- bon in Arctic Reservoirs Vulnerability Experiment (CARVE) flights for 2012–2014 are incorporated into the Commane et al. (2017) optimized CO2 fluxes used in our analysis be- low. The NOAA/US Coast Guard collaborative Alaska Coast Guard (ACG) flights have also made aircraft CO2 concen- tration measurements in the region, but these coastal flights observe only limited spatial coverage of the North Slope, and we do not use them here. tributions, and environmental drivers that best characterize the observed spatial and temporal distribution of biogenic CO2 in the atmosphere across the region. By developing re- gional CO2 budgets constrained by both atmospheric obser- vations and ecosystem environmental responses, we can bet- ter project how Arctic tundra ecosystems will respond to cli- mate change on annual and decadal timescales. 1 Introduction However, a comparison of process-based models of the North Slope found large variability in the sign and magnitude of the net CO2 flux with an approximately neutral regional an- nual net CO2 flux multi-model mean of −3.5 ± 67 TgCyr−1 (Fisher et al., 2014). In a more recent study, Tao et al. (2021) found an annual net CO2 flux range of −9 to 12 TgCyr−1 for the years 2010–2016, with only 2014 being an annual net CO2 source. Extrapolating from site-level CO2 flux mea- surements to regional budgets is difficult due to the extreme heterogeneity of tundra ecosystems in the North Slope region as well as a lack of spatial and seasonal representativeness by existing flux monitoring sites (Pallandt et al., 2022). In this study, we compare bottom-up flux estimates with top-down atmospheric observations from aircraft and a tall tower using an integrated modeling approach to quantify the CO2 budget sign and magnitude of the Alaska North Slope. Our framework first applies a bottom-up approach to under- stand Arctic tundra ecosystem CO2 fluxes, constrained by site-level observations, using an empirical model ensemble of CO2 fluxes derived from eddy flux measurements rep- resenting varied tundra ecosystems within the region. We then apply top-down information gained from regional CO2 concentration enhancement observations measured by a tall tower and aircraft, which sample the atmosphere–biosphere exchange throughout the Alaska North Slope, to evaluate the range of potential CO2 fluxes identified by the bottom- up model ensemble for 2012–2017. This evaluation also identifies the ecosystem parameterizations, vegetation dis- Tundra ecosystems, characterized by frozen soils covered in low shrubs, sedges, grasses, and mosses, make up approx- imately 50 % of the Arctic landscape (Raynolds et al., 2019). Due to the lack of trees, the magnitude of net CO2 uptake in https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 2.2 Observed atmospheric CO2 concentration enhancement calculation Time periods with highly variable CO concentrations (1CO > 40 ppb) indicate complex mixing of more remote combustion sources and are also removed (Chang et al., 2014). The re- maining grouped sampling points correspond to the available Weather Research and Forecasting–Stochastic Time-Inverted Lagrangian Transport (WRF-STILT) modeling system simu- lations (Henderson et al., 2015; see below): ARM-ACME V points are calculated every 50 m vertically below 1 km, ev- ery 100 m vertically above 1 km, and every 10 km horizon- tally from 1 s observations, and ABoVE Arctic-CAP points are matched every 20 s from averaged 10 s observations. To ensure these points observe the Alaska North Slope, we only use points with at least 70 % of the total 10 d WRF-STILT- simulated surface influence occurring in our regional do- main. We calculate the observed top-down atmospheric CO2 con- centration enhancement (1CO2) for the North Slope region for every land-sector hour at the NOAA BRW tower and for every 50 m of vertical distance transited during the airborne campaigns (ARM-ACME V and ABoVE Arctic-CAP). The observed 1CO2 (in units of ppm) generated by the North Slope ecosystem is calculated relative to the background con- centration without influence from this region such that observed 1CO2 = observed [CO2]−background [CO2] (1) following previous work (Sweeney et al., 2016; Commane et al., 2017; Jeong et al., 2018). following previous work (Sweeney et al., 2016; Commane et al., 2017; Jeong et al., 2018). The background CO2 concentrations at the NOAA BRW tower are determined by smoothing the 10 d mean of the observed ocean-sector concentrations using spline fitting to produce a daily CO2 background concentration. We calculate the uncertainty of these background concentrations by both (1) varying the starting hour of the 10 d mean calculation prior to spline fitting and (2) randomly sub-selecting 50 % of the ocean-sector concentrations 1000 times. The interval that contains 95 % of these 240 000 fits represents our daily background uncertainty. Figure S2 shows the ocean-sector concentrations, the resulting background concentration, and the uncertainty described here. To determine the background CO2 concentrations for the ARM-ACME V and ABoVE Arctic-CAP aircraft campaigns, we isolate aircraft observations without surface influence from the North Slope using the WRF-STILT footprints, as done for larger regions in Chang et al. (2014) and Commane et al. (2017). 2.1.2 Eddy covariance CO2 flux tower observations We also use up to 5 years (2013–2017) of year-round ob- servations of net CO2 flux from eight eddy covariance tower sites (for 32 total site-years) representing an array of tundra ecosystems throughout the Alaska North Slope (Figs. 1b, S1; Table S2). These half-hourly eddy flux measurements of net CO2 flux are not gap filled to avoid introducing additional un- certainties. Three of the sites are located near Imnavait Creek along a wetness gradient from valley to hilltop: wet sedge tundra (Imnavait Creek sedge – ICS), moist acidic tussock tundra (Imnavait Creek tussock – ICT), and dry heath tun- dra (Imnavait Creek heath – ICH) (Euskirchen et al., 2017, 2012). The other sites include tussock tundra at Ivotuk (IVO), wet polygonized tundra at Atqasuk (ATQ), and three sites near Utqia˙gvik: wetland tundra (Biocomplexity Experiment, South – BES), wet polygonized tundra (Barrow Environmen- tal Observatory – BEO), and moist tundra (Climate Monitor- ing and Diagnostics Laboratory – CMDL) (Zona et al., 2016; Arndt et al., 2020). L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5956 2.2 Observed atmospheric CO2 concentration enhancement calculation These observed CO2 concentrations represent the state of the air before it interacts with the surface in the study region. The regional backgrounds vary by the direc- tion from which the air enters the domain. For example, the backgrounds from the south and from over land generally experience CO2 drawdown prior to those from over the Arc- tic Ocean. The time- and direction-dependent backgrounds that we use are shown in Fig. S3. We apply the uncertainty from the NOAA BRW tower background to the aircraft back- grounds as a reasonable representation of the variability as- sociated with available background CO2 concentration data. 2.2 Observed atmospheric CO2 concentration enhancement calculation from those directions during the growing season (defined here as May–August). For the ARM-ACME V and ABoVE Arctic-CAP aircraft campaign observations, we group aver- aged sampling points into 50 m vertical bins after remov- ing data influenced by combustion sources such as anthro- pogenic activity and biomass burning events. These combus- tion sources of CO2 are expected to be small (< 1 TgCyr−1 on the North Slope; see Table S1 in the Supplement) dur- ing our study period. They are not accounted for in bio- genic CO2 flux models, however, and must be removed from our analysis when observed. We remove time peri- ods with an elevated carbon monoxide (CO) concentration (above 150 ppb), as in Chang et al. (2014) and Commane et al. (2017), which indicates local combustion sources. Time periods with highly variable CO concentrations (1CO > 40 ppb) indicate complex mixing of more remote combustion sources and are also removed (Chang et al., 2014). The re- maining grouped sampling points correspond to the available Weather Research and Forecasting–Stochastic Time-Inverted Lagrangian Transport (WRF-STILT) modeling system simu- lations (Henderson et al., 2015; see below): ARM-ACME V points are calculated every 50 m vertically below 1 km, ev- ery 100 m vertically above 1 km, and every 10 km horizon- tally from 1 s observations, and ABoVE Arctic-CAP points are matched every 20 s from averaged 10 s observations. To ensure these points observe the Alaska North Slope, we only use points with at least 70 % of the total 10 d WRF-STILT- simulated surface influence occurring in our regional do- main. from those directions during the growing season (defined here as May–August). For the ARM-ACME V and ABoVE Arctic-CAP aircraft campaign observations, we group aver- aged sampling points into 50 m vertical bins after remov- ing data influenced by combustion sources such as anthro- pogenic activity and biomass burning events. These combus- tion sources of CO2 are expected to be small (< 1 TgCyr−1 on the North Slope; see Table S1 in the Supplement) dur- ing our study period. They are not accounted for in bio- genic CO2 flux models, however, and must be removed from our analysis when observed. We remove time peri- ods with an elevated carbon monoxide (CO) concentration (above 150 ppb), as in Chang et al. (2014) and Commane et al. (2017), which indicates local combustion sources. 2.1.1 Atmospheric CO2 concentration observations We use a suite of CO2 concentration observations from various sources on the North Slope for our analysis. The United States (US) National Oceanic and Atmospheric Ad- ministration (NOAA) Barrow Atmospheric Baseline Ob- servatory (BRW) tall tower near Utqia˙gvik, Alaska, has made continuous in situ CO2 concentration measurements since 1973 (Sweeney et al., 2016). The US Department of Energy (DOE) Atmospheric Radiation Measurement Cli- mate Research Facility Airborne Carbon Measurements V (ARM-ACME V) airborne campaign measured CO2 con- For the NOAA BRW tower, we use hourly CO2 con- centration observations with wind direction from the land (135–202.5◦clockwise with respect to north) and ocean sec- tors (0–45◦), avoiding Utqia˙gvik anthropogenic activity, with wind speeds > 2.5 ms−1 (Fig. S2) (Commane et al., 2017; Sweeney et al., 2016). We only use land-sector observa- tions from the cold season (defined here as September– April) because seasonal wind patterns do not favor transport https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 2.4 Empirically simulated biogenic CO2 fluxes from tundra ecosystems We develop the TVPRM as an ensemble of ecosystem- resolved models that represent a more extensive range of potential tundra ecosystem functional relationships, environ- mental drivers, and scaling assumptions than available from other CO2 flux models. For this study, TVPRM generates a set of spatially and temporally varying CO2 flux maps for a 6-year period (2012–2017) at a 30 km × 30 km spatial and a 1 h temporal resolution for the Alaska North Slope. p The footprints are generated by the WRF-STILT atmo- spheric transport modeling system (Henderson et al., 2015). In this system, WRF meteorological fields are first gener- ated for the study region and time period (v3.5.1 for ARM- ACME V and NOAA BRW tower footprints used here, and v3.9.1 for ABoVE Arctic-CAP footprints). STILT then uses the WRF meteorology to estimate the contribution of sur- face fluxes to the atmospheric concentration at a specified time and place, called a receptor, by calculating the amount of time that air (represented by a distribution of particles) spends in the lower half of the boundary layer at a given location. The WRF-STILT model configurations from Hen- derson et al. (2015) have been extensively employed in nu- merous previous papers to study greenhouse gas fluxes us- ing observations from aircraft and towers in Alaska, includ- ing on the North Slope (e.g., Chang et al., 2014; Miller et al., 2016; Zona et al., 2016; Commane et al., 2017; Karion et al., 2015; Hartery et al., 2018). An evaluation by Henderson et al. (2015) for WRF v.3.4.1 and v3.5.1 showed that their po- lar WRF configuration performs well against surface obser- vations of air temperature and wind speed in Alaska and that WRF-STILT can capture the shape and approximate depth of greenhouse gases in the column. Zona et al. (2016) note that WRF planetary boundary layer ventilation rates may be bi- ased in the fall (and winter) when heat fluxes are low, but this error is difficult to assess quantitatively. For this study, we use receptors set to correspond to the tower and aircraft CO2 concentration observations. The footprints (and their corre- sponding measurements) for these receptors sample air from throughout the North Slope but are concentrated more heav- ily toward the area around the NOAA BRW tower (Fig. 1c). L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope tion of the meteorological reanalysis products driving the model. The CO2 flux models used for comparison to the TVPRM ensemble are similarly treated using 0.5◦gridded 10 d WRF-STILT footprints, which are available on a cir- cumpolar grid poleward of 30◦N. The simulated CO2 fluxes from Luus et al. (2017), Natali and Watts et al. (2019), and Watts et al. (2021) are regridded to a 0.5◦spatial resolution. For the models by Natali and Watts et al. (2019) and Watts et al. (2021), which only estimate monthly CO2 fluxes, we apply a constant flux for that month. As the ends of our de- fined cold season (September–April) include transitional pe- riods when some biogenic plant activity does occur (hence belowground CO2 emissions ̸= NEE), we add in estimates of photosynthesis and plant respiration fluxes from the TVPRM ensemble for April and September for the Natali and Watts et al. (2019) and Watts et al. (2021) bottom-up scenarios. In this calculation, we multiply the hourly simulated CO2 flux (in units of µmolCO2 m−2 s−1) by the footprint (in units of ppm(µmolCO2 m−2 s−1)−1) for that hour starting at the observation point, backward in time for each hour up to 10 d, where the footprint quantifies the influence of the land sur- face on the concentration observed at a measurement point. The simulated 1CO2 is then the sum of these hours. We use expected CO2 fluxes based on a variety of bottom- up model approaches which represent North Slope ecosys- tems. Year-round bottom-up estimates of net CO2 fluxes (de- fined by the models as net ecosystem exchange, NEE) are ob- tained from the Tundra Vegetation Photosynthesis and Res- piration Model (TVPRM) ensemble as well as from exist- ing model output from Luus et al. (2017) and Commane et al. (2017). Independent bottom-up estimates of belowground CO2 emissions (equal to the NEE) for the cold season (net CO2 uptake = 0) were obtained from Natali and Watts et al. (2019) and Watts et al. (2021). The TVPRM model en- semble development process is described in Sect. 2.4, and the other CO2 flux models, including their native spatial and temporal resolutions, are listed in Table S3. 2.3 Simulated atmospheric CO2 concentration enhancement calculation To understand how landscape interactions with the atmo- sphere (through CO2 flux) influenced the observed CO2 con- centrations across space and time, we calculate the corre- sponding simulated 1CO2 (in units of ppm) by transporting bottom-up biogenic CO2 fluxes to each observation site such that simulated 1CO2 = simulated CO2 flux (2) (2) Biogeosciences, 19, 5953–5972, 2022 https://doi.org/10.5194/bg-19-5953-2022 5957 (7) NEE = Rsoil + Rplant −GPP. NEE = Rsoil + Rplant −GPP. Positive NEE values indicate a net source of CO2 into the at- mosphere, and negative NEE values indicate net movement of CO2 into the biosphere. We use NEE to be synonymous with net CO2 flux. Using SIF, which correlates to photosyn- thetic activity (Porcar-Castell et al., 2014; Yang et al., 2015), in the modeling framework provides an advantage over in- dices such as the enhanced vegetation index (EVI) due to the limited canopy and evergreen nature of tundra ecosystems (Luus et al., 2017). ( ) The parameter values (αs, βs, αa, βa, λ, and PAR0) for the site-level relationships used by TVPRM are determined first using the observed net CO2 fluxes from the eddy flux sites (see Sect. S1 in the Supplement). We determine the site- level parameters separately for each combination of reanaly- sis product (North American Regional Reanalysis – NARR, Mesinger et al., 2006, and the fifth-generation European Cen- tre for Medium-Range Weather Forecasts atmospheric re- analysis – ERA5, Hersbach et al., 2020), which provide Ta, Ts, and PAR, and the SIF product (Global Ozone Monitoring Experiment-2 – GOME-2, Joiner et al., 2016; Global OCO- 2, where OCO-2 is the Orbiting Carbon Observatory-2, SIF – GOSIF, Li and Xiao, 2019; and contiguous SIF – CSIF, Zhang et al., 2018) that will later be used to generate the re- gional TVPRM ensemble (Tables S4 and S5; see Sects. S2 and S3). Additional αs and βs parameters are determined using Ts from the remote-sensing-driven permafrost model (RS-PM; Yi et al., 2019, 2018) to test its implementation in TVPRM. RS-PM uses tailored input for Alaska permafrost zones, such as downscaled snow depth and aircraft-observed soil dielectric constants, and was developed and tested using Ts and active-layer thickness measurements from the North Slope. RS-PM also produces Ts at a higher vertical resolu- tion in the near-surface region than the reanalysis products in order to capture subsurface heterogeneity in unfrozen soil, which is important to represent the zero-curtain throughout the freezing and thawing periods in Alaska. 2.4 Empirically simulated biogenic CO2 fluxes from tundra ecosystems F l l i i l d 1CO f h TVPRM TVPRM is driven by parameterized functional relation- ships for soil respiration (Rsoil), plant respiration (Rplant), and photosynthesis (gross primary productivity, GPP), which are described by the following respective equations: Rsoil = αs × Ts + βs; (3) Rplant = αa × Ta + βa; (4) GPP = λ × Tscale × SIF × PAR × 1 1 + PAR PAR0 ; (5) Tscale = (Ta −Tmin)(Ta −Tmax) (Ta −Tmin)(Ta −Tmax) −(Ta −Topt)2 . (6) (5) (6) The simulated hourly CO2 fluxes (in units of µmolCO2 m−2 s−1) are determined as responses to light and heat: Rsoil is a function of near-surface soil tem- perature (Ts; ◦C); Rplant is a function of air temperature (Ta; ◦C); and GPP is a function of a temperature scalar (Tscale) and photosynthetically active radiation (PAR; µmolphotonm−2 s−1), with solar-induced chlorophyll fluorescence (SIF; mWm−2 nm−1 sr−1) used to define the seasonal cycle of photosynthetic capacity. Ts depths are determined by reanalysis product and listed in Table S4. Tscale ranges from zero to one based on the position of Ta on the continuum between minimum temperature (Tmin = 0 ◦C), maximum temperature (Tmax = 40 ◦C), and The simulated hourly CO2 fluxes (in units of µmolCO2 m−2 s−1) are determined as responses to light and heat: Rsoil is a function of near-surface soil tem- perature (Ts; ◦C); Rplant is a function of air temperature (Ta; ◦C); and GPP is a function of a temperature scalar (Tscale) and photosynthetically active radiation (PAR; µmolphotonm−2 s−1), with solar-induced chlorophyll fluorescence (SIF; mWm−2 nm−1 sr−1) used to define the seasonal cycle of photosynthetic capacity. Ts depths are determined by reanalysis product and listed in Table S4. Tscale ranges from zero to one based on the position of Ta on the continuum between minimum temperature (Tmin = 0 ◦C), maximum temperature (Tmax = 40 ◦C), and For calculating simulated 1CO2 from the TVPRM ensem- ble, we grid the distribution of WRF-STILT particles and their corresponding surface influence to the spatial resolu- https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 Biogeosciences, 19, 5953–5972, 2022 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5958 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope optimal temperature (Topt = 15 ◦C). NEE is then calculated as optimal temperature (Topt = 15 ◦C). NEE is then calculated as tional relationships for both tundra groups. These fluxes are weighted by the spatial distribution of inland and coastal tun- dra from three different vegetation maps (Circumpolar Arctic Vegetation Map – CAVM, Walker et al., 2005; Raster Cir- cumpolar Arctic Vegetation Map – RasterCAVM, Raynolds et al., 2019; and ABoVE land cover – ABoVE LC, Wang et al., 2020; the reader is also referred to Fig. S5, Table S6, and Sect. S5 in the Supplement) to produce the regionally scaled TVPRM net CO2 flux. By varying the choice of rep- resentative inland and coastal tundra sites, meteorological re- analysis product, vegetation map, and SIF product, we gen- erate 288 different simulations (members) of net CO2 flux (referred to here as the unconstrained TVPRM ensemble) for each grid box across the region for each of the 6 study years. Monthly and annual regional net CO2 flux values are calcu- lated as the area-weighted sum of all grid boxes simulated in our domain. Notable changes since the previous iteration of this empirical CO2 flux model (Commane et al., 2017; Luus et al., 2017) include the expansion of the model to include multiple ensemble members to account for variability and uncertainty in model formulation, the use of additional site- years of CO2 flux observations (with increased data coverage over the cold season), more inclusive data filtering methods, and much higher temporal (1, 4, and 8 d rather than monthly) and spatial (0.01◦and 0.05◦rather than 0.5◦) resolution SIF datasets. We compare TVPRM to the previous model version by Luus et al. (2017) and its CARVE-informed optimization by Commane et al. (2017) in Sect. 3.3. as (7) 3 Results from the two aircraft campaigns and at the tower (TVPRM Constrained) (Sect. 3.1 and 3.2). Then, noting the large range of potential cold-season CO2 fluxes, we compare our constrained TVPRM member with CO2 fluxes from pre- vious studies (Sect. 3.3). Finally, we suggest and quantify sources of the missing CO2 flux observed during the early cold season (defined here as September–December) and in- corporate those fluxes into our net CO2 budget (TVPRM Constrained + Additional Zero-Curtain Emissions and In- land Water Fluxes) (Sect. 3.4). This analysis provides a unique regional net CO2 flux quantification for the North Slope that is verified using atmospheric observations and can also be explained from an ecological and physical perspec- tive. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope For panels (a) and (b), observed values are vertically binned medians, and vertical lines contain the middle 95 % of 1CO2 values from all binned points for the constrained TVPRM member + ZC and IW. (d) Combined comparison of observed and simulated 1CO2 for all aircraft and tower points using the constrained TVPRM member + ZC and IW. The linear best fit (red line), the slope determined by ordinary least squares, and the coefficient of determination (R2) of all points (n = 455) are shown. The 1 : 1 is line shown in dark gray. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope ification of annual biogenic CO2 fluxes on the Alaska North Slope Figure 2. Aircraft and tower CO2 concentration measurements constrain year-round simulated CO2 fluxes on the Alaska North Slope. (a– c) Comparison of observed and simulated 1CO2 during the ARM-ACME V flight campaign (a), during the ABoVE Arctic-CAP flight campaign (b), and at the NOAA BRW tower (c) for air over the Alaska North Slope. Horizontal lines indicate the range of uncertainty in the NOAA BRW tower ocean-sector background calculation. Vertical boxes colored by month of the year represent 50 % and whiskers represent 95 % of the 1CO2 values from all members of the unconstrained TVPRM ensemble (see Sect. 2.4) from all binned points. Black points show values from the constrained TVPRM member with additional zero-curtain (ZC) emissions and inland water (IW) fluxes (see Sect. 3.4). For panels (a) and (b), observed values are vertically binned medians, and vertical lines contain the middle 95 % of 1CO2 values from all binned points for the constrained TVPRM member + ZC and IW. (d) Combined comparison of observed and simulated 1CO2 for all aircraft and tower points using the constrained TVPRM member + ZC and IW. The linear best fit (red line), the slope determined by ordinary least squares, and the coefficient of determination (R2) of all points (n = 455) are shown. The 1 : 1 is line shown in dark gray. Figure 2 Aircraft and tower CO2 concentration measurements constrain year-round simulated CO2 fluxes on the Alaska North Slope (a– Figure 2. Aircraft and tower CO2 concentration measurements constrain year-round simulated CO2 fluxes on the Alaska North Slope. (a– c) Comparison of observed and simulated 1CO2 during the ARM-ACME V flight campaign (a), during the ABoVE Arctic-CAP flight campaign (b), and at the NOAA BRW tower (c) for air over the Alaska North Slope. Horizontal lines indicate the range of uncertainty in the NOAA BRW tower ocean-sector background calculation. Vertical boxes colored by month of the year represent 50 % and whiskers represent 95 % of the 1CO2 values from all members of the unconstrained TVPRM ensemble (see Sect. 2.4) from all binned points. Black points show values from the constrained TVPRM member with additional zero-curtain (ZC) emissions and inland water (IW) fluxes (see Sect. 3.4). 2.5 Evaluation framework We use the atmospheric CO2 concentration observations to evaluate the many tundra ecosystem parameterizations, veg- etation distributions, and environmental drivers that represent the net CO2 flux on the North Slope over various spatial and temporal scales. For this assessment, we compare the ob- served 1CO2 values, which are the observed CO2 concen- tration changes driven by regional CO2 fluxes, with the sim- ulated 1CO2 values determined by combining the regional biogenic CO2 flux models with the atmospheric transport model. Using the median parameter value sets for each site, we simulate the TVPRM net CO2 flux for our study pe- riod at every site location to perform a cross-site evalua- tion (Fig. S1). These simulated net CO2 fluxes perform well against the net CO2 flux observations at their corresponding sites (Figs. 1d, S4; see Sect. S4). This process also identi- fies two distinct ecosystem groups – “inland”, predominately graminoid and shrub tundra (ICS, ICT, ICH, and IVO), and “coastal”, predominately wetland tundra (ATQ, BES, BEO, and CMDL) – based on the similar simulated CO2 flux re- sponses to the meteorology- and SIF-determined functional relationships within each group demonstrated by the cross- site evaluation (Fig. S1). To compare the regional observed 1CO2 and simulated 1CO2, we calculated the coefficient of determination (R2) as the square of the Pearson correlation coefficient for all points. The slope is determined by ordinary least squares us- ing the median of each 10 % bin of ordered observed and corresponding simulated net CO2 flux. The normalized mean bias (NMB) of all points is defined as P(simulated−observed) Pobserved . The root-mean-square error (RMSE) of all points is defined as p (simulated −observed)2. These comparisons enable us to constrain the regional net CO2 flux on the Alaska North Slope. First, we iden- tify the year-round empirically driven net CO2 fluxes from the TVPRM ensemble (TVPRM Unconstrained) which are most consistent with the CO2 concentration observations The net CO2 flux for each meteorological grid box in our study domain is then calculated using the site-level func- Biogeosciences, 19, 5953–5972, 2022 https://doi.org/10.5194/bg-19-5953-2022 5959 https://doi.org/10.5194/bg-19-5953-2022 3.1.2 Using tower-observed CO2 enhancements 3a), resulting in an overestimate of the re- gional 1CO2 in the late cold season. The CO2 flux response to Ts for ICT members is similar to that for ICS but lower in magnitude, and the simulated 1CO2 from members of nei- ther site performs well against the observations in both the early and late cold season. Therefore, ICS and ICT inland tundra responses to Ts are not representative of the regional 1CO2 observed at the NOAA BRW tower throughout the entire cold season, and we remove those members from our TVPRM ensemble. Given the large range of unconstrained representations of the regional CO2 flux, the accuracy in simulating the aircraft- observed 1CO2 varies between TVPRM ensemble mem- bers. For example, members using the RasterCAVM vege- tation map, which places less coastal tundra area cover in the south (Fig. S5), produce a smaller mean July net CO2 uptake flux (by ∼1 µmolm−2 s−1, Fig. S7a) throughout the south- ern North Slope than members using other vegetation maps (CAVM and ABoVE LC), and this placement consistently underestimates the net 1CO2 uptake during the growing sea- son by 5–10 ppm compared with the aircraft observations (Fig. S8). Also, members driven by SIF products that inte- grate additional remote sensing and/or meteorological data (GOSIF and CSIF) better reflect the timing and magnitude of the peak season carbon uptake in tundra ecosystems than members produced by interpolated SIF retrievals (GOME-2 SIF product), which underestimate the observed CO2 uptake during July (Fig. S8). The observed net CO2 fluxes at the IVO inland tundra and CMDL coastal tundra sites both show prolonged zero-curtain emissions (Fig. S1) and respond strongly to Ts in the early cold season (Fig. S9). The stronger response of CO2 fluxes to Ts from the early to late cold season at IVO (70 % de- crease by January; Fig. 3a) compared with at the Imnavait Creek sites produces TVPRM members that better represent the large regional decrease in 1CO2 observed on the North Slope (Fig. 3c). While all coastal tundra sites respond sim- ilarly to Ts during the cold season, we determine that the CO2 flux magnitude at CMDL is most consistent with the regional observations (Fig. S11). Ts values from ERA5 re- main warmer throughout the late cold season compared with those from NARR, which causes simulations using ERA5 Ts to overestimate CO2 release during that time (Fig. S11). 3.1.2 Using tower-observed CO2 enhancements during ARM-ACME V, and the ABoVE Arctic-CAP flights observe a strong CO2 source throughout the North Slope (+10 ppm) by November. The difference in observed 1CO2 during peak growing season uptake between 2015 and 2017 is likely similar to the uncertainty in the respective values and could be due to differences in areas of the North Slope sampled between years. As seen with the September–November aircraft data, the ob- served 1CO2 at the NOAA BRW tower (Fig. 2c) indicate that the CO2 source to the atmosphere increases substan- tially from September to peak in October and November (+12 ppm) before decreasing to near zero throughout the late cold season (January–April). p y The magnitude and timing of the observed net CO2 uptake throughout the growing season is generally well represented by the empirical net CO2 flux model ensemble (TVPRM Un- constrained; Figs. 2a, b, and S6). The median coefficients of determination (R2) and ordinary least squares slopes between the observed and simulated 1CO2 for this time are 0.54 and 0.41 for ARM-ACME V and 0.82 and 0.72 for ABoVE Arctic-CAP, respectively. Only for the July obser- vations during the ABoVE Arctic-CAP campaign do many members of the CO2 flux trend toward an underestimate of net CO2 uptake, with all points showing a much larger range of simulated values compared to ARM-ACME V. The net CO2 release tends to be overestimated by the TVPRM en- semble during the ABoVE Arctic-CAP seasonal transitions in May and September, but the observed Rsoil is consistently underestimated during November. Most of the TVPRM ensemble members substantially un- derestimate the observed 1CO2 in the early cold season (September–December) as the soils freeze, and some simula- tions produce too much CO2 in the late cold season when the soils are frozen (Fig. 2c). The cold-season CO2 flux differs greatest in magnitude and spatial extent between the ensem- ble members parameterized for the ICS and ICT inland tun- dra sites (Figs. 3a, S9, S10), with a net CO2 flux difference of ∼0.2 µmolm−2 s−1 throughout the region (Fig. S7b). While the magnitude of CO2 flux from ICS members bet- ter matches the observed 1CO2 in the early cold season than that from other sites (Figs. 3b, c, and S11), the response to Ts at ICS shows only a modest decrease in CO2 flux between the early and late cold season (32 % decrease between October and March; Fig. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 3.1.1 Using aircraft-observed CO2 enhancements The observed 1CO2 during the ARM-ACME V (June– September 2015) and ABoVE Arctic-CAP (May–November 2017) airborne campaigns show a strong seasonal uptake pat- tern throughout the growing season (Fig. 2a, b). The frequent flights during ARM-ACME V (multiple flights per week) ob- serve the transition from early to peak growing season uptake (observed 1CO2 = −11 ppm) and on into cold-season respi- ration, which results in net CO2 source conditions in Septem- ber (+5 ppm). While less frequent, the ABoVE Arctic-CAP flights begin at the end of the cold season, extend later into following cold season, and cover a larger area of the North Slope. Peak growing season uptake observed by the ABoVE Arctic-CAP flights (−14 ppm) is slightly stronger than for https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 5960 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope Yearly colored lines shown for September–April beginning in September 2012 and ending in April 2017. The same is shown for all eight eddy flux sites in Fig. S9. (b, c) Comparison of observed and simulated 1CO2 at the NOAA BRW tower for air over the North Slope using the median of all unconstrained TVPRM ensemble members for the inland tundra site parameterizations at ICS (b) and IVO (c). All points are colored by day of year. The slope determined by ordinary least squares and the coefficient of determination (R2) of all points (n = 191) are shown. The 1 : 1 line is also shown in dark gray. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5961 Figure 3. Cold-season CO2 emissions for the inland tundra site parameterizations and comparison to tower observations. (a) Time series of simulated daily mean Alaska North Slope net CO2 flux for the median of all unconstrained TVPRM ensemble members using each of the three inland tundra site parameterizations: ICS (red), ICT (orange), and IVO (green). Yearly colored lines shown for September–April beginning in September 2012 and ending in April 2017. The same is shown for all eight eddy flux sites in Fig. S9. (b, c) Comparison of observed and simulated 1CO2 at the NOAA BRW tower for air over the North Slope using the median of all unconstrained TVPRM ensemble members for the inland tundra site parameterizations at ICS (b) and IVO (c). All points are colored by day of year. The slope determined by ordinary least squares and the coefficient of determination (R2) of all points (n = 191) are shown. The 1 : 1 line is also shown in dark gray. Figure 3. Cold-season CO2 emissions for the inland tundra site parameterizations and comparison to tower observations. (a) Time series of simulated daily mean Alaska North Slope net CO2 flux for the median of all unconstrained TVPRM ensemble members using each of the three inland tundra site parameterizations: ICS (red), ICT (orange), and IVO (green). Yearly colored lines shown for September–April beginning in September 2012 and ending in April 2017. The same is shown for all eight eddy flux sites in Fig. S9. (b, c) Comparison of observed and simulated 1CO2 at the NOAA BRW tower for air over the North Slope using the median of all unconstrained TVPRM ensemble members for the inland tundra site parameterizations at ICS (b) and IVO (c). All points are colored by day of year. The slope determined by ordinary least squares and the coefficient of determination (R2) of all points (n = 191) are shown. The 1 : 1 line is also shown in dark gray. Figure 3. Cold-season CO2 emissions for the inland tundra site parameterizations and comparison to tower observations. (a) Time series of simulated daily mean Alaska North Slope net CO2 flux for the median of all unconstrained TVPRM ensemble members using each of the three inland tundra site parameterizations: ICS (red), ICT (orange), and IVO (green). 3.1.2 Using tower-observed CO2 enhancements Un- like during the growing season, cold-season CO2 fluxes are not sensitive to the vegetation distribution nor the SIF prod- ucts. Using these comparisons, we identify less-representative ensemble members that generally underestimate the ob- served 1CO2 uptake during the growing season (Raster- CAVM vegetation map and GOME-2 SIF product members). Removing these members from the TVPRM ensemble im- proves the collective performance of the remaining members during the growing season (Fig. S6), brings the median slope of agreement closer to 1 for both campaigns (improves from 0.53 to 0.64 and from 0.71 to 0.94 for ARM-ACME V and ABoVE Arctic-CAP, respectively), and reduces the median NMB (−0.34 to −0.03) and median RMSE (3.12 to 2.73) for ABoVE Arctic-CAP. Finally, we identify the TVPRM member that best matches the observed 1CO2: parameterized by IVO inland tundra and CMDL coastal tundra site responses, distributed by the ABoVE LC vegetation map, and driven by NARR reanaly- sis and the CSIF SIF product (referred to here as TVPRM Biogeosciences, 19, 5953–5972, 2022 https://doi.org/10.5194/bg-19-5953-2022 https://doi.org/10.5194/bg-19-5953-2022 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope Figure 4. Tall-tower atmospheric observations of the Alaska North Slope support early-cold-season emissions not driven by soil temper- ature (Ts) and present no evidence of elevated late-cold-season emissions. (a, b) Comparison of hourly cold-season (September–April) observed and simulated 1CO2 at the NOAA BRW tower for the constrained TVPRM member, where soil respiration (Rsoil) is determined only by Ts (a) and for the constrained TVPRM member + additional zero-curtain (ZC) emissions and inland water (IW) fluxes (b). Hori- zontal segments indicate the range of uncertainty in the NOAA BRW tower ocean-sector background calculation. The slope determined by ordinary least squares and the coefficient of determination (R2) of all points (n = 191) are shown. The 1 : 1 line is also shown in dark gray. (c) Monthly cold-season total Alaska North Slope net CO2 fluxes for various CO2 flux models. TVPRM-based simulations and Natali and Watts et al. (2019) show values for 2012–2017, Luus et al. (2017) show 2012–2014, and Watts et al. (2021) show September 2016–April 2017. Ribbons represent the range of all years, where applicable. The area of the North Slope domain used to calculate regional totals is 3.537 × 105 km2. Fi 4 T ll t t h i b ti f th Al k N th Sl t l ld i i t d i b il t Figure 4. Tall-tower atmospheric observations of the Alaska North Slope support early-cold-season emissions not driven by soil temper- ature (Ts) and present no evidence of elevated late-cold-season emissions. (a, b) Comparison of hourly cold-season (September–April) observed and simulated 1CO2 at the NOAA BRW tower for the constrained TVPRM member, where soil respiration (Rsoil) is determined only by Ts (a) and for the constrained TVPRM member + additional zero-curtain (ZC) emissions and inland water (IW) fluxes (b). Hori- zontal segments indicate the range of uncertainty in the NOAA BRW tower ocean-sector background calculation. The slope determined by ordinary least squares and the coefficient of determination (R2) of all points (n = 191) are shown. The 1 : 1 line is also shown in dark gray. (c) Monthly cold-season total Alaska North Slope net CO2 fluxes for various CO2 flux models. TVPRM-based simulations and Natali and Watts et al. (2019) show values for 2012–2017, Luus et al. (2017) show 2012–2014, and Watts et al. (2021) show September 2016–April 2017. Ribbons represent the range of all years, where applicable. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope The area of the North Slope domain used to calculate regional totals is 3.537 × 105 km2. constrained CO2 fluxes to several other representations of gridded CO2 flux on the North Slope (Table S3) and find that difficulty in simulating the magnitude and timing of the ob- served 1CO2 throughout the cold season is not unique to the constrained fluxes from our study. the implementation of a multilayer fit driven by soil column temperature from RS-PM, but neither of these instances of remote-sensing-informed Ts substantially improve the agree- ment of the 1CO2 at the NOAA BRW tower during the early cold season. Attempts to use alternative Rsoil formulations based on Ts, including Q10 relationships, also fail to repro- duce the observed elevated CO2 fluxes during the cold sea- son. The net CO2 fluxes from Luus et al. (2017) are similar to the constrained TVPRM member during the growing sea- son (Fig. S16) but release more than 3 times as much CO2 into the atmosphere throughout the late cold season (Fig. 4c). This large late-cold-season CO2 flux leads to a large overes- timate compared with the observed 1CO2 (Fig. S14b). The optimization employed by Commane et al. (2017) increases the September–October CO2 flux to a range that matches our observations at the NOAA BRW tower. However, Commane et al. (2017) did not optimize the cold-season fluxes from November to March, instead reverting to Luus et al. (2017) 3.2 Alternative Ts products and Rsoil parameterizations Constrained; Figs. S6, S12). This constrained simulation estimates a mean regional CO2 flux of 0.05 µmolm−2 s−1 for the late cold season in 2012–2015 and reproduces the observed 1CO2 during this time well (Fig. 4a). The late- cold-season NMB and RMSE against the observations at the NOAA BRW tower are reduced from 4.91 to 2.04 and from 1.94 to 1.30, respectively, for the constrained simula- tion compared with the median of the entire TVPRM ensem- ble (Fig. S12). However, the early-cold-season CO2 emis- sions, with a mean regional CO2 flux of 0.25 µmolm−2 s−1 for September–December (Fig. S13a), are still underesti- mated, with the simulated 1CO2 lower than the observed 1CO2 by ∼5 ppm (Fig. 4a). To test the impact of reanalysis Ts on the early-cold-season CO2 fluxes, we implement Ts values that are more specifi- cally developed to represent Alaska tundra permafrost soils during freeze–thaw processes than the reanalysis products driving our constrained TVPRM member. A single layer of Ts at 8 cm depth from RS-PM (Fig. S14a) captures the mag- nitude and temporal behavior of the observed early-cold- season CO2 fluxes slightly better than the constrained mem- ber (Figs. 4a, S12), which uses NARR reanalysis Ts and does not incorporate permafrost-model-derived Ts. The RS-PM Ts extends CO2 emission fluxes further into the cold season by up to a month, which is consistent with a better representa- tion of the zero-curtain period; however, emissions remain higher throughout the late cold season than our atmospheric- observation-constrained CO2 fluxes (Fig. S15). We also test https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slop 5962 3.4 Sources of missing CO2 fluxes The median Alaska North Slope annual net CO2 flux from the TVPRM ensemble (−5 TgCyr−1) for 2012–2017 is con- sistent with the previous multi-model comparison (Fisher et al., 2014), but we find a much smaller range of regional CO2 flux values (26 to −29 TgCyr−1 for 95 % of TVPRM mem- bers; Fig. S18). The largest contribution to this ensemble range comes from the difference in parameterizations deter- mined for the ICS and ICT inland tundra sites, with TVPRM members using ICS trending toward a net CO2 source, while ICT trends toward net CO2 uptake. The distribution of in- land and coastal tundra throughout the region represented by the vegetation maps also has a noticeable impact on the sign of the net CO2 flux, with members using the Raster- CAVM more likely to release net CO2 into the atmosphere than members using the other maps. There is also little inter- annual variability in the unconstrained TVPRM ensemble, with only 2014 moving toward a net CO2 source, consistent with Tao et al. (2021) for these years. None of the flux products discussed above, including our TVPRM ensemble, account for any potential CO2 fluxes dur- ing the zero-curtain period that are not driven by Ts or are from areas on the terrestrial–aquatic interface. To account for these processes, we first add an additional CO2 flux with zero-curtain timing to our constrained CO2 flux (TVPRM) member from both inland and coastal tundra areas that con- sists of 0.25 µmolm−2 s−1 for October with a reduction to 0 by the end of December. This peak additional CO2 flux is within the daily variability in the observed CO2 flux at the IVO and CMDL eddy flux sites during the zero-curtain pe- riod (Fig. S9), and the reduction into December is consis- tent with these observations. The additional zero-curtain flux improves the ability of the model to reproduce the observed 1CO2 at the NOAA BRW tower (slope = 0.46, R2 = 0.41). We also apply the coastal tundra site ecosystem parame- terization used in our constrained TVPRM member to all areas of inland water on the North Slope, which account for 4 % of the domain according to the ABoVE LC map (Fig. S5) and were previously set to zero CO2 flux. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope fluxes during this time, and thus produced late-cold-season fluxes that are too large. Overall, Commane et al. (2017) pro- jected a regional total cold-season CO2 source of 37–40 TgC for 2012–2014, which is more than twice as high as our con- strained TVPRM member CO2 flux (15–18 TgC) for those years. coastal tundra fluxes to inland water areas also improves the performance of our model (slope = 0.32 and R2 = 0.29 against NOAA BRW tower observations). The magnitude of additional zero-curtain flux suggested here and the portion of inland water represented with coastal tundra site parameteri- zations produce the best statistical comparison for a range of choices tested (Fig. S17). Carbon dioxide fluxes from work by Natali and Watts et al. (2019), a cold-season model developed for the global high-latitude permafrost region, are similar to our con- strained TVPRM member in September, but the fluxes re- main high throughout the cold season (Fig. 4c), similarly to Luus et al. (2017), for a range of total cold-season CO2 flux of 40–43 TgC for 2012–2017. This sustained CO2 re- lease also leads to an overestimation in the 1CO2 in the late cold season for this region (Fig. S14c). Tao et al. (2021) also show that the cold-season CO2 fluxes of Natali and Watts et al. (2019) are high compared with their model. More recent work by Watts et al. (2021), using observations from new Soil Respiration Station monitoring sites in Alaska, produces cold-season CO2 fluxes more similar to our constrained CO2 fluxes, with an underestimate in the simulated 1CO2 during the early cold season (Fig. S14d), for a total cold-season CO2 flux of 18 TgC for September 2016 to April 2017. Together, adding these zero-curtain (ZC) and inland wa- ter (IW) CO2 fluxes to our constrained simulation (referred to as TVPRM Constrained + ZC and IW) increases the mean regional CO2 flux in the early cold season by 70 % (0.18 µmolm−2 s−1; Fig. S13b) and results in a large im- provement to our comparison of 1CO2 at the NOAA BRW tower (slope = 0.54, R2 = 0.40; Figs. 4b, S12) and across the region using airborne data, especially during the Novem- ber ABoVE Arctic-CAP flights (Figs. 2, S6). The year-round comparison using all available aircraft and tower observa- tions shows that these net CO2 fluxes are now representative of the region (slope = 0.90, R2 = 0.80; Fig. 2d). L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope As a result, the North Slope regional total cold-season CO2 flux increases by 6 TgC (∼38 %) to 20–24 TgC for 2012–2017 compared with the constrained empirical CO2 flux model member. 3.3 Evaluation of other CO2 flux models during the cold season More early-cold-season (September–December) CO2 flux into the atmosphere is observed at the NOAA BRW tower than is emitted by our constrained empirical simulation member, and these observations also indicate low late-cold- season (January–April) CO2 emissions. We compare our https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 5963 3.4 Sources of missing CO2 fluxes Purple squares indicate the middle 95 % of all TVPRM ensemble members. Figure 5. Annual and seasonal Alaska North Slope net CO2 flux constrained by aircraft and tower observations. (a) Annual, (b) late-cold- season (January–April), (c) growing-season (May–August), and (d) early-cold-season (September–December) total Alaska North Slope net CO2 fluxes for various CO2 flux models for 2012–2017, as in Fig. 4. Purple squares indicate the middle 95 % of all TVPRM ensemble members. North Slope to be a strong annual net CO2 source for 2012– 2014 (+9 to +15 TgCyr−1; Fig. S18) and are inconsistent with our results. Our results are more consistent with Tao et al. (2021), but we find a smaller range in the magnitude of net CO2 flux over the same years and more years trending toward a net CO2 source. cates that the tundra ecosystems of the Alaska North Slope respond to light and heat, as quantified by PAR, Ts, and Ta, and that the net CO2 flux is largely controlled by the simple Rsoil, Rplant, and GPP relationships in the empirical model over this time. The growing season of each year determines the sign of the regional annual net CO2 flux during our study period, with 2013 and 2015 being strong net sinks and 2014 being the strongest net source. The relative magnitude of each com- ponent of the net CO2 flux during the growing season (i.e., Rsoil, Rplant, and GPP) varies from year to year (Table S7) and helps explain the interannual variability in the net source or sink status of the North Slope. The growing season in 2015 was very warm, dry, and sunny in Alaska and resulted in ex- treme biomass burning activity outside of the North Slope (Table S1). High regional mean Ta and PAR (Table S8) and low accumulated precipitation (Table S9) in NARR confirm this was the case for North Slope as well, with high Ta and PAR contributing to a very high GPP. The growing season SIF signal from the CSIF product, which determines the sea- sonal cycle and relative magnitude of photosynthetic activ- ity, is also large in 2015 (Table S8), further enhancing GPP. 3.4 Sources of missing CO2 fluxes This year and others with a larger GPP component of NEE correspond to growing seasons with stronger SIF signals, which is an indicator of increased productivity and consis- We find that the regional net growing season CO2 up- take and the cold-season emissions on the North Slope are comparable in magnitude, so the net balance could depend on small perturbations in either flux. However, the regional cold-season CO2 emissions for these years were relatively similar from year to year: 18–21 TgC for the early cold sea- son (Fig. 5d), diminishing to only 2–3 TgC for the late cold season (Fig. 5b). Therefore, the interannual variability in the regional carbon balance is largely driven by fluctuating net growing season CO2 fluxes during these years: greater net growing season uptake in 2013 and 2015 than in 2012, 2014, 2016, and 2017 (Fig. 5c). 3.4 Sources of missing CO2 fluxes Repre- senting these aquatic areas with biogenic CO2 fluxes con- sistent with coastal tundra ecosystems is one simple way to bridge the terrestrial–aquatic gap in tundra ecosystem mod- els, where portions of aquatic systems on the land–water gra- dient (i.e., the edges) may be more likely to respond to the en- vironment as coastal tundra than with the zero-flux assumed by water area. The ice phenology for areas of inland water producing CO2 flux is then considered to be similar to that of the freeze–thaw timing in coastal tundra soils. Adding these Our best quantification of the annual net CO2 flux for the North Slope informed by atmospheric observa- tions, TVPRM Constrained + ZC and IW, indicates that the region is a small net sink for 2013 (−5 TgCyr−1) and 2015 (−6 TgCyr−1) and a small net source for 2012 (+6 TgCyr−1), 2014 (+6 TgCyr−1), 2016 (+2 TgCyr−1), and 2017 (+2 TgCyr−1) (Fig. 5a). We estimate a 10 % un- certainty in the net annual CO2 flux based on the slope from our final comparison with the year-round observa- tions (Fig. 2d). The year-round net CO2 fluxes from Luus et al. (2017) (driven with NARR meteorology, monthly GOME-2 SIF, and the CAVM vegetation map) indicate the https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 5964 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope Figure 5. Annual and seasonal Alaska North Slope net CO2 flux constrained by aircraft and tower observations. (a) Annual, (b) late-cold- season (January–April), (c) growing-season (May–August), and (d) early-cold-season (September–December) total Alaska North Slope net CO2 fluxes for various CO2 flux models for 2012–2017, as in Fig. 4. Purple squares indicate the middle 95 % of all TVPRM ensemble members. Figure 5. Annual and seasonal Alaska North Slope net CO2 flux constrained by aircraft and tower observations. (a) Annual, (b) late-cold- season (January–April), (c) growing-season (May–August), and (d) early-cold-season (September–December) total Alaska North Slope net CO2 fluxes for various CO2 flux models for 2012–2017, as in Fig. 4. Purple squares indicate the middle 95 % of all TVPRM ensemble members. Figure 5. Annual and seasonal Alaska North Slope net CO2 flux constrained by aircraft and tower observations. (a) Annual, (b) late-cold- season (January–April), (c) growing-season (May–August), and (d) early-cold-season (September–December) total Alaska North Slope net CO2 fluxes for various CO2 flux models for 2012–2017, as in Fig. 4. 4.2 Regional-scale cold-season CO2 emissions While varying topography and soil inundation throughout the North Slope means that each of these site relationships is likely to be rep- resentative of many other locations in the region with similar conditions, the early- to late-cold-season reduction in CO2 fluxes at these sites is not consistent with the observed re- gional atmospheric trend, and we remove the members pa- rameterized by them from the ensemble. Individual eddy flux site parameterizations may reproduce regional CO2 fluxes for a given season, but it is important to consider their response to drivers across multiple seasons when scaling from the site- level to regional domains. g g y The regional net CO2 flux is highly sensitive, however, to the distribution of tundra vegetation types (upland vs. coastal) throughout the North Slope during the growing sea- son. Coastal tundra takes up more CO2 for a given unit PAR compared with inland tundra, based on the relationships be- tween observed site-level net CO2 flux and PAR in this study (TVPRM parameters; Fig. S1), which could be evidence of an adaptation to lower light levels. This difference is consis- tent with Luus et al. (2017), who calculated greater uptake at “wetland” sites like Atqasuk and Barrow than at “graminoid tundra” sites like Ivotuk and Imnavait when all driver in- puts are constant, and with Mbufong et al. (2014), who also found that peak growing season net uptake for constant light is greater at Barrow than at Ivotuk. The stronger CO2 up- take response of coastal tundra to light is important to con- sider due to the fact that the vegetation distributions assessed here, with more coastal tundra to the south (CAVM, Walker et al., 2005; ABoVE LC, Wang et al., 2020), better agree with the atmospheric observations. When considering the ability of coastal tundra to take up CO2 when moved toward the south, Patankar et al. (2013) saw that tundra plants exposed to additional intense light did not respond with additional up- take. Therefore, while the ecosystem response of the south- ern North Slope is more consistent with coastal ecosystems, it seems possible that these areas are misclassified in either our simplified two-tundra-type scheme or in the vegetation maps themselves. 4.2 Regional-scale cold-season CO2 emissions Extremely low Ts causes this very low Rsoil, which, relative to moderate Ta and PAR, is likely a result of an above-average lingering snowpack into May (Table S9). This lingering snowpack is perhaps surprising given that the mean snowpack for the proceeding cold season was not par- ticularly deep. The important impact that snow cover and the timing of snowmelt has on Ts and carbon response in tun- dra ecosystems has recently been emphasized (e.g., Kim et al., 2021) and is also supported by our work, which shows that the prevalence of snow in the spring may determine the sign of the regional net CO2 for an entire year. Observations across scales, at the in situ eddy flux towers, the NOAA BRW tower, and from aircraft, consistently show signs of large early-cold-season CO2 emissions from ecosys- tems on the Alaska North Slope. However, there is no evi- dence of widespread elevated emissions in this region dur- ing the late cold season, contrary to other studies (Commane et al., 2017; Natali and Watts et al., 2019). The TVPRM en- semble parameterizations using terrestrial eddy flux sites and the fluxes from other terrestrial CO2 models cannot repro- duce both the observed magnitude and across-season timing of these cold-season CO2 emissions. 2 The largest differences in the net CO2 flux between TVPRM ensemble members result from the contrasting site conditions driving the ICS and ICT Rsoil parameterizations during the cold season. When taken separately by cold- season segment, ICS members perform quite well against ob- servations at the NOAA BRW tower for the early cold sea- son and ICT members perform well for the late cold sea- son. The contrasting performance between site parameteri- zations is due to the topographic and hydrologic conditions, which are quite heterogeneous over a short distance and in- fluence the plant communities and carbon storage, at each site. The ecosystems sampled by the ICS tower are season- ally inundated and retain a deep layer of organic soil that can be respired in greater amounts longer into the early cold sea- son, whereas the well-drained hillslope at ICT does not al- low for the accumulation of organic matter in the same way (Euskirchen et al., 2017; Larson et al., 2021). 4.2 Regional-scale cold-season CO2 emissions tent with previous studies (e.g., Magney et al., 2019; Sun et al., 2017). While fairly high Ta and Ts in 2015 also result in high Rsoil and Rplant, respectively, this elevated respira- tion is not enough to offset the very high GPP and results in a large net CO2 sink. In contrast, the summer of 2014 was cool, wet, and cloudy, and the North Slope experienced a very low Ta, PAR, and SIF signal, producing very low GPP. Lower-than-normal Ta also results in very low Rplant, but (as for 2015) this is not enough to offset the extremely low up- take, resulting in a large net CO2 source for 2014. In 2013, the other growing season with a strong net CO2 sink, mod- erately high GPP combines with moderately low Rplant and very low Rsoil. Extremely low Ts causes this very low Rsoil, which, relative to moderate Ta and PAR, is likely a result of an above-average lingering snowpack into May (Table S9). This lingering snowpack is perhaps surprising given that the mean snowpack for the proceeding cold season was not par- ticularly deep. The important impact that snow cover and the timing of snowmelt has on Ts and carbon response in tun- dra ecosystems has recently been emphasized (e.g., Kim et al., 2021) and is also supported by our work, which shows that the prevalence of snow in the spring may determine the sign of the regional net CO2 for an entire year. tent with previous studies (e.g., Magney et al., 2019; Sun et al., 2017). While fairly high Ta and Ts in 2015 also result in high Rsoil and Rplant, respectively, this elevated respira- tion is not enough to offset the very high GPP and results in a large net CO2 sink. In contrast, the summer of 2014 was cool, wet, and cloudy, and the North Slope experienced a very low Ta, PAR, and SIF signal, producing very low GPP. Lower-than-normal Ta also results in very low Rplant, but (as for 2015) this is not enough to offset the extremely low up- take, resulting in a large net CO2 source for 2014. In 2013, the other growing season with a strong net CO2 sink, mod- erately high GPP combines with moderately low Rplant and very low Rsoil. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5965 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 4.1 Tundra ecosystem growing season net CO2 fluxes The good performance of the TVPRM ensemble against the atmospheric observations during the growing season indi- https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope 5966 L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope our TVPRM ensemble determined by North Slope eddy flux tower measurements. therefore, the North Slope was not a consistent net source through 2017. Accordingly, care must be taken to accurately represent CO2 fluxes from Arctic ecosystems during both the early and late cold season when calculating the annual net CO2 budget. TVPRM could be used with projections of me- teorology and SIF to calculate the future net CO2 balance for this region, but we caution against overuse of the model us- ing current parameters, as the flux–driver relationships in the rapidly warming Arctic ecosystems are changing so quickly that we would not assume accuracy into the future. While we can constrain the annual net CO2 budget with existing data, the Arctic is rapidly changing and needs constant mon- itoring. The following recommendations would provide more detailed spatial and seasonal constraints and up-to-date infor- mation on the processes driving CO2 fluxes across the region. therefore, the North Slope was not a consistent net source through 2017. Accordingly, care must be taken to accurately represent CO2 fluxes from Arctic ecosystems during both the early and late cold season when calculating the annual net CO2 budget. TVPRM could be used with projections of me- teorology and SIF to calculate the future net CO2 balance for this region, but we caution against overuse of the model us- ing current parameters, as the flux–driver relationships in the rapidly warming Arctic ecosystems are changing so quickly that we would not assume accuracy into the future. While we can constrain the annual net CO2 budget with existing data, the Arctic is rapidly changing and needs constant mon- itoring. The following recommendations would provide more detailed spatial and seasonal constraints and up-to-date infor- mation on the processes driving CO2 fluxes across the region. We find that the atmospheric observations are best matched by biogenic CO2 fluxes that include an additional CO2 source from tundra ecosystems during the zero-curtain period that are independent of Ts variability and year-round net CO2 fluxes from areas of inland water. 4.3.1 Future observation efforts Our results motivate the need for a more extensive network of CO2 eddy flux towers operating year-round, alongside sen- sors for soil moisture and Ts profiles throughout the active layer to better understand the mechanisms driving year-round and especially early-cold-season CO2 fluxes. Noting that au- tomated or semiautomated monitoring systems for aquatic environments currently do not exist for the North Slope or other high-latitude regions, this sensor network should be distributed throughout poorly sampled ecosystem types, par- ticularly along wetness gradients that span mixed terrestrial– aquatic environments. The results of this study also support the need for additional continuous CO2 concentration mea- surements at tall towers across the North Slope (including away from the coast) to increase coverage of observed 1CO2 during all seasons and to better constrain the regional back- ground. Airborne measurements of both CO2 concentrations and CO2 fluxes remain valuable to sample areas less accessi- ble via ground-based measurements, but a large-scale flight campaign in the region has not occurred since 2017. Any additional flights should be targeted as early before, and as late after, the growing season as possible. Satellites that rely on reflected sunlight to detect CO2 have increasingly been used to constrain CO2 budgets in the northern latitudes (e.g., Byrne et al., 2022), but data are very limited in the cold sea- son, especially in far northern regions like the North Slope. 4.3 Future state of net CO2 flux on the Alaska North Slope 4.3 Future state of net CO2 flux on the Alaska North Slope As the Arctic warms rapidly, the competition between the growing and cold-season Arctic CO2 fluxes will determine the net biogenic CO2 flux into the atmosphere. Warming Ta warms soils, thaws permafrost, increases the active-layer thickness, and has extended the duration of the zero cur- tain from weeks to over 100 d (Romanovsky and Osterkamp, 2000; Schuur et al., 2015; Zona et al., 2016), all of which increase cold-season CO2 emissions. The warming may also increase net growing season uptake, but the severe light lim- itation at high northern latitudes limits the extent of the growing season, especially on the North Slope (Zhang et al., 2020). The future of CO2 fluxes from inland waters and wetlands in the Arctic is uncertain, but some studies suggest CO2 emissions from lakes may increase (Bayer et al., 2019). The culmination of these effects will likely push the North Slope into being a consistent net source in the future. How- ever, observations at the NOAA BRW tower during our study period do not show elevated late-cold-season CO2 emissions; L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope The additional zero-curtain flux represents large-scale emission events that are not directly timed to microbial activity and root respi- ration controlled by Ts but that could be related to the de- layed physical release of previously produced CO2 from soil through the snowpack as the soil layers remain unfrozen (Bowling and Massman, 2011). The Alaska North Slope also has many waterbodies distributed throughout the coastal tun- dra region, and the extent to which carbon cycles between small, shallow ponds and their surrounding terrestrial com- ponents is unclear (Magnússon et al., 2020). The biogenic CO2 fluxes in these areas are likely driven by ecosystem- scale CO2 fluxes from both coastal tundra and small ponds (Holgerson and Raymond, 2016; Tan et al., 2017), and their impact on the regional net CO2 flux, via both emissions and uptake, may be significant (Elder et al., 2018; Beckebanze et al., 2022). Only by adding fluxes that match observed zero- curtain CO2 emission pulses and by approximating net CO2 fluxes in aquatic areas can we reproduce the observed 1CO2 magnitude in both the early and late cold season. The re- sulting seasonal change between the early and late cold sea- son is consistent with the extended duration of the observed regional-scale zero curtain. The simplistic approximations suggested here are not inconsistent with the existing uncer- tainties in tundra CO2 flux modeling and demonstrate the im- portance of considering these additional CO2 fluxes and their mechanisms for future study. 4.2 Regional-scale cold-season CO2 emissions The large variability in net CO2 flux calcu- lated using the different maps supports the importance of ac- curate ecosystem type locations in upscaling eddy flux mea- surements and highlights the need for improved vegetation mapping and classification schemes in the Arctic ecology re- search community. The observed cold-season CO2 flux pattern on the North Slope may be unique to tundra ecosystems of this region. For example, the CO2 fluxes from Natali and Watts et al. (2019) and Watts et al. (2021) both incorporate measurements from the North Slope. However, Natali and Watts et al. (2019) used boosted regression trees trained on belowground respiration measurements from across the pan-Arctic tundra and boreal zones, which may not be representative of our study region. The fluxes from Watts et al. (2021) are based on respiration measurements from throughout only Alaska and northwest Canada and conform better to local conditions. The eval- uation of these CO2 fluxes against atmospheric CO2 mea- surements also produces results that are more consistent with https://doi.org/10.5194/bg-19-5953-2022 https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 5 Conclusions – NARR meteorology is available from https://psl.noaa.gov/ data/gridded/data.narr.html (NOAA PSL, 2018). – NARR meteorology is available from https://psl.noaa.gov/ data/gridded/data.narr.html (NOAA PSL, 2018). Observed atmospheric concentrations from aircraft and tow- ers are a powerful tool that provide a regional constraint on the many combinations of possible CO2 flux parameteriza- tions and distributions of tundra ecosystems on the North Slope of Alaska. We find that the annual regional net CO2 flux on the North Slope in not a consistent net source nor sink but instead varies between −6 and +6 TgCyr−1 for 2012–2017. We can also identify ecosystem relationships and driver combinations that best represent both local CO2 flux patterns and regional atmospheric CO2 enhancements. The simulated regional net CO2 flux is highly sensitive to the assumptions made while scaling up eddy flux observations, especially the ecosystem response to Ts of tundra during the cold season and the spatial distribution of tundra types across the North Slope. Additionally, scaling methods that average observations from multiple eddy covariance flux sites should consider which sites are most representative of the regional impact of the biosphere on the atmosphere using integrative top-down observations. – ERA5 meteorology is available from https://www.ecmwf. int/en/forecasts/dataset/ecmwf-reanalysis-v5 (Hersbach et al., 2017). – ERA5 meteorology is available from https://www.ecmwf. int/en/forecasts/dataset/ecmwf-reanalysis-v5 (Hersbach et al., 2017). – GOME-2 SIF is available from https://doi.org/10.3334/ORNLDAAC/2083 (Joiner et al., 2022). – GOSIF is available from https://globalecology.unh.edu/data/ GOSIF.html (Li and Xiao, 2019). – CSIF is available from https://doi.org/10.6084/m9.figshare.6387494 (Zhang, 2018). – The CAVM vegetation map is available from https://www. geobotany.uaf.edu/cavm/ (CAVM Team, 2003). – The CAVM vegetation map is available from https://www. geobotany.uaf.edu/cavm/ (CAVM Team, 2003). – The RasterCAVM vegetation map is available from https://doi.org/10.17632/c4xj5rv6kv.1 (Raynolds and Walker, 2019). – The ABoVE LC vegetation map is available from https://doi.org/10.3334/ORNLDAAC/1691 (Wang et al., 2019). This work shows that year-round measurements of atmo- spheric CO2 concentrations and fluxes across heterogeneous terrestrial and aquatic ecosystems are needed to represent the drivers of CO2 fluxes from Arctic regions. Arctic ecosystems have the potential to accelerate warming if vast stores of car- bon are released or to buffer warming if increasing carbon uptake from vegetation occurs. All components of Arctic tun- dra ecosystems must be fully incorporated into Earth system models to improve projections of future climate warming and associated carbon cycle feedbacks. – RS-PM Ts is available from the authors upon request. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope – ABoVE Arctic-CAP aircraft observations are available from https://doi.org/10.3334/ORNLDAAC/1658 (Sweeney and McKain, 2019). L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope – TVPRM NEE for all ensemble simulations is available from https://doi.org/10.3334/ORNLDAAC/1920 (Schiferl and Commane, 2022). – TVPRM NEE for all ensemble simulations is available from https://doi.org/10.3334/ORNLDAAC/1920 (Schiferl and Commane, 2022). consistent with regional-scale fluxes, rather than incorporat- ing data from all available sites. Consistency and accuracy in classification schemes used in vegetation maps must also be addressed. As we have shown with the atmospheric ob- servations, not all model scenarios have equal likelihood to be true, and the mean of the model ensemble is not necessar- ily the most likely or most consistent with the atmosphere. Using these atmospheric observations is uncertain, however, due to potential errors in the transport modeling, which are difficult to quantify. Atmospheric modeling of remote areas such as the Alaska North Slope requires further evaluation and improvement. Moreover, increasing the model tempo- ral resolution should be considered, as the importance of the zero-curtain and snow cover to the net CO2 flux of tundra ecosystems is recognized, both of which vary on the order of days and weeks, rather than months. consistent with regional-scale fluxes, rather than incorporat- ing data from all available sites. Consistency and accuracy in classification schemes used in vegetation maps must also be addressed. As we have shown with the atmospheric ob- servations, not all model scenarios have equal likelihood to be true, and the mean of the model ensemble is not necessar- ily the most likely or most consistent with the atmosphere. Using these atmospheric observations is uncertain, however, due to potential errors in the transport modeling, which are difficult to quantify. Atmospheric modeling of remote areas such as the Alaska North Slope requires further evaluation and improvement. Moreover, increasing the model tempo- ral resolution should be considered, as the importance of the zero-curtain and snow cover to the net CO2 flux of tundra ecosystems is recognized, both of which vary on the order of days and weeks, rather than months. – ICS, ICT, and ICH eddy flux tower observations are available from http://aon.iab.uaf.edu/data (Euskirchen and Edgar, 2019). – IVO, ATQ, BES, BEO, and CMDL eddy flux tower observa- tions are available from https://doi.org/10.18739/A2X34MS1B (Zona, 2019). – NOAA BRW tower observations are available from https: //gml.noaa.gov/aftp/data/barrow/co2/in-situ/ (Thoning et al., 2018). – ARM-ACME V aircraft observations are available from https://www.arm.gov/research/campaigns/aaf2015armacmev (Biraud et al., 2016). – ABoVE Arctic-CAP aircraft observations are available from https://doi.org/10.3334/ORNLDAAC/1658 (Sweeney and McKain, 2019). 4.3.2 Future modeling efforts The large initial range of potential regional net CO2 flux val- ues that we found for the Alaska North Slope indicates a large sensitivity to the choices and assumptions made when scal- ing eddy flux observations from the site to the regional scale. The most important of these choices are the representation of the upland tundra, particularly for the response of Rsoil to Ts during the cold season, and the distribution of vegetation types throughout the domain. Future tundra CO2 modeling efforts should focus on using site-level data that are the most https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 5967 References Arndt, K. A., Oechel, W. C., Goodrich, J. P., Bailey, B. A., Kalhori, A., Hashemi, J., Sweeney, C., and Zona, D.: Sensi- tivity of Methane Emissions to Later Soil Freezing in Arctic Tundra Ecosystems, J. Geophys. Res.-Biogeo., 124, 2595–2609, https://doi.org/10.1029/2019JG005242, 2019. Competing interests. The contact author has declared that none of the authors has any competing interests. Arndt, K. A., Lipson, D. A., Hashemi, J., Oechel, W. C., and Zona, D.: Snow melt stimulates ecosystem respiration in Arctic ecosystems, Glob. Change Biol., 26, 5042–5051, https://doi.org/10.1111/gcb.15193, 2020. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Bayer, T. K., Gustafsson, E., Brakebusch, M., and Beer, C.: Future Carbon Emission From Boreal and Per- mafrost Lakes Are Sensitive to Catchment Organic Car- bon Loads, J. Geophys. Res.-Biogeo., 124, 1827–1848, https://doi.org/10.1029/2018JG004978, 2019. Acknowledgements. We would like to acknowledge that the Alaskan North Slope is home to multiple Alaska Native nations, including the Nunamiut, Gwich’in, Koyukuk, and Iñupiaq peoples. We support and honor the place-based knowledge of Indigenous Peoples and recognize their ancestral and contemporary steward- ship of their homelands that we research. Luke D. Schiferl and Róisín Commane are supported by research funding from the De- partment of Earth and Environmental Sciences at Columbia Uni- versity and the NASA ABoVE grant no. NNX17AC61A. Luke D. Schiferl is additionally supported by the National Science Foun- dation (NSF) Office of Polar Programs grant no. 1848620. Erik J. L. Larson and J. William Munger are supported by NASA ABoVE grant no. NNX17AE75G. Jennifer D. Watts is supported by NASA ABoVE grant no. 80NSSC19M0209 and NASA grant no. NNH17ZDA001N-NIP. Part of the research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with NASA (contract no. 80NM0018D0004). Im- navait Creek flux towers are funded under grants from the NSF Office of Polar Programs (grant nos. 1503912 and 0632264). Re- sources supporting John M. Henderson and WRF-STILT mod- eling were provided by NASA grant nos. NNX17AE75G and NNX17AC61A as well as by the NASA High-End Computing (HEC) program through the NASA Advanced Supercomputing Beckebanze, L., Rehder, Z., Holl, D., Wille, C., Mirbach, C., and Kutzbach, L.: Ignoring carbon emissions from thermokarst ponds results in overestimation of tundra net carbon uptake, Biogeosciences, 19, 1225–1244, https://doi.org/10.5194/bg-19- 1225-2022, 2022. L. D. Schiferl et al.: Quantification of annual biogenic CO2 fluxes on the Alaska North Slope – Commane et al. (2017b) optimized fluxes are available from https://doi.org/10.3334/ORNLDAAC/1389 (Commane et al., 2017a). (NAS) Division at Ames Research Center. We thank the R Project community for analysis and plotting tools, especially the ggplot2, ggpattern, magick, anytime, lubridate, raster, and cowplot packages. NCEP Reanalysis data were provided by the NOAA/OAR/ESRL PSL, Boulder, Colorado, USA. Some of the data products used in this paper were acquired for CARVE, a NASA Earth Ventures Sub- orbital (EV-S1) investigation. – Natali and Watts et al. (2019) fluxes are available from https://doi.org/10.3334/ORNLDAAC/1683 (Watts et al., 2019). – Natali and Watts et al. (2019) fluxes are available from https://doi.org/10.3334/ORNLDAAC/1683 (Watts et al., 2019). – Watts et al. (2021) fluxes are available from https://doi.org/10.3334/ORNLDAAC/1935 (Watts et al., 2022). – Watts et al. (2021) fluxes are available from https://doi.org/10.3334/ORNLDAAC/1935 (Watts et al., 2022). Financial support. This research has been supported by the National Aeronautics and Space Administration (grant nos. NNX17AC61A, NNX17AE75G, 80NSSC19M0209, NNH17ZDA001N-NIP, and 80NM0018D0004) and the Na- tional Science Foundation, Office of Polar Programs (grant nos. 1848620, 1503912, and 0632264). Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/bg-19-5953-2022-supplement. Author contributions. LDS and RC designed the study. KAA, ESE, JPG, AK, WCO, and DZ provided eddy covariance flux tower data. SCB, KM, and CS provided aircraft concentration data. JMH and MEM provided WRF-STILT particle files and footprints. YY pro- vided RS-PM Ts data. JDW provided Watts et al. (2021) cold-season belowground CO2 fluxes. LDS developed and evaluated TVPRM net CO2 fluxes against observations. RC, EJLL, JWM, and JDW as- sisted the analysis. LDS wrote the paper. All co-authors contributed to the preparation of the manuscript. Review statement. This paper was edited by Paul Stoy and re- viewed by two anonymous referees. Review statement. This paper was edited by Paul Stoy and re- viewed by two anonymous referees. 5 Conclusions – NOAA BRW tower and ARM-ACME V aircraft campaign WRF-STILT footprints are available from https://doi.org/10.3334/ORNLDAAC/1431 (Henderson et al., 2017), and particle trajectories are available from https://doi.org/10.3334/ORNLDAAC/1430 (CARVE Science Team, 2017). – ABoVE Arctic-CAP aircraft campaign WRF-STILT footprints are available from https://doi.org/10.3334/ORNLDAAC/1896 (Henderson et al., 2021a), and particle trajectories are available from https://doi.org/10.3334/ORNLDAAC/1895 (Henderson et al., 2021b). Data availability. Data that support the findings of this study are listed below: – Luus et al. (2017) fluxes are available from https://doi.org/10.3334/ORNLDAAC/1314 (Luus and Lin, 2017). https://doi.org/10.5194/bg-19-5953-2022 Biogeosciences, 19, 5953–5972, 2022 5968 L. D. 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English

Fast and sensitive mapping of nanopore sequencing reads with GraphMap

Nature communications

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ARTICLE Received 30 Dec 2015 | Accepted 11 Mar 2016 | Published 15 Apr 2016 Received 30 Dec 2015 | Accepted 11 Mar 2016 | Published 15 Apr 2016 DOI: 10.1038/ncomms11307 Results Overview of the GraphMap algorithm. The GraphMap algorithm is structured to achieve high-sensitivity and speed using a five-stage ‘read-funneling’ approach as depicted in Fig. 1a. The underlying design principle is to have efficiently computable stages that conservatively reduce the set of candidate locations based on progressively defined forms of the read-to-reference alignment. For example, in stage I, GraphMap uses a novel adaptation of gapped spaced seeds15 to efficiently reduce the search space (Fig. 1b) and then clusters seed hits as a form of coarse alignment (Fig. 1c). These are then refined in stage II using graph-based vertex-centric processing of seeds to efficiently (allowing seed-level parallelism) construct alignment anchors (Fig. 1d). GraphMap then chains anchors using a kmer version of longest common subsequence construction (stage III; Fig. 1e), refines alignments with a form of L1 linear regression (stage IV; Fig. 1e) and finally evaluates the remaining candidates to select the best location to construct a final alignment (stage V). GraphMap computes a BLAST-like E-value as well as a mapping quality for its alignments. Further details about each of these stages, the design choices and how they impact GraphMap’s performance can be found in the Methods section. q p q Read mapping and alignment tools are critical building blocks for many such applications. For mapping, reads from nanopore sequencing are particularly challenging due to their higher and non-uniform error profiles5. For example, one-demensional (1D) reads from the MinION sequencer have raw base accuracy o65–75%; higher quality two-dimensional (2D) reads (80–88% accuracy) comprise a fraction of all 2D reads and the total data set, with overall median accuracy being between 70 and 85% (refs 1,6–9). Reads from other short read (for example, Illumina; o1%) and long read (for example, PacBio; B10%) sequencing technologies have lower overall and mismatch (o1%) error rates. The increased read lengths in nanopore sequencing should facilitate mapping, reducing the ambiguity in location that is the major challenge for short read mappers. However, with current mappers, high-error rates result in a large fraction of reads and bases (10–30%) remaining unmapped or unused (for example, 1D reads) for downstream applications1,6,7. This is further compounded when comparing two error-prone reads to each other or mapping to an imperfect or distant reference. Thus, retaining sensitivity while accommodating high error or divergence rates is the key difficulty for current mapping methods. Results MinION error rates and profiles (that is, ratio of insertions, deletions and substitutions) can vary across chemistries, sequencing runs, read types and even within a read. Furthermore, other nanopore and single-molecule sequencing technologies may present a different distribution of error rates and profiles. Therefore, a general solution to mapping that is applicable to different error characteristics would have high utility for both current and future applications. GraphMap maps reads accurately across error profiles. GraphMap was designed to be efficient while being largely agnostic of error profiles and rates. To evaluate this feature a wide range of synthetic data sets were generated that capture the diversity of sequencing technologies (Illumina, PacBio, ONT 2D, ONT 1D) and the complexity of different genomes (Fig. 2, Supplementary Fig. 1a). GraphMap’s precision and recall was then measured in terms of identifying the correct read location and in reconstructing the correct alignment to the reference (Methods section). These were evaluated separately as, in principle, a mapper can identify the correct location but compute an incorrect alignment of the read to the reference. To provide for a gold-standard to compare against, BLAST (ref. 16) was used as a representative of a highly sensitive but slow aligner which is sequencing technology agnostic. On synthetic Illumina and PacBio data, GraphMap’s results were found to be comparable to BLAST (Supplementary Note 1) as well as other mappers (Supplementary Data 1). On synthetic ONT data, we noted slight differences (o3%) between BLAST and GraphMap, but notably, GraphMap improved over BLAST in finding the right mapping location in some cases (for example, for N. meningitidis ONT 1D data; Fig. 2a). GraphMap’s precision and recall in selecting the correct mapping location were consistently 494%, even with high-error rates in the simulated data. Unlike other mappers, GraphMap’s results were obtained without tuning parameters to the specifics of the sequencing technology. While alignment algorithms have been widely studied, gold-standard solutions such as dynamic programming (or even fast approximations such as BLAST) are too slow in practice for aligning high-throughput sequencing reads. To address this need, a range of read mapping tools have been developed that exploit the characteristics of second-generation sequencing reads (relatively short and accurate) by trading-off a bit of sensitivity for dramatic gains in speed10,11. The design decisions employed in these mappers are often tuned for specific error characteristics of a sequencing technology, potentially limiting their utility across technologies and error profiles. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 ARTICLE T T he release of Oxford Nanopore Technologies (ONT) MinION sequencers in 2014 ushered in a new era of cheap and portable long-read sequencers. Nanopore sequencers have transformative potential for research, diagnostic and low-resource applications. While some initial nanopore sequencing based applications have been reported (for example, scaffolding and resolution of repeats in genomes1 and variant detection in clonal haploid samples2), many others remain to be explored. In particular, diploid and rare-variant calling3, de novo genome assembly4, metagenome assembly and pathogen identification are all promising applications that will likely require new development of in silico techniques. several real and synthetic data sets demonstrate that GraphMap is a more sensitive mapper than BWA-MEM, DALIGNER, BLASR and LAST, while reporting accurate alignments with nanopore sequencing data. This benefits all downstream applications of mapping, as highlighted here with a few natural proof-of-concept applications for a low cost, long read, portable sequencer, that is, single-nucleotide polymorphism calling in complex regions of the human genome, structural variants (SVs; insertions and deletions) detection and real-time pathogen identification. NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications Fast and sensitive mapping of nanopore sequencing reads with GraphMap Ivan Sovic´1,2,*, Mile Sˇikic´3,4,*, Andreas Wilm1, Shannon Nicole Fenlon1,5, Swaine Chen Ivan Sovic´1,2,*, Mile Sˇikic´3,4,*, Andreas Wilm1, Shannon Nicole Fenlon1,5, Swaine Chen1,6 & Niranjan Nagarajan1 Realizing the democratic promise of nanopore sequencing requires the development of new bioinformatics approaches to deal with its specific error characteristics. Here we present GraphMap, a mapping algorithm designed to analyse nanopore sequencing reads, which progressively refines candidate alignments to robustly handle potentially high-error rates and a fast graph traversal to align long reads with speed and high precision (495%). Evaluation on MinION sequencing data sets against short- and long-read mappers indicates that GraphMap increases mapping sensitivity by 10–80% and maps 495% of bases. GraphMap alignments enabled single-nucleotide variant calling on the human genome with increased sensitivity (15%) over the next best mapper, precise detection of structural variants from length 100 bp to 4 kbp, and species and strain-specific identification of pathogens using MinION reads. GraphMap is available open source under the MIT license at https://github.com/isovic/graphmap. 1 Computational & Systems Biology, Genome Institute of Singapore, 60 Biopolis Street, #02-01 Genome, Singapore 138672, Singapore. 2 Centre for Informatics and Computing, RuXer Bosˇkovic´ Institute, Bijenicˇka 54, 10000 Zagreb, Croatia. 3 Faculty of Electrical Engineering and Computing, Department of Electronic Systems and Information Processing, University of Zagreb, Unska 3, 10000 Zagreb, Croatia. 4 Bioinformatics Institute, Singapore 138671, Singapore. 5 Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton SO16 6YD, UK. 6 Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074, Singapore. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to N.N. (email: [email protected]). 1 NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 ARTICLE Region selection Graph mapping Align Candidate positions LCSk + L1 Reference bases Seed base Unused reference base (unless coloured) ‘Don't care’ base Reference bases: Selected bases: Indexed seed: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Index construction Query bases: Insertion seed (base 7 or 8): Deletion seed (between base 6 and 7): 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Index lookup 0 Read coordinates Reference coordinates Read sequence Reference bins with no seed hits Reference bins with seed hits Seeds Diagonals of seed hits Alignment graph 0 1 2 3 4 5 6 - - Reference: Query: 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 - - Reference: Query: Initial graph 0 1 2 3 4 5 6 Selected regions 1 2 3 4 5 6 8 9 10 11 12 13 1 2 3 4 5 6 8 9 10 11 12 13 1 2 3 4 5 6 8 9 10 11 12 13 1 2 3 4 5 6 9 11 12 13 1 2 3 4 5 6 8 9 10 11 12 Final anchor graph All anchors Anchors LCSK Filtered with L1 regression 1e6 7.286 7.284 7.282 7.280 7.278 7.276 7.274 7.272 7.286 7.284 7.282 7.280 7.278 Reference coordinates (bp) Reference coordinates (bp) 7.276 7.274 7.272 1e6 Second LCSK after L1 Selected anchors 10 14 7 (Mis)match seed (base 7): GC CT TA AA AA AG GA G A |||x||| C CTA AGA CTA AGA G AAAGA CT CT |||x||| ACAGA GC CT TA AA AA AG GA CTA AGA Read coordinates (bp) Read coordinates (bp) 0 1,450 2,900 4,350 5,800 7,250 0 1,450 2,900 4,350 5,800 7,250 a b c d e Figure 1 | A schematic representation of stages in GraphMap. (a) Order of stages in the ‘read-funneling’ approach used in GraphMap to refine alignments and reduce the number of candidate locations to one. (b) Structure of spaced seeds used for index construction and index look-up. For each position in the reference one seed is inserted into the index and for each position in the query, three seeds are looked up corresponding to the differen error scenarios (c) Region selection by clustering of candidate seeds on the reference. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 (a) Order of stages in the ‘read-funneling’ approach used in GraphMap to refine Figure 1 | A schematic representation of stages in GraphMap. (a) Order of stages in the ‘read-funneling’ approach used in GraphMap to refine alignments and reduce the number of candidate locations to one. (b) Structure of spaced seeds used for index construction and index look-up. For each position in the reference one seed is inserted into the index and for each position in the query, three seeds are looked up corresponding to the different error scenarios (c) Region selection by clustering of candidate seeds on the reference. Diagonals with sufficient number of seed hits are used to identify regions for further processing. (d) Generation of alignment anchors through a fast graph based ordering of seeds (Graph Mapping). After construction of the initial graph based on the reference, seeds from the query (2mers here; starting from the green seed) are looked up, and information in the graph propagated, to construct a maximal walk that serves as an anchor. (e) Filtering of seed matches using LCSk search and L1 regression. Anchors are chained into a monotonically increasing sequence, with outliers trimmed using L1 regression, to get an approximate alignment that helps select the correct mapping location. es in the ‘read-funneling’ approach used in GraphMap to re have different settings optimized for different sequencing technologies (Supplementary Fig. 1b). Despite this, BWA-MEM’s performance degrades rapidly even in the ONT setting (Fig. 2b), providing precision and recall o25% for mapping to the human genome (Supplementary Data 1). DALIGNER (ref. 17), a highly sensitive overlapper which additionally supports read mapping, also provided precision and recall that degraded quickly with read error rate and genome size (Fig. 2b, Supplementary Data 1). LAST, originally designed for aligning genomes, fared better in these settings, but still exhibits lower recall for large genomes (30% reduction compared with GraphMap; Fig. 2b) and precision o54% for mapping to the human genome (Supplementary Data 1). The use of a realigner (marginAlign) generally improved alignment precision and recall but results for finding the correct genomic location were similar to that of the original mapper (marginAlign uses LAST by default). GraphMap was the only programme that uniformly provided high sensitivity and recall (Fig. 2b), even for mapping to the human genome, while scaling linearly with genome size (Supplementary Fig. Results The less than ideal results reported in early studies using MinION data12 could therefore be in part due to the use of mappers (for example, BWA-MEM (ref. 6), BLASR (ref. 13) or LAST (ref. 14)) that are not suited to its error characteristics. In this work, we present GraphMap, the first mapping algorithm designed for high sensitivity with current nanopore sequencing data. In solving the mapping problem for the potentially variable error profile of ONT MinION sequencers, GraphMap furthermore generally accommodates variable error characteristics, without the need for parameter tuning, while retaining high sensitivity and precision. Therefore, GraphMap allows uniform mapping of sequencing reads from disparate technologies (for example, Illumina, PacBio or ONT) with BLAST-like sensitivity and improved runtime. Experiments with Constructing the correct alignment was more challenging for synthetic ONT data sets and correspondingly the percentage of correctly aligned bases with GraphMap (B70%) is similar to the number of correct bases in the input data. The use of alternate alignment algorithms and parameters did not alter results significantly (Supplementary Table 1), though the use of a NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Diagonals with sufficient number of seed hits are used to identify regions for further processing. (d) Generation of alignment anchors through a fast graph based ordering of seeds (Graph Mapping). After construction of the initial graph based on the reference, seeds from the query (2mers here; starting from the green seed) are looked up, and information in the graph propagated, to construct a maximal walk that serves as an anchor. (e) Filtering of seed matches using LCSk search and L1 regression. Anchors are chained into a monotonically increasing sequence, with outliers trimmed using L1 regression, to get an approximate alignment that helps select the correct mapping location. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Region selection Graph mapping Align Candidate positions LCSk + L1 a Reference bases Seed base Unused reference base (unless coloured) ‘Don't care’ base Reference bases: Selected bases: Indexed seed: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Index construction Query bases: Insertion seed (base 7 or 8): Deletion seed (between base 6 and 7): 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Index lookup 1 2 3 4 5 6 8 9 10 11 12 13 1 2 3 4 5 6 8 9 10 11 12 13 1 2 3 4 5 6 8 9 10 11 12 13 1 2 3 4 5 6 9 11 12 13 1 2 3 4 5 6 8 9 10 11 12 10 14 7 (Mis)match seed (base 7): b 0 Read coordinates Reference coordinates Read sequence Reference bins with no seed hits Reference bins with seed hits Seeds Diagonals of seed hits Selected regions c c b a Read coordinates Reference coordinates Reference coordinates All anchors Anchors LCSK Filtered with L1 regression 1e6 7.286 7.284 7.282 7.280 7.278 7.276 7.274 7.272 7.286 7.284 7.282 7.280 7.278 Reference coordinates (bp) Reference coordinates (bp) 7.276 7.274 7.272 1e6 Second LCSK after L1 Selected anchors Read coordinates (bp) Read coordinates (bp) 0 1,450 2,900 4,350 5,800 7,250 0 1,450 2,900 4,350 5,800 7,250 e Alignment graph - - Reference: Query: 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 - - Reference: Query: Initial graph Final anchor graph G A |||x||| C CTA AGA CTA AGA G AAAGA CT CT |||x||| ACAGA d 6 and 7): Alignment graph 0 1 2 3 4 5 6 - - Reference: Query: 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 - - Reference: Query: Initial graph 0 1 2 3 4 5 6 Final anchor graph GC CT TA AA AA AG GA G A |||x||| C CTA AGA CTA AGA G AAAGA CT CT |||x||| ACAGA GC CT TA AA AA AG GA CTA AGA d All anchors Anchors LCSK 1e6 7.286 7.284 7.282 7.280 7.278 7.276 7.274 7.272 Reference coordinates (bp) Selected anchors e 6 and 7): 0 1 2 3 4 5 6 0 1 2 3 4 5 6 GC CT TA AA AA AG GA GC CT TA AA AA AG GA CTA AGA d d e Filtered with L1 regression 7.286 7.284 7.282 7.280 7.278 Reference coordinates (bp) R 7.276 7.274 7.272 1e6 Second LCSK after L1 Read coordinates (bp) Read coordinates (bp) 0 1,450 2,900 4,350 5,800 7,250 0 1,450 2,900 4,350 5,800 7,250 Read coordinates (bp) ntation of stages in GraphMap. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 a 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 86 88 90 92 94 96 98 100 Location 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 60 65 70 75 80 Alignment 90 92 94 96 98 100 Location ONT 2D ONT 1D E. coli (4.6 Mbp) S. cerevisiae (12.1 Mbp) C. elegans (100 Mbp) H. sapiens chr3 (198 Mbp) N. meningitidis (2.2 Mbp) Precision - BLAST Recall – BLAST Precision - GraphMap Recall - GraphMap a b 0 20 40 60 80 100 GraphMap marginAlign-GraphMap LAST marginAlign BWA-MEM DALIGNER BLASR Precision 0 20 40 60 80 100 Recall H. sapiens (3 Gbp) H. sapiens chr3 (198 Mbp) C. elegans (100 Mbp) S. cerevisiae (12.1 Mbp) E. coli (4.6 Mbp) b Figure 2 | Evaluating GraphMap’s precision and recall on synthetic ONTdata. (a) GraphMap (shaded bars) performance in comparison to BLAST (solid bars) on ONT 2D and 1D reads. Genomes are ordered horizontally by genome size from smallest to largest. For each data set, the graph on the left shows performance for determining the correct mapping location (within 50 bp; y axis on the left) and the one on the right shows performance for the correct alignment of bases (y axis on the right; Methods section). (b) Precision and recall for determining the correct mapping location (within 50 bp) for various mappers on synthetic ONT 1D reads. having low consensus quality even in a high coverage setting (Fig. 3a). Across data sets, GraphMap mapped the most reads and aligned the most bases, improving sensitivity by 10–80% over LAST and even more compared with other tools (Fig. 3b; Supplementary Fig. 2; Supplementary Note 2). This led to fewer uncalled bases compared with LAST, BWA-MEM, BLASR, DALIGNER and marginAlign even in an otherwise high-coverage data set (Fig. 3c,d). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 a b 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 70 75 80 85 90 Alignment 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 86 88 90 92 94 96 98 100 Location 90 92 94 96 98 100 Location 60 65 70 75 80 Alignment 60 65 70 75 80 Alignment 90 92 94 96 98 100 Location 0 20 40 60 80 100 GraphMap marginAlign-GraphMap LAST marginAlign BWA-MEM DALIGNER BLASR Precision 0 20 40 60 80 100 Recall ONT 2D ONT 1D E. coli (4.6 Mbp) S. cerevisiae (12.1 Mbp) C. elegans (100 Mbp) H. sapiens chr3 (198 Mbp) N. meningitidis (2.2 Mbp) Precision - BLAST Recall – BLAST Precision - GraphMap Recall - GraphMap H. sapiens (3 Gbp) H. sapiens chr3 (198 Mbp) C. elegans (100 Mbp) S. cerevisiae (12.1 Mbp) E. coli (4.6 Mbp) Figure 2 | Evaluating GraphMap’s precision and recall on synthetic ONTdata. (a) GraphMap (shaded bars) performance in comparison to BLAST (solid bars) on ONT 2D and 1D reads. Genomes are ordered horizontally by genome size from smallest to largest. For each data set, the graph on the left shows performance for determining the correct mapping location (within 50 bp; y axis on the left) and the one on the right shows performance for the correct alignment of bases (y axis on the right; Methods section). (b) Precision and recall for determining the correct mapping location (within 50 bp) for various mappers on synthetic ONT 1D reads. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 1c, Supplementary maximum-likelihood based realigner (marginAlign2) improved both alignment precision and recall (Supplementary Data 1). The use of marginAlign as a realigner did not improve on GraphMap’s ability to identify the correct genomic location (Supplementary Data 1). These results highlight GraphMap’s ability to identify precise genomic locations based on robust alignments without the need for customizing and tuning alignment parameters to the unknown error characteristics of the data. For read-to-reference alignment, programs such as BLAST provide high sensitivity and can be feasible for small genomes, but can quickly become infeasible for larger genomes (for example, runtime for C. elegans or the human genome; Supplementary Data 1). Read mappers such as BWA-MEM and BLASR provide a different tradeoff, scaling well to large genomes but with low sensitivity and precision for high-error rates (Fig. 2b, Supplementary Data 1). This could partly be due to specific parameter settings as is the case for BLASR, which was designed for PacBio data. Mappers such as BWA-MEM on the other hand, 3 ARTICLE NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 In addition, GraphMap analysis resulted in 410-fold reduction in errors on the lambda phage and E. coli genome (Fig. 3c) and reported o40 errors on the E. coli genome compared with more than a 1,000 errors for LAST and BWA-MEM (Fig. 3d). With B80  coverage of the E. coli genome, GraphMap mapped B90% of the reads and called consensus bases for the whole genome with o1 error in 100,000 bases (Q50 quality). The next best aligner, that is, LAST did not have sufficient coverage (20  ) on 47,000 bases and reported consensus with a quality of BQ36. BWA-MEM aligned o60% of the reads and resulted in the calling of 4200 deletion errors in the consensus genome. Similar results were replicated in other genomes and data sets as well (Supplementary Fig. 2). Data 1). Experiments with a range of read lengths and error rates also demonstrate that GraphMap scales well across these dimensions (runtime and memory usage; Supplementary Table 2), though mapping to large genomes currently requires the use of large memory systems (B100 GB for human genome). Extrapolating this, mapping data from a MinION run of 100,000 reads to the human genome should take o5 h and o$7 on an Amazon EC2 instance (r3.4  large) using GraphMap. Sensitivity and mapping accuracy on nanopore sequencing data. GraphMap was further benchmarked on several published ONT data sets against mappers and aligners that have previously been used for this task (LAST, BWA-MEM and BLASR; Methods section), as well as a highly sensitive overlapper for which we tuned settings (DALIGNER; Methods section). In the absence of ground truth for these data sets, mappers were compared on the total number of reads mapped (sensitivity), and their ability to provide accurate (to measure precision of mapping and align- ment) as well as complete consensus sequences (as a measure of recall). Overall, as seen in the simulated data sets, LAST was the closest in terms of mapping sensitivity compared with Graph- Map, though GraphMap showed notable improvements. The differences between GraphMap and LAST were apparent even when comparing their results visually, with LAST alignments g pp y g As another assessment of mapping and alignment accuracy, error profiles of 1D and 2D ONT reads were computed for GraphMap and compared with those for LAST and marginAlign. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 coli K12 (R7.3) d d d c Insertions Deletions SNPs Uncalled bases Insertions Deletions SNPs Uncalled bases Insertions Deletions SNPs Uncalled bases Insertions Deletions SNPs Uncalled bases Figure 3 | Sensitivity and mapping accuracy on nanopore sequencing data. (a) Visualization of GraphMap and LAST alignments for a lambda phage MinION sequencing data set12 (using integrative genomics viewer (IGV) (ref. 36)). Grey columns represent confident consensus calls while coloured columns indicate lower quality calls. (b) Mapped coverage of the lambda phage12 and the E. coli K-12 genome31 (R7.3 data) using MinION sequencing data and different mappers. (c) Consensus calling errors and uncalled bases using a MinION lambda phage data set12 and different mappers. (d) Consensus calling errors and uncalled bases using a MinION E. coli K-12 data set (R7.3) and different mappers. Figure 3 | Sensitivity and mapping accuracy on nanopore sequencing data. (a) Visualization of GraphMap and LAST alignments for a lambda phage MinION sequencing data set12 (using integrative genomics viewer (IGV) (ref. 36)). Grey columns represent confident consensus calls while coloured columns indicate lower quality calls. (b) Mapped coverage of the lambda phage12 and the E. coli K-12 genome31 (R7.3 data) using MinION sequencing data and different mappers. (c) Consensus calling errors and uncalled bases using a MinION lambda phage data set12 and different mappers. (d) Consensus calling errors and uncalled bases using a MinION E. coli K-12 data set (R7.3) and different mappers. mappers, though GraphMap’s alignments resulted in lower mismatch rate estimates compared with LAST (Supplementary Fig. 3). GraphMap’s distributions were also more similar to those of marginAlign (used as a reference standard), indicating that GraphMap mapping and alignments are at least as accurate as those from LAST. Overall, deletion and mismatch rates for ONT data were observed to be higher than insertion rates, a pattern distinct from the low mismatch rates seen in PacBio data18 and explaining why mappers tailored for PacBio data may not work well for ONT data (Supplementary Fig. 3). LAST revealed characteristics of reads that are more amenable to GraphMap analysis. In particular, these reads were found to be slightly shorter on average (3.4 versus 5.7 kbp), more likely to have windows with higher than average error rate (27 versus 14%), and have a greater proportion of 1D reads (90 versus 76%; E. coli R7.3 data set). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Overall, GraphMap provided improved sensitivity of mapping on all ONT data sets (Supplementary Note 2), without sacrificing alignment accuracy, and this was further confirmed in the applications discussed below. Note that the consensus calling results reported here are not comparable to those for programs such as Nanopolish4 and PoreSeq19, which solve the harder problem of correcting the consensus in the presence of assembly and sequencing errors. To account for a ‘reference bias’, where an error-free reference may preferentially enable some programs to report alignments that give an accurate consensus, consensus calling was repeated on a mutated reference (Methods section). Overall, GraphMap was observed to have similar behaviour as other mappers in terms of reference bias, with comparable number of errors (single- nucleotide polymorphisms (SNPs), insertions and deletions) in mutated and non-mutated positions (Supplementary Table 3). These results further confirm that GraphMap’s high sensitivity does not come at the expense of mapping or alignment accuracy. In terms of runtime requirements, GraphMap was typically more efficient than BWA-MEM and slower than LAST on these data sets (Supplementary Table 4). Memory requirements were typically o5 GB, with GraphMap and BWA-MEM being inter- mediate between LAST/BLASR (least usage) and marginAlign/ DALIGNER (most usage; Supplementary Data 2). SNV calling in the human genome with high precision. Diploid variant calling using ONT data has multiple potential hurdles including the lack of a dedicated read mapper or diploid variant caller for it19. Not surprisingly, a recent report for calling single-nucleotide variants (SNVs) from high-coverage targeted sequencing of the diploid human genome reported that existing variant callers were unable to call any variants and a naive approach requiring 1/3 of the reads to support an allele could lead to many false-positive variants20. To evaluate if improved read mappings from GraphMap could increase sensitivity and precision, data reported in Ammar et al.20 was reanalysed using a rare-variant caller (LoFreq (ref. 3)) that is robust to high-error rates, and compared against a set of gold- standard calls21 for this sample (NA12878). Targeted nanopore sequencing reads were mapped by GraphMap to the correct location on the human genome with high specificity, despite the presence of very similar decoy locations (94% identity between CYP2D6 and CYP2D7 (ref. 20; Supplementary Fig. 4). GraphMap provided the most on-target reads, aligning 15–20% more reads than the next best mapper (BWA-MEM) for the three amplified genes (CYP2D6, HLA-A and HLA-B; Supplementary Fig. 4). NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 As observed before2, substantial variability in the shape and modes of error rate distributions were seen across different NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 LAST GraphMap 0 0 1 0 0 1 13 0 383 10 16 21 28 470 7 17 6,801 9 9,877 1 4 16 64 256 1,024 4,096 16,384 Insertions Deletions SNPs Uncalled bases 0 0 0 2 0 4 6 5 220 103 89 10,500 24 1,063 1,435 45 402 6,763 0 7,250 37,254 4,208,879 13,270 602,089 1 8 64 512 4,096 32,768 262,144 2,097,152 Insertions Deletions GraphMap LAST BWA-MEM BLASR marginAlign DALIGNER GraphMap LAST BWA-MEM BLASR marginAlign DALIGNER SNPs Uncalled bases 0 × 500 × 1,000 × 1,500 × 2,000 × 2,500 × 3,000 × 0 × 20 × 40 × 60 × 80 × GraphMap LAST BWA-MEM BLASR 10 kb 20 kb 20 kb 10 kb marginAlign DALIGNER Lambda phage coverage E. coli coverage E. coli K12 (R7.3) Lambda phage a b d c Figure 3 | Sensitivity and mapping accuracy on nanopore sequencing data. (a) Visualization of GraphMap and LAST alignments for a lambda phage MinION sequencing data set12 (using integrative genomics viewer (IGV) (ref. 36)). Grey columns represent confident consensus calls while coloured columns indicate lower quality calls. (b) Mapped coverage of the lambda phage12 and the E. coli K-12 genome31 (R7.3 data) using MinION sequencing data and different mappers. (c) Consensus calling errors and uncalled bases using a MinION lambda phage data set12 and different mappers. (d) Consensus calling errors and uncalled bases using a MinION E. coli K-12 data set (R7.3) and different mappers. LAST GraphMap 10 kb 20 kb 20 kb 10 kb a b 0 × 500 × 1,000 × 1,500 × 2,000 × 2,500 × 3,000 × 0 × 20 × 40 × 60 × 80 × GraphMap LAST BWA-MEM BLASR marginAlign DALIGNER Lambda phage coverage E. coli coverage b b a 0 0 1 0 0 1 13 0 383 10 16 21 28 470 7 17 6,801 9 9,877 1 4 16 64 256 1,024 4,096 16,384 I ti D l ti SNP U ll d b GraphMap LAST BWA-MEM BLASR marginAlign DALIGNER Lambda phage c 0 0 0 2 0 4 6 5 220 103 89 10,500 24 1,063 1,435 45 402 6,763 0 7,250 37,254 4,208,879 13,270 602,089 1 8 64 512 4,096 32,768 262,144 2,097,152 Insertions Deletions GraphMap LAST BWA-MEM BLASR marginAlign DALIGNER SNPs Uncalled bases E. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 The ability to sensitively and precisely call SNVs with GraphMap, provides the foundation for reconstructing haplotypes with long reads, and opens up the investigation of complex and clinically important regions of the human genome using nanopore sequencing. These were then used to call heterozygous variants in these challenging regions of the human genome with high precision (96% with GraphMap; Table 1). GraphMap alignments identified many more true-positive SNVs than other mappers, with comparable or higher precision (76% improvement compared with BWA-MEM and LAST) and a 15% increase in sensiti- vity over DALIGNER, which has slightly lower precision (93%; Table 1). While the use of a custom variant caller for marginAlign (marginCaller) improved its results in terms of sensitivity, it came at the expense of low precision (36%; Table 1). Subsampling GraphMap mappings to the same coverage as BWA-MEM provided comparable results (42 versus 47 true positives and 2 versus 2 false positives) indicating that Graph- Map’s improved mapping sensitivity (2  compared with other mappers) played a role in these results. The ability to sensitively and precisely call SNVs with GraphMap, provides the foundation for reconstructing haplotypes with long reads, and opens up the investigation of complex and clinically important regions of the human genome using nanopore sequencing. GraphMap enables sensitive and accurate SV calling. Long reads from the MinION sequencer are, in principle, ideal for the identification of large SVs in the genome22, but existing mappers have not been systematically evaluated for this application1. Read alignments produced by mappers are a critical input for SV callers. To compare the utility of various mappers, their ability to produce spanning alignments or split alignments indicative of a structural variation (insertions or deletions) was evaluated using real E. coli data mapped to a mutated reference (Methods section). As shown in Table 2, mappers showed variable performance in their ability to detect SVs through spanning alignments. In comparison, GraphMap’s spanning alignments readily detected insertions and deletions over a range of event sizes (100 bp–4 kbp), providing perfect precision and a 35% improvement in recall over the next best mapper (BLASR; Table 2). LAST alignments were unable to detect any events under a range of parameter settings but post-processing with marginAlign improved recall slightly (5%; Table 2). BWA-MEM alignments natively provided 10% recall at 67% precision. Post- processing BWA-MEM alignments with LUMPY improved recall to 45%, using information from split reads to predict events. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Table 1 | Comparison of various mappers for SNV calling. LAST marginAlign BWA-MEM BLASR DALIGNER GraphMap Precision (%) 94 100 (36) 96 100 93 96 True positives 49 1 (107) 47 43 75 86 Results are based on amplicon sequencing data for a human cell line (NA12878) for the genes CYP2D6, HLA-A and HLA-B. Precision values are likely to be an underestimate of what can be expected genome-wide due to the repetitive nature of the regions studied and the incompleteness of the gold-standard set. Results for marginAlign using marginCaller are shown in parentheses. Table 1 | Comparison of various mappers for SNV calling. Results are based on amplicon sequencing data for a human cell line (NA12878) for the genes CYP2D6, HLA-A and HLA-B. Precision values are likely to be genome-wide due to the repetitive nature of the regions studied and the incompleteness of the gold-standard set. Results for marginAlign using marginCa Table 2 | Comparison of various mappers for structural variant calling. LAST marginAlign BWA-MEM BLASR DALIGNER GraphMap Precision (%) 0 50 67 (90) 94 0 100 Recall (%) 0 5 10 (45) 75 0 100 F1 score (%) 0 9 17 (60) 83 0 100 Results are based on mapping a MinION data set for E. coli K-12 (R7.3) on a mutated reference containing insertion and deletions in a range of sizes ((100 bp, 300 bp, 500 bp, 1 kbp, 1.5 kbp, 2 kbp, 2.5 kbp, 3 kbp, 3.5 kbp and 4 kbp); 20 events in total). Bold values indicate the best results for each metric. The F1 score is given by a weighted average of precision and recall. Values in parentheses for BWA-MEM show the results using LUMPY. Table 2 | Comparison of various mappers for structural variant calling. Sensitive and specific pathogen identification with ONT data. Due to its form factor and real-time nature, an application of MinION sequencing that has garnered interest in the community is in the identification of pathogens in clinical samples. Sequen- cing errors (particularly in 1D data) and the choice of read mapper could significantly influence results in such an applica- tion and lead to misdiagnosis. GraphMap’s high specificity in read mapping as seen in the results for Ammar et al. (Supplementary Fig. 4) suggested that it could be useful in this setting. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Clonal sequencing data on the MinION and a database of microbial genomes was used to create several synthetic bench- marks to evaluate the performance of various mappers for this application (Methods section). For species level identification, all mappers reported high precision (typically 495%) but recall varied over a wide range from 20 to 90% (Table 3). GraphMap had the highest recall and F1 score in all data sets, providing an improvement of 2–18% over other mappers. The improvement was more marked when a perfect reference was not part of the database (for example, S. enterica Typhi, Table 3) For this application, BWA-MEM was the next best mapper while LAST and BLASR exhibited 425% reduced recall compared with GraphMap (Table 3). Not surprisingly, strain-level identification using MinION data appears to be much more difficult and in some cases a closely related strain can attract more reads than the correct strain (Fig. 4c). However, in the data sets tested, Graph- Map assigned most reads to a handful of strains that were very similar to the correct strain (Fig. 4c–e; 99.99% identity for E. coli K-12 and BW2952). Moreover, the use of strain-specific sequences was able to unambiguously identify the correct strain from this subset (for example, there were no reads mapping to NC_012759.1:4.13–4.17 Mbp, a region unique to BW2952), indicating that this approach could be used to systematically identify pathogens at the strain level. These were then used to call heterozygous variants in these challenging regions of the human genome with high precision (96% with GraphMap; Table 1). GraphMap alignments identified many more true-positive SNVs than other mappers, with comparable or higher precision (76% improvement compared with BWA-MEM and LAST) and a 15% increase in sensiti- vity over DALIGNER, which has slightly lower precision (93%; Table 1). While the use of a custom variant caller for marginAlign (marginCaller) improved its results in terms of sensitivity, it came at the expense of low precision (36%; Table 1). Subsampling GraphMap mappings to the same coverage as BWA-MEM provided comparable results (42 versus 47 true positives and 2 versus 2 false positives) indicating that Graph- Map’s improved mapping sensitivity (2  compared with other mappers) played a role in these results. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 GraphMap produced spanning alignments natively that accurately demarcated the alignment event and did this without reporting any false positives (Fig. 4a,b and Table 2). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Analysis of reads that were only mapped by GraphMap when compared with those that were mapped by both GraphMap and 5 ARTICLE NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 b a 200 bp deletion 3,876,400 bp 3,876,600 bp 3,876,800 bp 3,877,000 bp b a 112 kb 4 kbp deletion detected by GraphMap c d e 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 GraphMap BWA-MEM LAST BLASR DALIGNER 0 500 1,000 1,500 2,000 2,500 3,000 E. coli str. K-12 E. coli BW2952 E. coli ATCC 8739 E. coli BL21(DE3) E. coli B str. REL606 E. coli HS E. coli UMN026 E. coli E24377A E. coli SE11 E. coli O130:H2 str. 12009 S. enterica Paratyphi A str. ATCC 9150 S. enterica Typhi str. CT18 S. enterica Paratyphi A str. AKU_12601 S. enterica Typhi str. Ty2 S. enterica str. CT18 plasmid pHCM2 S. enterica Heidelberg str. SL476 S. enterica Paratyphi C strain RKS4594 S. enterica Agona str. SL483 S. enterica Paratyphi B str. SPB7 S. typhimurium LT2 E. coli UTI89 E. coli APEC O1 E. coli S88 E. coli BL21(DE3) E. coli 536 E. coli CFT073 E. coli ED1a E. coli O127:H6 str. E2348/69 E. coli BW2952 E. coli UTI89 plasmid pUTI89 S. enterica Typhi E. coli UTI89 E. coli K-12 d 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 GraphMap BWA-MEM LAST BLASR DALIGNER S. enterica Typhi c 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 E. coli K-12 d e c 0 E. coli UTI89 E. coli APEC O1 E. coli S88 E. coli BL21(DE3) E. coli 536 E. coli CFT073 E. coli ED1a E. coli O127:H6 str. E2348/69 E. coli BW2952 E. coli UTI89 plasmid pUTI89 Figure 4 | Variant calling and species identification using nanopore sequencing data and GraphMap. (a) An IGV view of GraphMap alignments that enabled the direct detection of a 200-bp deletion (delineated by red lines). (b) GraphMap alignments spanning a B4-kbp deletion (delineated by red lines). Number of reads mapping to various genomes in a database (sorted by GraphMap counts and showing top 10 genomes) using different mappers (GraphMap, BWA-MEM, LAST, DALIGNER and BLASR) and three MinION sequencing data sets for (c) E. coli K-12 (R7.3) (d) S. enterica Typhi and (e) E. coli UTI89. Note that GraphMap typically maps the most reads to the right reference genome (at the strain level) and the S. Discussion The design choices in GraphMap, including the use of new algorithmic ideas such as gapped spaced seeds, graph mapping and longest common subsequence in k Length substrings (LCAk), provide a new tradeoff between mapping speed and sensitivity that is well-suited to long nanopore reads. For mapping error- prone synthetic long reads to the human genome, GraphMap was the only mapper that exhibited BLAST-like sensitivity, while being orders of magnitude faster than BLAST. On nanopore sequencing data from the MinION system, GraphMap was unmatched in terms of sensitivity, mapping 490% of reads and NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 ARTICLE b e 110 kb 112 kb 114 kb 4 kbp deletion detected by GraphMap a b c d e 200 bp deletion 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 GraphMap BWA-MEM LAST BLASR DALIGNER 0 500 1,000 1,500 2,000 2,500 3,000 E. coli str. K-12 E. coli BW2952 E. coli ATCC 8739 E. coli BL21(DE3) E. coli B str. REL606 E. coli HS E. coli UMN026 E. coli E24377A E. coli SE11 E. coli O130:H2 str. 12009 S. enterica Paratyphi A str. ATCC 9150 S. enterica Typhi str. CT18 S. enterica Paratyphi A str. AKU_12601 S. enterica Typhi str. Ty2 S. enterica str. CT18 plasmid pHCM2 S. enterica Heidelberg str. SL476 S. enterica Paratyphi C strain RKS4594 S. enterica Agona str. SL483 S. enterica Paratyphi B str. SPB7 S. typhimurium LT2 E. coli UTI89 E. coli APEC O1 E. coli S88 E. coli BL21(DE3) E. coli 536 E. coli CFT073 E. coli ED1a E. coli O127:H6 str. E2348/69 E. coli BW2952 E. coli UTI89 plasmid pUTI89 3,876,400 bp 3,876,600 bp 3,876,800 bp 3,877,000 bp 110 kb 112 kb 114 kb S. enterica Typhi E. coli UTI89 E. coli K-12 4 kbp deletion detected by GraphMap Figure 4 | Variant calling and species identification using nanopore sequencing data and GraphMap. (a) An IGV view of GraphMap alignments that enabled the direct detection of a 200-bp deletion (delineated by red lines). (b) GraphMap alignments spanning a B4-kbp deletion (delineated by red lines). Number of reads mapping to various genomes in a database (sorted by GraphMap counts and showing top 10 genomes) using different mappers (GraphMap, BWA-MEM, LAST, DALIGNER and BLASR) and three MinION sequencing data sets for (c) E. coli K-12 (R7.3) (d) S. enterica Typhi and (e) E. coli UTI89. Note that GraphMap typically maps the most reads to the right reference genome (at the strain level) and the S. enterica Typhi data set is a mixture of sequencing data for two different strains for which we do not have reference genomes in the database. Results for marginAlign were nearly identical to that of LAST (within 1%) and have therefore been omitted. ARTICLE The HT defines a mapping from image points into an accumulator space, called the Hough space. In the case of line detection, if a given set of points in Cartesian space are collinear, then their relation can be expressed with a linear equation with common slope m and intercept c: y ¼ mx þ c; ð1Þ ð1Þ where (x, y) are the coordinates of a point in 2D space. HT attempts to determine parameters m and c of a line that describes the given set of points. Note that the system is generally over-determined and thus the problem can be solved using linear regression techniques. However, the HT uses an evidence-gathering approach, which can be used to detect an arbitrary number of lines in the image instead of only one best (Fig. 1c). Equation (1) can be converted into its dual in parameter space: c ¼  mx þ y: ð2Þ ð2Þ The intuition is as follows: given a point (x, y) in Cartesian space, its parameter space representation defines a line. If multiple Cartesian space points are given, each transforms into a different line in the parameter space. Their intersections specify potential lines in the original, Cartesian space. HT defines an accumulator space, in which m and c are rasterized so as to take only a finite range of values. HT then simply counts all the potential solutions in the accumulator space by tracing all the dual lines for each point in the Cartesian space, and increasing the vote count for each (m, c) coordinate. All HT space coordinates with count above a defined threshold can then be considered as candidate lines in the original Cartesian space. In principle, the approach used in GraphMap could be adapted for the problem of computing overlaps and alignments between reads. As was recently shown, nanopore sequencing reads can be used to construct high-quality assemblies de novo4 and sensitive hashing techniques have been used for the assembly of large genomes24. GraphMap’s sensitivity and specificity as a mapper could thus serve as the basis for fast computation of overlap alignments and de novo assemblies in the future. A single-seed hit can be represented with a ‘k-point’ (q, t) in 2D space, where q is the seed’s position on the read, and t is the position of the seed hit on the reference. ARTICLE (3) A key where the DC base as well as the following base is skipped. This key allows for at most one insertion and one match/mismatch (for example, ‘11100111’; see ‘Insertion seed’ in Fig. 1b). In total, for each shape d3 keys are constructed, where d is the number of DC bases. GraphMap uses two complementary shapes for the region selection process: ‘1111110111111’ (or the 6-1-6 shape) and ‘11110111101111’ (or the 4-1-4-1-4 shape), where 1 marks the inclusive bases and 0 the DC positions. This shape combination was selected based on empirical evaluation of a range of combinations, due to the computational intractability of computing the optimal shape for the Levenshtein distance25,26 (see Supplementary Data 3 for results for each shape and the combination). For each shape, a separate index is used in GraphMap. At every seed position, both shapes are looked up, and all hits are used in the next step for binning. In general, GraphMap’s improved sensitivity should benefit a range of applications for nanopore data and a few of these were explored in this study. In particular, variant calling and species identification with error-prone data can be affected by errors in mapping and alignment. Despite the lack of custom variant callers, read mappings from GraphMap were shown to provide sensitive and precise SNV calls on complex regions of the human genome. In addition, GraphMap alignments readily spanned insertions and deletions over a wide range of sizes (100 bp–4 kbp) allowing for the direct detection of such events, without assembly or split read analysis. With the development of new nanopore- specific variant calling tools, GraphMap’s improved sensitivity should continue to provide a useful starting point for these applications. Furthermore, GraphMap alignments were used to identify the species-level origin of reads with high precision and recall. The sensitivity of mapping with GraphMap can be a key advantage in applications where MinION sequencing reads are used in real-time to identify pathogens23, particularly in combination with rapid protocols for generating 1D reads on the MinION. With further downstream processing, these read mappings could be used for strain-level typing and chara- cterization of antibiotic resistance profiles23, meeting a critical clinical need. To derive a general approach for binning seed hits, we draw on the concept of a Hough transform (HT), a method commonly used in image processing for detection of shapes such as lines, circles and ellipses. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 structures (for example, FM-index) can significantly reduce this requirement (for a modest increase in runtime) and this option is currently under implementation. positions its weight. Gapped q-grams allow for DC positions within a shape to also contain insertions and deletions (indels). The approach in GraphMap for implementing Levenshtein gapped q-grams is based on constructing a hash index of the reference sequence, where the q-gram positions are hashed by the keys constructed from the shape’s layout—only inclusive bases are taken for constructing the key, while the DC bases are simply skipped (Fig. 1b). During the lookup step, multiple keys are constructed for each shape and used for retrieval. For each DC base, three look-up keys are constructed: GraphMap’s speed and sensitivity do not come at the expense of location and alignment precision, as demonstrated by extensive experiments with synthetic and real data sets. For determining the correct genomic location, GraphMap’s precision is typically 498% and it is able to distinguish between candidate locations that are 494% identical on the human genome. For alignment precision, GraphMap’s performance scales according to sequen- cing error rate, is comparable to BLAST and other mappers (BWA-MEM, LAST, BLASR and DALIGNER), and was observed to be robust to the choice of alignment algorithms and parameters. GraphMap mappings provided a better starting point for the realigner marginAlign2 and should do so for consensus calling algorithms such as Nanopolish4 and PoreSeq19 as well. (1) A key constructed in the same manner as during the indexing process, which captures all seed positions and with a DC base being a match or a mismatch (for example, ‘1110111’; see ‘(Mis)match seed’ in Fig. 1b), p g (2) A key where the DC base is not skipped. This key captures up to one deletion (as indels are frequently 1 bp long) at the specified position (for example, ‘111111’; see ‘Deletion seed’ in Fig. 1b), and p g (2) A key where the DC base is not skipped. This key captures up to one deletion (as indels are frequently 1 bp long) at the specified position (for example, ‘111111’; see ‘Deletion seed’ in Fig. 1b), and (3) A key where the DC base as well as the following base is skipped. This key allows for at most one insertion and one match/mismatch (for example, ‘11100111’; see ‘Insertion seed’ in Fig. 1b). ARTICLE In the case a read is completely error-free and extracted from the exact reference, its set of k-points would be perfectly collinear in such defined space. Moreover, under these ideal conditions, they would all lie on a line tilted at a 45 angle (slope m ¼ 1). This collinearity also corresponds to the main diagonal in the dynamic programming alignment matrix. Since m is known, only the intercept parameter c needs to be determined to find the accurate mapping position. As c corresponds to the (already discrete) coordinates on the reference sequence, a simple integer array of the length of the reference can be used for counting votes (Fig. 1c). For each k-point, its c parameter value is determined with a simple expression: NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 enterica Typhi data set is a mixture of sequencing data for two different strains for which we do not have reference genomes in the database. Results for marginAlign were nearly identical to that of LAST (within 1%) and have therefore been omitted. Table 3 | Precision and recall for species identification using MinION reads. E. coli K-12 (R7.3) S. enterica Typhi E. coli UTI89 Prec. Rec. F1 Prec. Rec. F1 Prec. Rec. F1 BLASR 93 22 36 99 28 44 98 55 70 LAST 94 37 53 97 34 51 95 65 78 DALIGNER 80 10 17 99 28 43 98 55 71 BWA-MEM 94 47 63 98 45 61 98 85 91 GraphMap 95 51 67 97 56 72 99 88 93 Bold values indicate the best results for each data set and metric. Results for marginAlign were nearly identical to that of LAST (within 1%) and have therefore been omitted. Table 3 | Precision and recall for species identification using MinION reads. ble 3 | Precision and recall for species identification using MinION reads. 95% of bases on average. Compared with other mappers, this lead to a 10–80% increase in mapped bases (for example, 18% increase on a recent MinION MkI data set; Supplementary Note 2). This is a significant improvement—typically mapping programs are highly optimized and increase in sensitivity of even a few percentage points can be hard to achieve. Additionally, sensitivity is a key requirement for mapping tools and mapping-based analysis, as reads that cannot be mapped are unavailable for use in downstream applications. A drawback of the current implementation of GraphMap is the requirement of large- memory machines for mapping to large genomes (B100 GB for the human genome). The use of more memory-efficient index 7 NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications ARTICLE Next, a subset of anchors which are located within dL1 ¼ e  T ffiffiffi 2 p =2 from either side of the L1 line is selected, where e is the expected error rate (by default, ffiffiffi p conservatively set to 45%), T is the target (read) length and the factor ffiffiffi 2 p =2 is used to convert the distance from target coordinate space to a distance perpendicular to the L1 line. A confidence interval c ¼ 3  PN i¼1 di=N is calculated, where di is the distance from a selected anchor i to the L1 line (the constant 3 was chosen to mimic a 3s rule). LCSk is then repeated once again but only on the anchors which are located within the distance ±c from the L1 line to compensate for possible gaps caused by anchor filtering (Fig. 1e). The use of L1 filtering was observed to improve the precision of alignment start and end coordinates for many reads, though the overall impact on performance was less significant in comparison to the LCSk stage (Supplementary Data 3). Graph-based vertex-centric construction of anchors. In this stage, candidate regions from stage I are refined by constructing alignment chains or anchors from short seeds matches. To do this, GraphMap uses the notion of a ‘kmer mapping graph’. Given a pair of sequences (target and query), it starts by constructing a kmer mapping graph from the target sequence. In the current implementation, the read was chosen to be the target sequence to reduce memory consumption. The vertices of the kmer mapping graph are the kmers of the target sequence of length T (Fig. 1d). Unlike in a de Bruijn graph, identical kmers are not truncated into the same vertex of the graph but are kept as separate individual vertices (Fig. 1d). For every vertex vi 8i 2 0 . . . T  k ð Þ ð Þ, l directed outbound edges are added which connect vi to vertices vi þ 1,vi þ 2,y,vi þ l (Fig. 1d). The rationale for such a design is as follows: in case l ¼ 1 and if the query is a subset of the target with no differences or errors, the target’s mapping graph would contain the same kmers in the exact same order as in the query sequence. Thus, an exact walk exists in both sequences. ARTICLE Walks that are too short (defaulto12 bases, that is, smaller than the seeds from stage I) are excluded to avoid a large search space. Vertex-centric walks allow GraphMap to quickly construct longer alignments in the presence of higher substitution error rates, as seen in nanopore sequencing data. In the presence of low-substitution error rates (o2%, as is the case for Illumina as well as PacBio reads), a single walk can cover most of, if not the entire read. For ONT reads we observed shorter walks that we refer to here as anchors (Fig. 1d). (extensions to read ends are done without gap penalty). Clustering is done by collecting neighbouring anchors where the ratio of distances in the read and reference coordinates is oe/2 (as before, e is the expected error rate in the data, and the factor of 2 allows for more stringent clustering). Clusters with very few bases (o30 or 2% of read length) were discarded for this purpose as they were found to reduce alignment accuracy. GraphMap allows users to output all equally or similarly good secondary alignments by specifying an ambiguity factor F in the range [0,1] and using that to select regions which have nkmersZ(1  F)  nkmers,best, where nkmers,best is the number of kmers of the region with the maximum f value. We denote the count of regions with nkmers above the ambiguity threshold as Na. g Extending anchors into alignments using LCSk. Each anchor reported by GraphMap in stage II represents a shared segment (or subsequence) between the target and the query sequence with known start and end positions in both sequences. Due to the presence of repeats, the set of anchors obtained is not necessarily monotonically increasing in both the target and query coordinates. For this reason, a subset of anchors that satisfy the monotonicity condition needs to be selected. The problem of identifying such a subset can be expressed as finding the Longest Common Subsequence in k Length Substrings27 (LCSk). Note that this is distinct from just finding the longest common subsequence as that ignores the information determined in the anchors and can favour alignments that have many more indels. Recently, an efficient and simple algorithm for solving a variant of the LCSk problem has been proposed28. In our implementation we follow this paradigm and instead of using substrings of fixed size k, we allow for variable length substrings. ARTICLE However, in realistic conditions, variations and sequencing errors exist in reads. Although the majority of kmers might still be in the same order, a simple exact linear walk through the reference’s and read’s mapping graphs cannot be found due to the differing kmers present. Instead, the walk is fragmented into several smaller ones and this is particularly severe when the error rate is high, as seen in nanopore sequencing. To address this issue, the additional (l  1) edges act as a bridge between vertices in the mapping graph. Thus GraphMap allows a linear walk to be found not only by following consecutive kmers in the graph, but to jump-over those that produce poorer solutions. Figure 1d depicts such an example. GraphMap uses l ¼ 9 by default as it was empirically found to enable anchor construction for most ONT reads. After filtering, five empirically derived scores that describe the quality of the region are calculated. They include: the number of exact kmers covered by the anchors nkmers, the s.d. s of anchors around the L1 line, the length of the query sequence which matched the target (distance from the first to the last anchor) mlen, the number of bases covered by anchors (includes only exact matching bases) ncb and the read length. The last four scores are normalized to the range [0,1] with the following equations (4–7): fs ¼ max 0; 1  s eTffiffi 2 p ! ; ð4Þ fmlen ¼ mlen N ; ð5Þ fcb ¼ min ncb mlen  1  e ð Þ ; 1   ; ð6Þ fT ¼ min ln T ln Q ; 1   ; ð7Þ ð4Þ ð6Þ fT ¼ min ln T ln Q ; 1   ; ð7Þ For graph construction, GraphMap uses an index constructed from the target on the fly, using a smaller continuous seed for sensitivity (default k ¼ 6, similar to the kmer used for MinION base-calling). In principle, any indexing method can be used and for runtime efficiency GraphMap uses perfect kmer hashing when ko10 and suffix arrays otherwise. To do graph traversal, for each consecutive kmer in the query, a list of hits on the target sequence is obtained from the index. ARTICLE The vertex- centric walk then works as follows: for a chosen vertex, collect information from input edges, choose the ‘best’ edge and update the information it contains, and transmit this information to all outbound edges simultaneously. The ‘best’ edge is defined here to be the one belonging to the longest walk. The information that is transmitted through the edges contains the walk length, the position of the starting kmer in both the target and the read, and the number of covered bases and kmers in both sequences. Thus the runtime complexity of the vertex-update operation is O(1). ð7Þ where Q is the length of the reference sequence (query in our previous definition). The overall quality of the alignment in a region is then calculated as the product of the normalized scores: f ¼ fs  fmlen  fcb  fT: ð8Þ ð8Þ Construction of final alignment. After all selected regions have been processed they are sorted by the f value. The region with the highest value fmax is selected for the final alignment. The default settings for GraphMap use an implementation of 29 g g Myers’ bit-vector algorithm for fast alignment29. GraphMap also allows users a choice of aligners, including an implementation of Gotoh’s semi-global alignment algorithm30, as well as an option to construct anchored alignments. Specifically, in the anchored approach, anchors from the LCSk step are clustered and alignments within and between cluster end points computed using Myers’ bit-vector alignment (extensions to read ends are done without gap penalty). Clustering is done by collecting neighbouring anchors where the ratio of distances in the read and reference coordinates is oe/2 (as before, e is the expected error rate in the data, and the factor of 2 allows for more stringent clustering). Clusters with very few bases (o30 or 2% of read length) were discarded for this purpose as they were found to reduce alignment accuracy. Myers’ bit-vector algorithm for fast alignment29. GraphMap also allows users a choice of aligners, including an implementation of Gotoh’s semi-global alignment algorithm30, as well as an option to construct anchored alignments. Specifically, in the anchored approach, anchors from the LCSk step are clustered and alignments within and between cluster end points computed using Myers’ bit-vector alignment After all kmers from the query have been processed, a list of walks in the graph is collected. ARTICLE These outliers are caused by repeats or sequencing errors, but they still satisfy the monotony condition. Similar to the observation presented for region selection, the LCSk list of anchors should ideally be collinear in the 2D query-target coordinate space, with a slope of 45. All deviations from this line are caused by indel errors, and can be viewed as noise. The filtering of outlier anchors begins by fitting a 2D line with a 45 slope in the query-target space under the least absolute deviation criteria (L1). Next, a subset of anchors which are located within dL1 ¼ e  T ffiffiffi 2 p =2 from either side of the L1 line is selected, where e is the expected error rate (by default, conservatively set to 45%), T is the target (read) length and the factor ffiffiffi 2 p =2 is used to convert the distance from target coordinate space to a distance perpendicular to the L1 line. A confidence interval c ¼ 3  PN i¼1 di=N is calculated, where di is the distance from a selected anchor i to the L1 line (the constant 3 was chosen to mimic a 3s rule). LCSk is then repeated once again but only on the anchors which are located within the distance ±c from the L1 line to compensate for possible gaps caused by anchor filtering (Fig. 1e). The use of L1 filtering was observed to improve the precision of alignment start and end coordinates for many reads, though the overall impact on performance was less significant in comparison to the LCSk stage (Supplementary Data 3). y p q g y y y condition. Similar to the observation presented for region selection, the LCSk list of anchors should ideally be collinear in the 2D query-target coordinate space, with a slope of 45. All deviations from this line are caused by indel errors, and can be viewed as noise. The filtering of outlier anchors begins by fitting a 2D line with a 45 slope in the query-target space under the least absolute deviation criteria (L1). NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 location. Additionally, using an accumulator array that is of size equal to the size of the reference sequence can cause high memory consumption, especially in the case of processing large sequences in multithreaded environments. construct approximate alignments that help identify the correct mapping location. Removing this stage reduced GraphMap’s precision and recall by 10–30% without significantly affecting its runtime or memory usage (Supplementary Data 3). construct approximate alignments that help identify the correct mapping location. Removing this stage reduced GraphMap’s precision and recall by 10–30% without significantly affecting its runtime or memory usage (Supplementary Data 3). g g To address both the error-rate and memory consumption issues, GraphMap rasterizes the reference sequence into partitions of length L/3 (where L is the read length), so that at least one partition is fully covered by the read. For each seed hit to a bin, it increments the value of the bin corresponding to its c parameter value determined using equation (3). Bins are then sorted in descending order of the number of hits. To limit the search to the most likely bins, only bins with count 475% of the max count are selected for further processing. A region is then defined as a portion of the reference that expands the corresponding bin’s start and end location by an additional read length, to compensate for potential indel errors and ensure that the entire alignment area enters the next step of mapping. In the case that the reference genome is specified as being circular by the user, GraphMap allows the region to be constructed by concatenating the beginning and the end of the reference. Regions are then processed separately until the last step of the method, when the highest scoring region is selected for alignment. Refining alignments using L1 linear regression. The alignments obtained using LCSk tend to be largely accurate but since its definition lacks constraints on the distance between substrings, the alignments obtained may include outlier matches and incorrect estimation of overall alignment length (Fig. 1e). These outliers are caused by repeats or sequencing errors, but they still satisfy the monotony Refining alignments using L1 linear regression. The alignments obtained using LCSk tend to be largely accurate but since its definition lacks constraints on the distance between substrings, the alignments obtained may include outlier matches and incorrect estimation of overall alignment length (Fig. 1e). Methods D i ti Description of the GraphMap algorithm. Region selection. GraphMap starts by roughly determining regions on the reference genome where a read could be aligned. This step is performed to reduce the search space for the next step of the algorithm, while still providing high sensitivity. As a first step, region selection relies on finding seeds between the query sequence and the reference, before clustering them into candidate regions. For seed finding, commonly used approaches such as maximal exact matches (MEMs; as used in BWA-MEM (ref. 10)) or Hamming distance based spaced seeds24,25 c ¼ t  q: ð3Þ ð3Þ c ¼ t  q: g p (as used in LAST (ref. 14)) were found to be either not sensitive enough or not specific enough in the presence of error rates as high as is feasible in nanopore data (for example, see ‘Fixed seed k ¼ 13’ for ONT 1D data in Supplementary Data 3). Instead, a form of gapped spaced seeds was employed, similar to gapped q-gram filters for Levenshtein distance15. Specifically, the approach proposed in Burkhardt and Ka¨rkka¨inen15 was extended to use both one- and two-gapped q-grams (Fig. 1b) as detailed below. The index of the accumulator array with the highest count is the exact mapping position of the read on the reference. In this simple form, this approach mirrors the techniques used in other aligners (for example, FASTA). However, the concept of the HT allows us to extend and generalize this notion. count for substitution and indel errors in this framework as follo We account for substitution and indel errors in this framework as follows: substitution errors cause only the reduction in the maximum vote count for the correct c value and induce noise votes in other locations on the reference. Such type of errors can be addressed using appropriate thresholding on the hit count (see below). On the other hand, indels are of special interest because they shift the alignment diagonal and cause more substantial reduction of votes for the correct Gapped q-grams are a seeding strategy that allow for fast and very sensitive lookup of inexact matches, with variations allowed in predefined ‘do not care’ (DC) positions of the seed. Consistent with existing terminology, the concrete layout of the inclusive and DC bases is referred to here as a shape and the number of used NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Data sets. For evaluating GraphMap and other tools, we used eight publicly available MinION sequencing data sets, 49 synthetic data sets and MinION sequencing reads for an E. coli UTI89 sample as detailed below. bases was subtracted from the reported alignment position to compensate for the shift. (2) (2) Reporting the correct alignment at a per-base-pair level—a base was considered correctly aligned if it was placed in exactly the same position as it was simulated from. Unaligned reads and soft- or hard-clipped portions of aligned reads were not taken into account for precision calculation. Recall was calculated with respect to all simulated bases in reads. q g p MinION library preparation. Genomic DNA was extracted from Escherichia coli UTI89 using the QIAamp DNA mini kit (Qiagen). Extracted DNA (1 mg) was then sheared in a total volume of 80 ml using a Covaris g-TUBE according to the manufacturer’s instructions with centrifugation for 1 min at 6,000 r.p.m. Sheared DNA was end repaired and A-tailed using the GeneRead DNA Library Prep I Kit from Qiagen according to the manufacturer’s protocol. The reaction was purified using 1  volume of Agencourt Ampure XP beads and eluted in 30 ml nuclease- free water. Subsequent steps of DNA sequencing library preparation were carried out using Oxford Nanopore’s MinION Genomic DNA Sequencing Kit (SQK-MAP003) according to the manufacturer’s recommended protocol, including the addition of purified BSA (NEB) to Agencourt Ampure XP beads and Elution buffer. Parameter settings for mappers. BWA-MEM was evaluated with the nanopore setting (-  ont2d) unless otherwise stated (version:bwa-0.7.12-r1034, commit: 1e29bcc). BLASR was evaluated with the options ‘-sam -bestn 1’ (version: 1.3.1, commit: f7bf1e5) and in addition for the database search we set more stringent parameters (‘-minMatch 7 -nCandidates 1’). LAST was run with a commonly used nanopore setting31 (‘-q 1 -r 1 -a 1 -b 1’; version: 475). BLAST (version: ncbi-blast- 2.2.30 þ -  64-linux) was run with default settings for Illumina data and a more suitable nanopore setting33 ‘-reward 5 -penalty -4 -gapopen 8 -gapextend 6 -dust no’ for ONT and PacBio data. GraphMap (version: v0.21, commit: 0bd0503) was run with default settings. In addition, for circular genomes we used the -C option, anchored alignments for calling structural variations (‘-a anchor’) and E-value filtering (‘-z 1e0’) for database search and variant calling. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 (8) Amplicon sequencing of human HLA-A, HLA-B and CYP2D6 genes20. The data set contains 36,779 reads in total. As a reference, chromosomes 6 and 22 from hg19 GRCh37 H. sapiens reference were used20. (8) Amplicon sequencing of human HLA-A, HLA-B and CYP2D6 genes20. The data set contains 36,779 reads in total. As a reference, chromosomes 6 and 22 from hg19 GRCh37 H. sapiens reference were used20. Synthetic data sets. Synthetic Illumina reads were generated using the ART simulator32 (150 bp single-end) and PacBio continuous long reads (CLR) reads using the PBSIM simulator18 (with default settings). For synthetic MinION data we adopted PBSIM (as no custom ONT simulators exist currently) and used parameters learnt from LAST alignments (to avoid bias towards GraphMap) with E. coli K-12 R7.3 data (Supplementary Table 5). Reads were simulated (n ¼ 1,000) for six reference sequences: N. meningitidis serogroup A strain Z2491 (1 chromosome, 2.2 Mbp, NC_003116.1), E. coli K-12 MG1655 (1 chromosome, 4.6 Mbp, U00096.2), S. cerevisiae S288C (16 chromosomes, 12 Mbp), C. elegans (6 chromosomes, 100 Mbp), H. sapiens Chr3 (198 Mbp, GRCh38, CM000665.2) and the entire H. sapiens genome (3.1 Gbp, GRCh38). Synthetic data sets. Synthetic Illumina reads were generated using the ART simulator32 (150 bp single-end) and PacBio continuous long reads (CLR) reads using the PBSIM simulator18 (with default settings). For synthetic MinION data we adopted PBSIM (as no custom ONT simulators exist currently) and used parameters learnt from LAST alignments (to avoid bias towards GraphMap) with E. coli K-12 R7.3 data (Supplementary Table 5). Reads were simulated (n ¼ 1,000) for six reference sequences: N. meningitidis serogroup A strain Z2491 for six reference sequences: N. meningitidis serogroup A strain Z2491 (1 chromosome, 2.2 Mbp, NC_003116.1), E. coli K-12 MG1655 (1 chromosome, 4.6 Mbp, U00096.2), S. cerevisiae S288C (16 chromosomes, 12 Mbp), C. elegans (6 chromosomes, 100 Mbp), H. sapiens Chr3 (198 Mbp, GRCh38, CM000665.2) and the entire H. sapiens genome (3.1 Gbp, GRCh38). Single-nucleotide variant calling. All 2D reads from Ammar et al.20 were mapped to the human genome (GRCh37.p13; chr 6 and 22) and for each read only the alignment with the highest AS was kept. To avoid chimeric reads as reported in the original study only reads that fully spanned the amplicon regions were used for this analysis. Variants were called using LoFreq (ref. 3; version: 2.1.2) with the parameters ‘-a 0.01 -q 0 -Q 0—no-default-filter’. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 marginAlign was run with the ‘—em’ option on each data set to estimate the correct parameters since data quality varied across data sets (commit: 10a7a41). In the case of simulations, the model parameters were first calculated for every simulated data type using a sample data set, and then marginAlign was run using corresponding models. Furthermore, since marginAlign is a realigner and uses a mapper for seeding the alignment position, we forked and expanded marginAlign to create a version that uses GraphMap instead of LAST as its seed mapper. Our modified version of marginAlign is available on GitHub: https://github.com/isovic/marginAlign (commit: d69264d). The modified version of marginAlign was also used with the ‘—em’ option, with the additional parameter ‘—graphmap’ to use GraphMap. We also compared against DALIGNER (commit: d4aa487). For synthetic data, DALIGNER was tested using three combinations of parameters: default, ‘-e.7 -k10’ and ‘-e.7 -k9’. As ‘-e.7 -k10’ was found to have the best results for synthetic ONT data (Supplementary Data 1), it was used for all tests on real nanopore data. MinION sequencing of E. coli UTI89. Immediately before sequencing, 12 ml of the DNA library was combined with 134 ml EP buffer and 4 ml Fuel Mix and mixed by inversion 10 times. The flow cell was primed by washing with two aliquots of 150 ml of EP buffer, with 10 min in between washes. Prepared DNA Library (150 ml) was then loaded onto the flow cell and the Genomic DNA 48 h sequencing run programme was selected. Fresh sample was loaded onto the flow cell at 12 h intervals throughout the run. Publicly available sequencing data sets. Eight publicly available MinION sequencing data sets were used for evaluation. These include a lambda phage data set, three E. coli data sets (each produced with a different version of MinION chemistry), reads for S. enterica Typhi, A. bayalyi ADP1 and B. fragilis BE1, and a data set consisting of three amplicons from the human genome, as detailed below: (1) Lambda phage burn-in data set12. The data set consists of 40,552 reads in total (211 Mbp of data), generated using an early R6 chemistry. The reference genome (NC_001416) is 49-kbp-long giving an expected coverage of 44,300  . (1) Lambda phage burn-in data set12. The data set consists of 40,552 reads in total (211 Mbp of data), generated using an early R6 chemistry. ARTICLE Concretely, the size of each substring is equal to the length of the corresponding anchor in both sequences. As a result, the reconstruction of LCSk is obtained in the form of a list of consecutive anchors in the target and the query sequence. The LCSk stage was observed to be key to GraphMap’s ability to Mapping quality. Since the region filtering process in GraphMap maintains a large collection of possible mapping positions on the given reference, it enables meaningful calculation of the mapping quality directly from its definition: ð9Þ Q ¼  10  log p; where p is the probability of the read being mapped to the wrong position. We calculate p simply as p ¼ maxð10  4; 1  1 NaÞ, that is, max Q ¼ 40, and report quality values according to the sequence alignment/map (SAM) format specification. E-value. For each reported alignment, GraphMap calculates the E-value which is given as a custom ‘ZE’ parameter in the output SAM file. Following the approach used in BLAST, we rescore alignments and use pre-calculated Gumbel parameters to compute E-values in the same way as in BLAST (default parameters from BLAST: match ¼ 5, mismatch ¼  4, gapopen ¼  8 and gapextend ¼  6). where p is the probability of the read being mapped to the wrong position. We calculate p simply as p ¼ maxð10  4; 1  1 NaÞ, that is, max Q ¼ 40, and report quality values according to the sequence alignment/map (SAM) format specification. where p is the probability of the read being mapped to the wrong position. We calculate p simply as p ¼ maxð10  4; 1  1 NaÞ, that is, max Q ¼ 40, and report quality values according to the sequence alignment/map (SAM) format specification. l h d l h l l h l h h E-value. For each reported alignment, GraphMap calculates the E-value which is given as a custom ‘ZE’ parameter in the output SAM file. Following the approach used in BLAST, we rescore alignments and use pre-calculated Gumbel parameters to compute E-values in the same way as in BLAST (default parameters from 9 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 The reference genome (NC_001416) is 49-kbp-long giving an expected coverage of 44,300  . (2) E. coli K-12 MG1655 R7 data set31. The data set has 111,128 reads (668 Mbp) providing 144  coverage of a 4.6-Mbp genome (U00096.2). 31 Consensus calling using MinION data. Consensus was called using a simple majority vote of aligned bases, insertion and deletion events (insertion sequences were taken into account while counting events) and positions with o20  coverage were not called. Our consensus caller is implemented in a script ‘consensus.py’ that is freely available at https://github.com/isovic/samscripts. All reads were mapped to just the corresponding reference and analysed to determine consensus sequences. The E. coli K-12 reference was mutated using Mutatrix (https://github.com/ekg/mutatrix) with parameters ‘—snp-rate 0.0006— population-size 1—microsat-min-len 0—mnp-ratio 0—indel-rate 0.0067—indel- max 10’ to emulate the draft nanopore-only assembly reported by Loman et al.4 (B3,750 SNPs and B42,500 indels). Real nanopore reads were mapped to the mutated reference, and consensus variants (from ‘consensus.py’) were used to construct a consensus sequence with GATK’s FastaAlternateReferenceMaker tool (GATK version 3.4–46). Consensus sequences were compared with the original reference using nucmer and dnadiff34 (MUMmer 3.0). Positions±2 bp from the mutated position were also considered in calculating consensus errors in mutated positions to account for alignment uncertainty in homopolymer sequences. g g g (3) E. coli K-12 MG1655 R7.3 data set31. The data set has 70,531 reads (311 Mbp) providing 67  coverage of the genome (U00096.2). (4) E. coli K-12 MG1655 SQK-MAP006-1 data set. The data set consists of 116,635 reads (1.06 Gbp) providing 228  coverage of the genome (U00096.2). Sequencing was performed in four runs: two with natural DNA, and two with a low-input library that includes a PCR step. The data set used in this paper consists of the first natural DNA run (MAP006-1; http://lab.loman.net/ 2015/09/24/first-sqk-map-006-experiment/). (4) E. coli K-12 MG1655 SQK-MAP006-1 data set. The data set consists of 116,635 reads (1.06 Gbp) providing 228  coverage of the genome (U00096.2). Sequencing was performed in four runs: two with natural DNA, and two with a low-input library that includes a PCR step. The data set used in this paper consists of the first natural DNA run (MAP006-1; http://lab.loman.net/ 2015/09/24/first-sqk-map-006-experiment/). (5) S. enterica Typhi data set1. The data set is composed of two runs of strain H125160566 (16,401 and 6,178 reads, respectively) and one run of strain 08-04776 (10,235 reads). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 When combined, this data set consists of 32,814 reads (169 Mbp) which amounts to 35  coverage of a closely related reference sequence, S. enterica Typhi Ty2 (NC_004631.1; 4.8 Mbp genome). 8 (5) S. enterica Typhi data set1. The data set is composed of two runs of strain H125160566 (16,401 and 6,178 reads, respectively) and one run of strain 08-04776 (10,235 reads). When combined, this data set consists of 32,814 reads (169 Mbp) which amounts to 35  coverage of a closely related reference sequence, S. enterica Typhi Ty2 (NC_004631.1; 4.8 Mbp genome). 8 q yp y p g (6) A. baylyi ADP1 data set8. The data set consists of 66,492 reads (205 Mbp) providing 57  coverage of a 3.6-Mbp genome (NC_005966.1). 7 (6) A. baylyi ADP1 data set8. The data set consists of 66,492 reads (205 Mbp) providing 57  coverage of a 3.6-Mbp genome (NC_005966.1). 7 (7) B. fragilis BE1 data set7. The data set consists of 21,900 reads (141 Mbp) providing 27  coverage of a 5.2-Mbp genome (LN877293.1 assembly scaffold). (7) B. fragilis BE1 data set7. The data set consists of 21,900 reads (141 Mbp) providing 27  coverage of a 5.2-Mbp genome (LN877293.1 assembly scaffold). Benchmarking mappers for pathogen identification. Bacterial genomes related to a list of water-borne pathogens were selected from NCBI’s bacterial database to construct a database of 259 genomes (550 Mbp; Supplementary Data 4). MinION sequencing data sets from cultured isolates were used as proxy for sequencing of pathogen-enriched clinical samples (using data for E. coli K-12 R7.3, S. enterica Typhi and E. coli UTI89, as specified earlier). This is a simple test case as real samples are likely to have contamination from other sources as well (for example, human DNA). We mapped these three read data sets to the database of bacterial genomes using each of the mappers to find unique alignments and test if these could help identify the correct species and strain. For BWA-MEM, LAST, marginAlign and DALIGNER, we chose the best alignment based on alignment score (AS; as long as AS and mapping quality were 40) and for GraphMap and BLASR we used the unique reported alignment (mapping quality40). Since marginAlign and DALIGNER do not report the AS in their output, we rescored their alignments (parameters match ¼ 1, mismatch ¼  1, gapopen ¼  1 and gapextend ¼  1) to make them comparable. NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications Additional information 13. Chaisson, M. J. & Tesler, G. Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory. BMC Bioinformatics 13, 238 (2012). Accession codes: The MinION sequencing of E. coli UTI89 was deposited in the European Nucleotide Archive under the accession code ERX987748. Accession codes: The MinION sequencing of E. coli UTI89 was deposited in the European Nucleotide Archive under the accession code ERX987748. Accession codes: The MinION sequencing of E. coli UTI89 was deposited in the European Nucleotide Archive under the accession code ERX987748. y f 14. Kielbasa, S. M., Wan, R., Sato, K., Horton, P. & Frith, M. C. Adaptive seeds tame genomic sequence comparison. Genome Res. 21, 487–493 (2011). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 15. Burkhardt, S. & Ka¨rkka¨inen, J. in Combinatorial Pattern Matching Vol. 2373 (eds Apostolico, A. & Takeda, M.) 225–234 (Springer, 2002). Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/. p p g 16. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990). g J , ( ) 17. Myers, G. in Algorithms in Bioinformatics Vol. 8701 (eds Brown, D. & 17. Myers, G. in Algorithms in Bioinformatics Vol. 8701 (eds Brown, D. & Morgenstern B ) 52 67 (Springer 2014) , G. in Algorithms in Bioinformatics Vol. 8701 (eds Brown, D. & 17. Myers, G. in Algorithms in Bioinformatics Vol. 8701 (eds Brown, D. & Morgenstern, B.) 52–67 (Springer, 2014). How to cite this article: Sovic´, I. et al. Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Nat. Commun. 7:11307 doi: 10.1038/ncomms11307 (2016). 18. Ono, Y., Asai, K. & Hamada, M. PBSIM: PacBio reads simulator—toward accurate genome assembly. Bioinformatics 29, 119–121 (2013). 19. Szalay, T. & Golovchenko, J. A. De novo sequencing and variant calling with nanopores using PoreSeq. Nat. Biotechnol. 33, 1087–1091 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Structural variation detection. We modified the E. coli K-12 MG1655 reference by inducing 20 SV events (10 insertions and 10 deletions) of different sizes: 100, 300, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500 and 4,000 bp. All 2D reads from both E. coli K-12 data sets (R7 and R7.3) were combined and mapped. SVs were detected by simple consensus vote of indel events reported in spanning alignments (Z20 bases to avoid sequencing errors). In the absence of a sophisticated SV caller for nanopore data we used a simple rule that identifies windows where 415% of the reads at each position report an insertion (or deletion) event (at least five reads). To avoid fragmented events due to a local drop in allele frequency, windows which were less than window-length apart (max of the two windows) were merged. A detected event was considered a true positive if its size was within a 25% margin of the true size and its start and end locations were o25% of event size away from the true locations. LUMPY (ref. 35; version: 0.2.11) was used for testing the use of split read alignments. The script ‘extractSplitReads_BwaMem’ provided with LUMPY was used to extract split reads from BWA-MEM alignments. As the default setting (‘minNonOverlap ¼ 20’) did not report any results, the script was run with the setting ‘minNonOverlap ¼ 0’ to allow split alignments to be adjacent on the read. Structural variation detection. We modified the E. coli K-12 MG1655 reference by inducing 20 SV events (10 insertions and 10 deletions) of different sizes: 100, 300, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500 and 4,000 bp. All 2D reads from both E. coli K-12 data sets (R7 and R7.3) were combined and mapped. SVs were detected by simple consensus vote of indel events reported in spanning alignments (Z20 bases to avoid sequencing errors). In the absence of a sophisticated SV caller for nanopore data we used a simple rule that identifies windows where 415% of h d h i i i i ( d l i ) ( l fi 22. Patel, A., Schwab, R., Liu, Y. T. & Bafna, V. Amplification and thrifty single- molecule sequencing of recurrent somatic structural variations. Genome Res. 24, 318–328 (2014). 23. Cao, M. D. et al. Real-time strain typing and analysis of antibiotic resistance potential using Nanopore MinION sequencing. doi: http://dx.doi.org/10.1101/ 019356 (2015). 24. Berlin, K. et al. Author contributions I.S., M.S. and N.N. conceived the project and designed GraphMap. I.S. implemented GraphMap and conducted all experiments with assistance and guidance from A.W., M.S. and N.N. MinION sequencing was performed by S.N.F. with guidance from S.C. The manuscript was written by I.S. and N.N. with input from all authors. 10. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinformatics 25, 1754–1760 (2009). 10. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows Wheeler transform. Bioinformatics 25, 1754–1760 (2009). Wheeler transform. Bioinformatics 25, 1754–1760 (2009). d l b d d l h Wheeler transform. Bioinformatics 25, 1754–1760 (2009). 11. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012). 12. Mikheyev, A. S. & Tin, M. M. Y. A first look at the Oxford Nanopore MinION sequencer. Mol. Ecol. Res. 14, 1097–1102 (2014). References 1. Ashton, P. M. et al. 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A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat. Methods 12, 733–735 (2015). 36. Thorvaldsdottir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013). 5. Wang, Y., Yang, Q. & Wang, Z. The evolution of nanopore sequencing. Front. Genet. 5, 449 (2014). Acknowledgements 6. Laver, T. et al. Assessing the performance of the Oxford Nanopore Technologies MinION. Biomol. Detect. Quantif. 3, 1–8 (2015). This work was supported by the IMaGIN platform (project no. 102 101 0025), through a grant from the Science and Engineering Research Council, funding to the Genome Institute of Singapore from the Agency for Science, Technology and Research This work was supported by the IMaGIN platform (project no. 102 101 0025), through a grant from the Science and Engineering Research Council, funding to the Genome Institute of Singapore from the Agency for Science, Technology and Research This work was supported by the IMaGIN platform (project no. 102 101 0025), through a grant from the Science and Engineering Research Council, funding to the Genome Institute of Singapore from the Agency for Science, Technology and Research (A*STAR), Singapore, and funding from the Croatian Science Foundation (Project no. 7353—Algorithms for Genome Sequence Analysis). Technologies MinION. Biomol. Detect. Quantif. 3, 1–8 (2015) 7. Risse, J. et al. A single chromosome assembly of Bacteroides fragilis strain 7. Risse, J. et al. A single chromosome assembly of Bacteroides fragilis strain BE1 from Illumina and MinION nanopore sequencing data. Gigascience 4, 60 (2015). BE1 from Illumina and MinION nanopore sequencing data. Gigascience 4, 60 (2015). g g y gy (A*STAR), Singapore, and funding from the Croatian Science Foundation (Project no. 7353—Algorithms for Genome Sequence Analysis). (A*STAR), Singapore, and funding from the Croatian Science Foundation (Project no. 7353—Algorithms for Genome Sequence Analysis). 8. Madoui, M.-A. et al. Genome assembly using Nanopore-guided long and error- free DNA reads. BMC Genomics 16, 327 (2015). 9. Ip, C. L. C. et al. MinION Analysis and Reference Consortium: Phase 1 data release and analysis. F1000Research 4, 1075 (2015). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 Assembling large genomes with single-molecule sequencing and locality-sensitive hashing. Nat. Biotechnol. 33, 623–630 (2015). y g 25. Ma, B., Tromp, J. & Li, M. PatternHunter: faster and more sensitive homology search. Bioinformatics 18, 440–445 (2002). search. Bioinformatics 18, 440–445 (2002) 26. Li, M., Ma, B., Kisman, D. & Tromp, J. PatternHunter II: highly sensitive and fast homology search. Genome Inform. 14, 164–175 (2003). 27. Benson, G., Levy, A. & Shalom, B. R. in Similarity Search and Applications Vol. 8199 (eds Brisaboa, N., Pedreira, O. & Zezula, P.) 257–265 (Springer, 2013). default setting (‘minNonOverlap ¼ 20’) did not report any results, the script was run with the setting ‘minNonOverlap ¼ 0’ to allow split alignments to be adjacent on the read. 28. Pavetic, F., Zuzic, G. & Sikic, M. LCSk þ þ : Practical similarity metric for long strings. Preprint at http://arxiv.org/abs/1407.2407 (2014). 29. Myers, G. A fast bit-vector algorithm for approximate string matching based on dynamic programming. J. ACM 46, 395–415 (1999). 30. Gotoh, O. An improved algorithm for matching biological sequences. J. Mol. Biol. 162, 705–708 (1982). Code availability. GraphMap is available open source under the MIT license at https://github.com/isovic/graphmap. Scripts to reproduce all results in this study can be found at https://github.com/isovic/graphmap-reproduce. 31. Quick, J., Quinlan, A. R. & Loman, N. J. A reference bacterial genome dataset generated on the MinION portable single-molecule nanopore sequencer. Gigascience 3, 22 (2014). 32. Huang, W., Li, L., Myers, J. R. & Marth, G. T. ART: a next-generation sequencing read simulator. Bioinformatics 28, 593–594 (2012). ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11307 A custom caller for marginAlign (marginCaller) was also used to call SNVs. The detected SNVs were then compared with known variants from dbSNP and a high-confidence set for NA12878 (ref. 21; the sequenced sample; ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_ 9606_b141_GRCh37p13/VCF/All.vcf.gz; ftp-trace.ncbi.nih.gov/giab/ftp/data/ NA12878/variant_calls/NIST/NISTIntegratedCalls_14datasets_131103_allcall_ UGHapMerge_HetHomVarPASS_VQSRv2.18_all.primitives.vcf.gz) to identify true positives and false positives. To estimate GraphMap’s scalability with respect to error rate and read length, 25 additional data sets were simulated from the S. cerevisiae S288C reference, To estimate GraphMap’s scalability with respect to error rate and read length, 25 additional data sets were simulated from the S. cerevisiae S288C reference, q has been added each pair (e, L) of error rate eA{5, 10, 15, 20 and 25}% and read lengths LA{1, 2, 3, 4 and 5} kbp (n ¼ 10,000). q has been added each pair (e, L) of error rate eA{5, 10, 15, 20 and 25}% and read lengths LA{1, 2, 3, 4 and 5} kbp (n ¼ 10,000). q has been added each pair (e, L) of error rate eA{5, 10, 15, 20 and 25}% and read lengths LA{1, 2, 3, 4 and 5} kbp (n ¼ 10,000). Evaluation methods. Performance on synthetic data. Mappers were evaluated for precision and recall in meeting two goals: (1) Finding the correct mapping location—a read was considered correctly mapped if its mapping position was within ±50 bp of the correct location. In case an alignment contained soft- or hard-clipped bases, the number of clipped 10 NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications Additional information The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 20. Ammar, R., Paton, T. A., Torti, D., Shlien, A. & Bader, G. D. Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes. F1000Research 4, 17 (2015). p yp 21. Zook, J. M. et al. Integrating human sequence data sets provides a resource of benchmark SNP and indel genotype calls. Nat. Biotechnol. 32, 246–251 (2014). 11 NATURE COMMUNICATIONS | 7:11307 | DOI: 10.1038/ncomms11307 | www.nature.com/naturecommunications

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D79 D79 JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) I. PENDAHULUAN D EWASA ini pembangunan proyek konstruksi meningkat secara masif dan signifikan, khususnya pada daerah perkotaan . Seperti halnya dengan salah satu proyek CBD (Central Bussiness District), Grand Kamala Lagoon di Kota Bekasi. Proyek CBD tersebut terdiri dari 4 tower, jalan kawasan, dan pusat komersialisasi. Isabella Tower merupakan salah satu dari keempat apartemen tersebut yang harus terselesaikan pada tahun 2020. Hal tersebut, tentunya membutuhkan adanya inovasi dalam metode pelaksanaan konstruksi. Penjadwalan proses pelaksanaan yang cepat dan tepat, biaya yang ekonomis, dan tidak mengesampingkan kestabilan dan tingkat keamanan struktur, menjadi syarat yang harus dipenuhi dalam proses pelaksanaan konstruksi. Dengan demikian penggunaan elemen beton pracetak dapat digunakan sebagai alternatif untuk memenuhi syarat proses pelaksanaan konstruksi tersebut. D Modifikasi Perencanaan Struktur Gedung Isabella Tower Bekasi Menggunakan Elemen Pracetak dan Hollow Core Slab dengan Sistem Ganda Diana Dwi Yunita, I Gusti Putu Raka, dan Faimun Departemen Teknik Sipil, Fakultas Teknik Sipil, Lingkungan, dan Kebumian, Institut Teknologi Sepuluh Nopember (ITS), Surabaya, 60111 e-mail: [email protected], [email protected] Abstrak—Isabella Tower Bekasi pada kondisi sebenarnya dibangun dengan menggunakan metode konvensional dengan ketinggian 20 lantai. Gedung tersebut difungsikan sebagai hunian atau tempat tinggal. Gedung hunian tersebut akan dilakukan perancangan menggunakan elemen pracetak. Perencanaan struktur gedung ini didesain menggunakan sistem ganda dengan kategori seismik E. Rangka utama didesain sebagai sistem rangka pemikul momen khusus dan dinding struktur beton khusus. Hasil analisis yang telah dilakukan diperoleh bahwa rangka atau frame utama gedung mampu menahan beban lateral X dan Y yang masing masing bernilai sebesar 29.95% dan 34.73%, sehingga persyaratan untuk sistem ganda terpenuhi. Perencanan elemen pracetak didasarkan pada tiga kondisi, yakni saat pengangkatan, sebelum komposit, dan setelah komposit. Digunakan tebal plat hollow core menggunakan tebal sebesar 15 cm. Dimensi balok terbesar ialah 50 x 70 cm dan dimensi kolom terbesar ialah 70 x 70 cm. Dalam perencanaan struktur gedung ini harus mengacu pada bagunan tahan gempa terbaru yakni, SNI 2847:2013, SNI 1727:2013, RSNI 2847:2018, RSNI 1726:2018, dan referensi lainnya. Pada tugas akhir ini membahas perancangan struktur gedung Isabella Tower Bekasi, sehingga akan menghasilkan perencanaan yang berisikan spesifikasi dan gambar yang sesuai dengan perencanaan struktur yang mengacu pada peraturan atau standarisasi yang berlaku. pemasangannya. Komponen pracetak diproduksi secara masif oleh sebuah industri pabrik untuk keperluan pembangunan gedung untuk waktu yang singkat dan biaya ekonomis [1]. Keunggulan beton pracetak dibandingkan dengan beton konvensional adalah waktu pembangunan proyek yang lebih cepat, terjaminnya quality control pada mutu beton, dapat meminimalkan framework maupun tenaga kerja, dan penghematan lahan. Desain untuk lantai dan atap pada sebuah struktur gedung juga memerlukan sebuah pertimbangan khusus. Hollow Core Slab mempunyai lubang longitudinal yang mempunyai fungsi utama untuk mengurangi berat beban untuk plat lantai. Plat tersebut biasanya digunakan untuk bangunan dengan bentang panjang, seperti perkantoran, rumah sakit, sekolah, pusat perbelanjaan dan industri. Selain itu biasanya digunakan juga dalam pembangunan gedung apartemen, karena dapat mengurangi biaya dan cepat dalam proses pemasangan [2]. Isabella Tower akan didesain menjadi 15 lantai, sehingga untuk menambah kekakuan frame struktur dalam menahan beban lateral gempa, tower tersebut juga akan didesain menggunakan sistem ganda. Kata Kunci—hollow core slab, pracetak, sistem ganda Pada perencanaan modifikasi ini, perlu adanya data sekunder berupa denah struktur gedung berupa informasi dan data umum, selanjutnya dilakukakan tinjuan pustaka mengenai sistem dan elemen struktur yang akan digunakan, yakni sebagai berikut: 3) Dimensi Kolom Penentuan dimensi minimal kolom direncanakan dengan menentukan total beban yang akan bekerja pada kolom. Sehingga akan diperoleh luasan minimal yang harus digunakan dalam perencanaan dimensi tersebut. Dimensi kolom direncanakan dalam 5 tipe yang berbeda, dengan dimensi terbesar yakni 70/70 yang terletak pada lantai basement.Berikut merupakan hasil preliminary design untuk elemen kolom A. Beton Pracetak Beton pracetak merupakan seluruh atau bagian dari sebuah elemen struktur yang dicetak atau diproduksi pada sutau tempat khusus yaitu pabrik industri. Yang selanjutnya akan dipasang pada struktur yang akan dibangun. Proses produksi yang dilakukan di pabrik biasanya diproduksi dalam jumlah massal dengan bentuk dan dimensi sesuai dengan pemesanan elemen pracetak tersebut [3]. Proses pembuatan yang berada di lokasi industri mengakibatkan beton pracetak dapat menyediakan kontrol mutu tinggi, efisien dalam mengkontruksi, dan penghematan waktu serta harga. Permasalahan utama dan bagian yang paling penting dalam beton precast adalah sistem sambungan yang menyatukan antara elemen-elemen beton precast yang terpisah menjadi suatu struktur bangunan yang utuh seperti halnya struktur beton yang monolit. Sehingga sambungan yang direncanakan Pracetak dapat didefinisikan sebagai sebuah konsep yang menggunakan standarisasi komponen struktural yang diproduksi di luar lokasi kontruksi dan komponen tersebut ditransportasikan dari tempat fabrikasi menuju pada lokasi JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) D80 pengulangan pada tahap preliminary design dan analisis struktur. Perencanaan struktur bawah dilanjutkan apabila desain struktur atas telah memenuhi kriteria desain yang disyaratkan. terutama untuk daerah rawan gempa harus ada jaminan bahwa sambungan tersebut harus mampu menerima beban gempa rencana, harus mampu memancarkan energi gempa dan harus mempunyai kemampuan berdeformasi secara inelastis [4]. 4) Dimensi Dinding Geser Setelah dilakukan tinjauan pustaka dilanjutkan dengan metodologi dengan mengacu pada standarisasi yang berlaku. Hasil analisis dan pembahasan yang pertama adalah preliminary design untuk masing masing elemen. Tahap kedua adalah tahap pembebanan dan analisis struktur yang menggunakan program bantu ETABS 2013. Setelah itu hasil output analisis struktur dikontrol dengan beberapa persyaratan, apabila memenuhi, maka tahap ketiga adalah, perencanaan struktur sekunder dan primer. Apabila tidak memenuhi persyaratan yang telah ditetapkan perlu adanya Perencanaan tebal dinding geser mengacu pada RSNI 2847:2018 pasal 11.3.3.1 dimana tebal dinding penumpu tidak boleh kurang dari l/25 tinggi atau panjang bentang tertumpu dan tidak kurang dari 100 mm [8]. Dalam perencanaan ini dinding geser dinding geser direncanakan meiliki ketebalan sebesar 50 cm Di mana hanya koneksi mekanis yang digunakan. Adanya perhitungan khusus pada sambungan kering difungsikan untuk mampu menahan momen dan gaya geser, menunjukkan perpindahan yang sama tetapi memiliki karakteristik disipasi energi lebih banyak dibandingkan dengan koneksi monolit. Gambar 1. Hollow Core Slabs 15 cm[9] D. Sistem Ganda Kombinasi antara dinding geser dan struktur rangka pemikul momen atau rangka bresing dapat menahan total gaya lateral dalam proporsi yang cukup kaku atau rigid. Dengan mempertimbangkan interaksi yang terjadi antar elemen tersebut pada tiap level lantai. Namun demikian, struktur rangka pemikul momen harus mampu menahan tidak kurang dari 25% dar gaya seismik horizontal [7]. 1) Dimensi Balok Perencanaan dimensi balok mengacu pada RSNI 2847:2018 9.3.3.1, yang tertera pada tabel 9.3.3.1 [8], sehingga diperoleh rekapitulasi sebagai berikut: Tabel 1. Rekapitulasi Dimensi Balok Induk No. Tipe Dimensi b (cm) h (cm) l (m) 1 BI 1 50 70 8 2 BI 2 40 60 6 3 BI 3 40 60 5 4 BI 4 40 60 4.2 5 BI 5 40 60 2.6 6 BI 1 50 70 8 7 BI 3 40 70 5 8 BI 6 40 70 4 Tabel 2. Rekapitulasi Dimensi Balok Anak No. Tipe Dimensi b (cm) h (cm) l (m) 1 BA 1 40 50 8 2 BA 2 30 50 6 3 BA 3 30 50 5 4 BA 4 30 50 4 5 BA 5 30 50 4.2 6 BA 6 30 50 2.6 ) Dimensi Plat Tabel 1. Rekapitulasi Dimensi Balok Induk No. Tipe Dimensi b (cm) h (cm) l (m) 1 BI 1 50 70 8 2 BI 2 40 60 6 3 BI 3 40 60 5 4 BI 4 40 60 4.2 5 BI 5 40 60 2.6 6 BI 1 50 70 8 7 BI 3 40 70 5 8 BI 6 40 70 4 Tabel 2. Rekapitulasi Dimensi Balok Anak No. Tipe Dimensi b (cm) h (cm) l (m) 1 BA 1 40 50 8 2 BA 2 30 50 6 3 BA 3 30 50 5 4 BA 4 30 50 4 5 BA 5 30 50 4.2 6 BA 6 30 50 2.6 Dimensi Plat A. Preliminary Design Hollow core slab merupakan salah satu elemen beton pracetak, yang menggunakan sistem beton pratekan. Tak hanya itu adanya celah menerus pada plat dapat digunakan untuk mengurangi berat sendiri plat dan biaya pelaksanaan konstruksi. Plat ini terbuat dari beton berkualitas tinggi yang merupakan salah satu elemen pra fabrikasi dengan proporsi rongga yang lebih besar. Dalam praktek biasanya hollow core slab disambungkan dengan elemen struktur lain menggunakan senyawa grouting. Berbeda dengan beton konvensional, pelat hollow core slab memiliki banyak keuntungan, seperti menghemat bahan, kecepatan pemasangan, menurunkan biaya konstruksi bangunan, memiliki tingkat kualitas pelat yang konsisten, ketahanan api yang baik, dan sifat isolasi suara. Sebagai tambahannya hollow core slab dapat diaplikasikan pada bentang panjang dan dengan ketebalan yang minimum. Hollow core slab dapat menggunakan strand pratekan, dengan ketebalan plat sebesar antara 400 dan 500 mm, dengan bentang 14 hingga 18 meter dengan lebar standar 900 mm dan 1200 mm yang biasanya digunakan dalam praktek lapangan [5]. C. Jenis Sambungan Elemen Pracetak Pada penelitian sebelumnya, yakni terhadap perilaku histeretik pada daerah sambungan mengklasifikasikan ke dalam dua kategori utama, yaitu [6] 2) Dimensi Plat Berdasarkan brosur PT. Beton Elemenindo Perkasa [9], digunakan hollow core slab dengan tebal 150 mm dengan jumlah wire 12 dan diameter wire sebesar 7 mm serta memiliki daya dukung sebagai berikut: 1) Sambungan basah (wet connections) Di mana beton atau proses grouting baru dilakukan langsung di lokasi proyek untuk menutupi tulangan yang terbuka di daerah sambungan Gambar 1. Hollow Core Slabs 15 cm[9] 2) Sambungan kering (dry connections) 2) Sambungan kering (dry connections) Di mana hanya koneksi mekanis yang digunakan. 1) Kontrol Perencanaan Hollow Core Slabs 1) Kontrol Perencanaan Hollow Core Slabs Kontrol perencanaan hollow core slab, terdiri dari tiga kontrol, yakni kontrol kapasitas, kekuatan lentur, dan kekuatan geser. Untuk kontrol kapasitas pembabanan ijin dengan bentang 5 m sebesar 890 𝐾𝑔 𝑚2 ⁄ dengan pembebanan yang terjadi sebesar 314.918 𝐾𝑔 𝑚2 ⁄ , sehingga memenuhi syarat kapasitas ijin. Selanjutnya dilakukan kontrol kuat lentur ijin (ɸMn) dan kuat geser ijin (ɸVcw) dengan masing masing kuat ijinnya sebesar 69.5598 ft-k/slab dan 16.7947 pounds, yang melebihi dari gaya momen (Mu) dan geser (Vu) yang terjadi sebesar, 18.794 ft-k/slab dan 4.4452 pounds. Sehingga hollow core slab setebal 15 cm dengan bentang 5m dapat digunakan. Gambar 2. Permodelan Struktur Menggunakan ETABS 2013 3) Perencanaan Tangga Perencanaan tangga diasumsikan menggunakan perletakan jepit dengan menggunakan program bantu SAP2000, sehingga diperoleh penulangan sebagai berikut: Berikut merupakan gaya geser dasar hasil analisis ragam yang diperoleh menggunakan program bantu ETABS 2013. Tabel 3. Hasil Analisis Gaya Geser Dasar Load Case/Combo FX (Kg) FY (Kg) EQX Max 764,280.24 706,335.38 EQY Max 753,996.61 762,171.13 Tabel 7. Rekapitulasi Penulangan Tangga Nama Struktur h (m) Tulangan Longitudinal Longitudinal Susut Plat Tangga Up 3.5 D10-250 D10-250 ɸ8-160 Plat Tangga Down D10-250 D10-125 ɸ8-160 Plat bordes D10-250 D10-250 ɸ8-160 1) Pembebanan Gravitasi Pembebanan yang dimasukkan ke dalam program bantu ETABS 2013 harus mendekati perhitungan pembebanan total yang telah dihitung manual dengan nilai toleransi sebesar 5%. Perhitungan manual diperoleh sebesar 21,952,865.57 Kg. sedangkan output yang diperoleh dari ETABS 2013 sebesar 22,806,439.66 Kg. Sehingga terdapat selisih beban sebesar 853,574.09 Kg atau sebesar 3.742689%. Selisih tersebut kurang dari batas toleransi selisih sebesar 5%. Sehingga pembebanan gravitasi pada ETABS 2013 dapat digunakan dalam perhitungan beban gempa. 2) Perencanaan Balok Anak Pracetak Berdasarkan analisis perhitungan yang telah dilakukan untuk perhitungan penulangan dalam kondisi setelah komposit, sebelum komposit, dan saat pengangkatan diperoleh kondisi yang paling kritis, yakni pada saat sebelum komposit dengan rekapitulasi penulangan lentur sebagai berikut: 2) Pembebanan Gempa Dinamis 2) Pembebanan Gempa Dinamis Pembebanan dinamis mengacu pada RSNI 1726:2018 [11], yang didalamnya memuat ketentuan dan persyaratan dalam perhitungan beban gempa. Nilai Ss dan Sds pada kelas situs kategori E di Bekasi Jawa Barat adalah sebesar 0.684g dan 0.607g. Berdasarkan kategori tersebut direncanakan menggunakan sistem ganda yaitu kombinasi antara dinding geser beton bertulang khusus dan sistem rangka pemikul momen khusus. Tabel 5. Penulangan Lentur Lapangan Balok ƿpakai As perlu n pakai d pakai s pakai (mm2) (mm) (mm) BA 1 0.017371942 1907.439259 4 25 58 BA 2 0.011389546 937.9291266 2 25 124 BA 3 0.007680806 632.5143522 2 25 124 BA 4 0.003535534 291.1512172 2 25 124 BA 5 0.00532174 438.2452691 2 25 124 BA 6 0.003535534 291.1512172 2 25 124 Tabel 6. Penulangan Lentur Tumpuan Balok ƿmin As min n pakai d pakai s pakai (mm2) (mm) (mm) BA 1 0.003536 388.2016229 2 25 224 BA 2 0.003536 291.1512172 2 25 124 BA 3 0.003536 291.1512172 2 25 124 BA 4 0.003536 291.1512172 2 25 124 BA 5 0.003536 291.1512172 2 25 124 BA 6 0.003536 291.1512172 2 25 124 3) Perencanaan Tangga Tabel 5. Berdasarkan hasil perhitungan periode fundamental struktur secara manual diperoleh sebesar 2.218 𝑠 yang memiliki nilai yang lebih kecil daripada hasil periode fundamental maksimum yang telah dihasilkan oleh program bantu ETABS 2013 yakni sebesar 3.229 s. Tabel 6. Sehingga, untuk memperoleh gaya gempa dinamik yang paling kritis, maka digunakan periode fundamental struktur yang paling kecil, yakni sebesar 2.218 𝑠 dan nilai Cs yang diambil sebesar 0.03588 Penulangan Lentur Tumpuan Nilai berat efektif bangunan yang didapatkan sebesar 21,302,090.75 Kg. Sehingga Gaya geser dasar yang telah diperoleh akan didistribusikan secara vertikal pada tiap lantai struktur yang berdasarkan RSNI 1726:2018, yakni sebesar 765,120.59 Kg B. Pembebanan dan Analisis Struktur Dalam perencanaan struktur gedung perlu adanya perhitungan terkait pembebanan gravitasi dan pembebanan JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) D81 Tabel 4. Reaksi Perletakan untuk Gempa X dan Y Pemikul Gaya Geser EQX EQY KN % KN % Shear Wall 6,132.77 70.05 6,176.88 65.27 SRPM 2,622.34 29.95 3,287.21 34.73 Tabel 4. gempa. Hal ini bertujuan agar elemen-elemen struktur mampu memikul beban-beban yang telah diperhitungkan sebelumnya. Sehingga dapat diperoleh struktur gedung yang kokoh. Pembebanan gravitasi mengacu pada SNI 1727:2013 [10]dan pembebanan gempa yang mengacu pada RSNI 1726:2018 [11]. Berdasarkan perolehan hasil tersebut, dapat disimpulkan bahwa presentase total untuk SRPM memiliki nilai sebesar lebih dari 25% [11], sehingga layout dan konfigurasi struktur gedung telah memenuhi persyaratan dual system. Gambar 2. Permodelan Struktur Menggunakan ETABS 2013 C. Perencanaan Struktur Sekunder 1) Perencanaan Balok Induk Berdasarkan analisis perhitungan yang telah dilakukan untuk perhitungan penulangan dalam kondisi setelah komposit, sebelum komposit, dan saat pengangkatan diperoleh kondisi yang paling kritis, yakni pada saat setelah komposit dengan pembebanan yang digunakan adalah kombinasi antara beban mati, beban mati tambahan, beban hidup dan beban gempa. Berikut merupakan rekapitulasi penulangan lentur: Gambar 5. Detail Penulangan Kolom (K5) Tabel 8. Penulangan Tumpuan Negatif Balok ƿpakai As perlu n pakai d pakai s pakai (mm2) (mm) (mm) BI 1 0.003953 1234.276499 4 25 91.33333 BI 2 0.003953 829.3073164 3 25 99.5 BI 3 0.003953 829.3073164 3 25 99.5 BI 4 0.003953 829.3073164 3 25 99.5 BI 5 0.003953 829.3073164 3 25 99.5 BI 6 0.003953 829.3073164 3 25 99.5 BI 7 0.003953 987.4211994 4 25 58 BI 8 0.003953 987.4211994 4 25 58 Tabel 9. Penulangan Lapangan Positif Balok ƿpakai As perlu n pakai d pakai s pakai (mm2) (mm) (mm) BI 1 0.004491 1402.211188 4 25 91.33333 BI 2 0.003953 829.3073164 3 25 99.5 BI 3 0.003953 829.3073164 3 25 99.5 BI 4 0.003953 829.3073164 3 25 99.5 BI 5 0.003953 829.3073164 3 25 99.5 BI 6 0.003953 829.3073164 3 25 99.5 BI 7 0.003953 987.4211994 4 25 58 BI 8 0.003953 987.4211994 4 25 58 Gambar 4. Detail Penulangan BI 1 2) Perencanaan Kolom Perencanaan kolom menggunakan program bantu SPColumn dalam pengecekan kebutuhan tulangan longitudinal dengan pembebanan yang digunakan berasal Tabel 8. Gambar 5. Detail Penulangan Kolom (K5) 3) Perencanaan Dinding Geser 3) Perencanaan Dinding Geser 3) Perencanaan Dinding Geser Perencanaan dinding geser menggunakan program bantu SPColumn dalam pengecekan kebutuhan tulangan longitudinal dengan pembebanan yang digunakan berasal dari Pier Forces yang berupa gaya aksial, momen arah x, dan momen arah y. Sehingga diperoleh penulangan longitudinal sebesar D25-120 Tabel 9. Gambar 6. Detail Penulangan Dindin Geser Tipe 1 Gambar 6. Detail Penulangan Dindin Geser Tipe 1 E. Perencanaan Sambungan 3) Kontrol Dual Systems y Berikut merupakan joint reaction yang diperoleh menggunakan program bantu ETABS 2013. JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) D82 Gambar 3. Detail Penulangan Tangga D P St kt P i dari Column Forces yang berupa gaya aksial, momen arah x, dan momen arah y. Sehingga diperoleh penulangan longitudinal sebesar 24D25 sedangkan untuk tulangan geser menggunakan 5D13 Tabel 10. Rekapitulasi Penulangan Lentur Kolom Kolom n D ɸMn Mux Muy Kontrol Momen K1 24 25 841.05 53.24 41.59 OK K2 24 25 870.71 116.3 86.12 OK K3 24 25 989.75 146.58 108.13 OK K4 24 25 1146.7 157.83 103.6 OK K5 24 25 1425.3 356.68 208.3 OK Tabel 11. Rekapitulasi Penulangan Geser Kolom Kolom Ash perlu n pakai D Ash pakai Kontrol mm2 mm mm2 K1 479.374424 5 13 663.6614481 OK K2 479.374424 5 13 663.6614481 OK K3 554.1265823 5 13 663.6614481 OK K4 550.2022901 5 13 663.6614481 OK K5 561.6 5 13 663.6614481 OK Gambar 5. Detail Penulangan Kolom (K5) Tabel 10. Rekapitulasi Penulangan Lentur Kolom Kolom n D ɸMn Mux Muy Kontrol Momen K1 24 25 841.05 53.24 41.59 OK K2 24 25 870.71 116.3 86.12 OK K3 24 25 989.75 146.58 108.13 OK K4 24 25 1146.7 157.83 103.6 OK K5 24 25 1425.3 356.68 208.3 OK Tabel 11. Rekapitulasi Penulangan Geser Kolom Kolom Ash perlu n pakai D Ash pakai Kontrol mm2 mm mm2 K1 479.374424 5 13 663.6614481 OK K2 479.374424 5 13 663.6614481 OK K3 554.1265823 5 13 663.6614481 OK K4 550.2022901 5 13 663.6614481 OK K5 561.6 5 13 663.6614481 OK Gambar 5. Detail Penulangan Kolom (K5) Tabel 10. Tabel 11. Tabel 11. k i l i l G Gambar 3. Detail Penulangan Tangga Gambar 3. Detail Penulangan Tangga Gambar 3. Detail Penulangan Tangga 1) Sambungan Balok-Kolom 1) Sambungan Balok-Kolom Dalam perencanaan tulangan atas dan bawah digunakan Modix Rebar Coupler tipe PM dan SM. Untuk kait standar juga menggunakan produk Peikko Group. Berikut merupakan hasil tipe produk yang digunakan untuk sambungan balok dengan kolom untuk tipe double sided connection: Gambar 4. Detail Penulangan BI 1 2) Perencanaan Kolom Perencanaan kolom menggunakan program bantu SPColumn dalam pengecekan kebutuhan tulangan longitudinal dengan pembebanan yang digunakan berasal Panjang Penyaluran Kait Standar dalam Tarik Panjang Penyaluran Kait Standar dalam Tarik JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) D83 peralatan crane untuk menunjang pemasangan balok induk. Sambungan tulangan pada bagian tarik menggunakan Modix Coupler SM25B-P-1650-SM25A-D-550 – SM25B-P- 1550. 4. Dalam meletakkan balok anak ke balok induk, digunakan konsol pendek pada balok induk, kemudian dirangkai menjadi satu kesatuan. Perencanaan konsol pada balok induk ini sama dengan perencanaan konsol kolom Panjang Penyaluran Kait Standar dalam Tekan Sambungan tulangan pada bagian tekan menggunakan Modix Coupler SM25B-P-1650-PM25-SM25A-D-550 – PM25-SM25B-P-1650 g p 5. Elemen plat diletakkan atau ditumpukan pada selimut beton sepanjang 5 cm. Setelah semua tulangan pada balok induk dan balok anak terpasang dilakukan pengecoran atau overtopping sebagai penutup tulangan. Gambar 7. Detail Sambungan BI 1-K3-BI1 2) Sambungan Balok Induk-Balok Anak Gambar 10. Rencana Denah Pondasi Sama halnya dengan sambungan balok-kolom, sambungan balok anak-balok induk pada perencanaan ini dapat dilihat sebagai berikut: Panjang Penyaluran Kait Standar dalam Tekan Panjang Penyaluran Kait Standar dalam Tekan Panjang Penyaluran Kait Standar dalam Tekan 1) Perencanaan Pondasi Pondasi pada gedung ini direncanakan menggunakan pondasi tiang pancang yang diproduksi oleh PT. WIKA Beton. Kombinasi beban yang digunakan dalam permodelan mengggunakan beban nominal dengan desain tegangan ijin sedangkan untuk poer menggunakan kombinasi beban terfaktor. Gambar 7. Detail Sambungan BI 1-K3-BI1 2) Perhitungan Tiang Pancang dan Poer Sambungan tulangan pada bagian tekan menggunakan Modix Coupler SM25B-P-1250-PM25-SM25A-D-500 – PM25-SM25B-P-1250 Tiang pancang yang digunakan untuk podasi tipe 1 yaitu 4D 60 dengan dimensi poer sebesar 2.7 m x 2.7 m x 1.5 m Penulangan poer direncanakan dengan menggunakan program bantu ETABS 2013 dengan gaya aksial satu tiang yang digunakan sebagai input pembebanan joint loads, sehingga diperoleh hasil output momen yang digunakan untuk analisis perhitungan penulangan. Berdasarkan hasil analisis tersebut diperoleh penulangan arah X dan Y sebesar D25-80 Gambar 8. Detail Sambungan BA 1- BI 1- BA 1 Gambar 8. Detail Sambungan BA 1- BI 1- BA 1 3) Sambungan Balok – Plat Transfer Length (Lt) = 1.2 x 350 mm = 420 mm Direncanakan menggunakan kait 2D16 Grade 300 Mpa Gambar 9. Detail Sambungan Balok-Plat Gambar 8. Detail Sambungan BA 1- BI 1- BA 1 Gambar 8. Detail Sambungan BA 1- BI 1- BA 1 Gambar 11. Detail Penulangan Pondasi Tipe 1 3) Sambungan Balok – Plat Transfer Length (Lt) = 1.2 x 350 mm = 420 mm Direncanakan menggunakan kait 2D16 Grade 300 Mpa Gambar 11. Detail Penulangan Pondasi Tipe 1 Gambar 9. Detail Sambungan Balok-Plat Panjang Penyaluran Kait Standar dalam Tarik Sambungan tulangan pada bagian tarik menggunakan sambungan manual seperti biasa tanpa menggunakan produk Peikko Gambar 10. Rencana Denah Pondasi Gambar 10. Rencana Denah Pondasi F. Metode Pelaksanaan Momen yang didapat menggunakan bantuan dari program bantu ETABS 2013 yaitu sebesar 1.3258 t.m, seperti pada gambar di bawah ini. Metode pelaksanaan ini merupakan uraian mengenai komponen dan material material pendukung yang digunakan dalam pelaksanaan metode beton pracetak 1. Proses produksi elemen pracetak dilakukan secara fabrikasi di dalam suatu industri beton pracetak Gambar 12. Momen akibat Tekanan Horizontal Tanah Sehingga digunakan tulangan lentur positif D13-150 dan tulangan lentur negatif D13-150 2. Jenis crane POTAIN MR 160 C dengan jarak jangkau maksimum 60 m dengan beban maksimum 7.5 ton 3. Setelah dilakukan pengecoran kolom, balok induk pracetak dipasang terlebih dahulu baru kemudian dilanjutkan dengan pemasangan balok anak. Diperlukan DAFTAR PUSTAKA [1] R. O. Asamoah, J. S. Ankrah, K. Offei-Nyako, and E. O. Tutu, “Cost analysis of precast and cast-in-place concrete construction for selected public buildings in Ghana,” J. Constr. Eng., vol. 2016, pp. 1–10, 2016. [2] I. M. Ezz El-Arab, “Web shear strengthening technique of deep precast prestressed hollow core slabs under truck loads,” J. Build. Constr. Plan. Res., vol. 05, no. 04, pp. 129–145, 2017. Sistem sambungan pada elemen balok dan kolom dan balok induk dengan balok anak menggunakan produk dari Peikko Group, yaitu dengan menggunakan Modix Rebar Coupler. [3] C. H. Najoan, J. Tjakra, and P. A. K. Pratasis, “Analisis metode pelaksanaan plat precast dengan plat konvensional ditinjau dari waktu dan biaya (studi kasus : markas komando daerah militer Manado),” J. Sipil Statik, vol. 4, no. 5, pp. 319–327, 2016. Dalam proses analisis struktur gedung Isabell Tower Bekasi Jawa Barat, menggunakan program bantu ETABS 2013. Hasil analisis diperoleh, yakni periode getar yang diambil adalah batas maksimum, toleransi berat struktur kurang dari 5%, gaya gempa dinamik yang lebih besar dari 100% gaya gempa statik, serta syarat dual system dengan rangka pemikul momen sebesar lebih dari 25% [4] J. Suherman, “Penggunaan block set connection (BSC) pada sambungan elemen beton precast,” Teknol. dan Kejuru., vol. 34, no. 2, pp. 217–227, 2011. pp [5] S. Simasathien and S.-H. Chao, “Shear strength of steel-fiber- reinforced deep hollow-core slabs,” PCI J., vol. 60, no. 4, pp. 85–101, 2015. [6] M. K. Joshi, C. V. R. Murty, and M. P. Jaisingh, “Cyclic behaviour of precast RC connections,” Indian Concr. J., vol. 79, no. 11, pp. 43–50, 2005. Momen nominal yang diperoleh (ɸMn) mampu menahan momen yang bekerja pada penampang (Mu), dengan kebutuhan tulangan longitudinal untuk balok induk sebesar 5D25 dan kolom sebesar 24D25. Geser nominal yang diperoleh (ɸVn) mampu menahan gaya geser yang bekerja pada penampang (Vu) dengan kebutuhan tulangan transversal untuk balok induk sebesar 3D13-90 dan kolom sebesar 5D13- 100. [7] L. Mamatha, G. S. Vijaya, and E. K. K. L, “Seismic analysis of R . C .dual frame systems with and without floating columns,” Int. Res. J. Eng. Technol., vol. 03, no. 09, pp. 1340–1346, 2016. [8] “Persyaratan Beton Struktural untuk Bangunan Gedung (RSNI 2847:2018),” Badan Standardisasi Indonesia. BSN, Bandung, pp. 1– 7, 2018. [9] “Hollow Core Slab Brosur.” PT. Beton Elemenindo Perkasa, Jakarta. [10] “Beban Minimum Untuk Perancangan Bangunan Gedung dan Struktur Lain (SNI 1727:2013),” Badan Standardisasi Indonesia. BSN, Bandung, 2013. IV. KESIMPULAN perhitungan tiang pancang berdiameter 60 cm produk WIKA Beton, dan perhitungan daya dukung tanah pada kedalaman 20 m dengan menggunakan tegangan ijin, serta penulangan pilecap berdasarkan pembebanan ultimate (kombinasi LRFD). Perhitungan mengacu pada peraturan RSNI 2847:2018, SNI 1726: 201X, sehingga modifikasi perencanaan dapat dikatakan aman dan memenuhi persyaratan yang berlaku Balok induk, balok anak, serta plat direncanakan menggunakan elemen pracetak, sedangkan kolom, dinding geser, tangga, plat basement, dna pile cap direncanakan menggunakan beton cast in situ untuk mempermudah pelaksanaan konstruksi dan mempersingkat waktu pelaksanaan JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 8, No. 2, (2019) ISSN: 2337-3539 (2301-9271 Print) D84 Gambar 12. Momen akibat Tekanan Horizontal Tanah Sehingga digunakan tulangan lentur positif D13-150 dan tulangan lentur negatif D13-150 DAFTAR PUSTAKA g [11] “Tata Cara Perencanaan Ketahanan Gempa untuk Struktur Bangunan Gedung dan Non Gedung (RSNI 1726:2018),” Badan Standardisasi Indonesia. BSN, Bandung, pp. 3–5, 2018. Gaya aksial nominal yang diperoleh (ɸPn) mampu menahan gaya aksial yang bekerja pada penampang (Pu). Pondasi yang direncanakan sesuai dengan ketentuan

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Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice

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UCSF UC San Francisco Previously Published Works Title Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice Permalink https://escholarship.org/uc/item/0hj9r0vj Journal PLOS ONE, 5(1) ISSN 1932-6203 Authors Yu, Siwang Khor, Tin Oo Cheung, Ka-Lung et al. Publication Date 2010 DOI 10.1371/journal.pone.0008579 Peer reviewed UCSF UC San Francisco Previously Published Works Title Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice Permalink https://escholarship.org/uc/item/0hj9r0vj Journal PLOS ONE, 5(1) ISSN 1932-6203 Authors Yu, Siwang Khor, Tin Oo Cheung, Ka-Lung et al. Publication Date 2010 DOI 10.1371/journal.pone.0008579 Peer reviewed UCSF UC San Francisco Previously Published Works Title Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice Permalink https://escholarship.org/uc/item/0hj9r0vj Journal PLOS ONE, 5(1) ISSN 1932-6203 Authors Yu, Siwang Khor, Tin Oo Cheung, Ka-Lung et al. Publication Date 2010 DOI 10.1371/journal.pone.0008579 Peer reviewed UCSF UC San Francisco Previously Pu Title Nrf2 Expression Is Regulated by Epigenetic M Permalink https://escholarship.org/uc/item/0hj9r0vj Journal PLOS ONE, 5(1) ISSN 1932-6203 Authors Yu, Siwang Khor, Tin Oo Cheung, Ka-Lung et al. Publication Date 2010 DOI 10.1371/journal.pone.0008579 Peer reviewed Abstract Recent studies from our laboratory and others have found that the expression levels of SOD, UGT1A1, NQO1 and several GST family genes were significantly suppressed in prostate tumors in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice [9–11]. Although the down-regulation of GST enzymes in human prostate cancer have been linked to the promoter hypermethylation of GST genes [5,12]; promoter DNA hypermethylation does not appear to cause GST gene repression in TRAMP tumors [9]. Instead, down-regulation of nuclear factor-erythroid 2p45 (NF-E2)- related factor 2 (Nrf2), a key regulator of cellular antioxidant enzymes, may be responsible for the transcriptional suppression of GSTs and other phase II detoxifying enzyme genes [11]. Powered by the California Digital Library University of California eScholarship.org Siwang Yu1.¤, Tin Oo Khor1., Ka-Lung Cheung1, Wenge Li1, Tien-Yuan Wu1, Ying Huang1, Barbara A. Foster2, Yuet Wai Kan3, Ah-Ng Kong1* 1 Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America, 2 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, United States of America, 3 Cardiovascular Research Institute and Departments of Laboratory Medicine and Medicine, University of California San Francisco, San Francisco, California, United States of America Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by the National Institutes of Health grants R01-CA118947 and R01-094828 to A.-N. T. Kong study design, data collection and analysis, decision to publish, or preparation of the manuscript. supported in part by the National Institutes of Health grants R01-CA118947 and R01-094828 to A.-N. T. Kong. The funders had no role in tion and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. . These authors contributed equally to this work. ¤ Current address: School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, People’s Republic of China [4]. Down-regulation of GST by DNA methylation appears to be quite common in human prostate cancer that it has been developed as a diagnostic marker [5]. The expression and the activities of SOD, catalase and GPx have been reported to be decreased in prostate carcinoma tissues as well as in plasma and erythrocytes [6–8]. Recent studies from our laboratory and others have found that the expression levels of SOD, UGT1A1, NQO1 and several GST family genes were significantly suppressed in prostate tumors in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice [9–11]. Although the down-regulation of GST enzymes in human prostate cancer have been linked to the promoter hypermethylation of GST genes [5,12]; promoter DNA hypermethylation does not appear to cause GST gene repression in TRAMP tumors [9]. Instead, down-regulation of nuclear factor-erythroid 2p45 (NF-E2)- related factor 2 (Nrf2), a key regulator of cellular antioxidant enzymes, may be responsible for the transcriptional suppression of GSTs and other phase II detoxifying enzyme genes [11]. [4]. Down-regulation of GST by DNA methylation appears to be quite common in human prostate cancer that it has been developed as a diagnostic marker [5]. The expression and the activities of SOD, catalase and GPx have been reported to be decreased in prostate carcinoma tissues as well as in plasma and erythrocytes [6–8]. Abstract Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-29-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer. Citation: Yu S, Khor TO, Cheung K-L, Li W, Wu T-Y, et al. (2010) Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice. PLoS ONE 5(1): e8579. doi:10.1371/journal.pone.0008579 Editor: Andy T. Y. Lau, University of Minnesota, United States of America Editor: Andy T. Y. Lau, University of Minnesota, United States of America Received September 13, 2009; Accepted December 6, 2009; Published January 5, 2010 Received September 13, 2009; Accepted December 6, 2009; Published January 5, 2010 Copyright:
2010 Yu et al. PLoS ONE | www.plosone.org Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice Siwang Yu1.¤, Tin Oo Khor1., Ka-Lung Cheung1, Wenge Li1, Tien-Yuan Wu1, Ying Huang1, Barbara A. Foster2, Yuet Wai Kan3, Ah-Ng Kong1* Khor1., Ka-Lung Cheung1, Wenge Li1, Tien-Yuan Wu1, Ying Huang1, Barbara A. o Khor1., Ka-Lung Cheung1, Wenge Li1, Tien-Yuan Wu1, Ying Huang1, Barbara A. an3, Ah-Ng Kong1* Hypermethylation of Specific CpG Sites in the CpG Island in Murine Nrf2 Gene Hypermethylation of Specific CpG Sites in the CpG Island in Murine Nrf2 Gene The genomic sequence of Nrf2 gene (NC_000068.6: 75544698- 75513576 Mus musculus chromosome 2, reference assembly (C57BL/6J), including 2 kb of its 59-upstream sequence) was analyzed for CpG island using CpG island Finder (http://people. usd.edu/,sye/cpgisland/CpGIF.htm). Since several mRNA vari- ants with different transcription start sites (TSS) have been reported in the literature [16,27,28], therefore in the present study we define the translation initiation site (TIS) as position 1 to avoid any possible confusion. A CpG island was identified between -1175 and +1240, with a GC content of 61.53%, CpG observed/expected ratio of 0.66 and a total of 150 CpGs. The CpG island includes the murine Nrf2 promoter, the first exon and part of the first intron (Figure 1A). Similar results were obtained when using other criteria and algorithm (http://www.uscnorris.com/cpgislands2/cpg.aspx, data not shown). 10 sets of bisulfite genomic sequencing (BGS) primers were designed using the BiSearch web server (http://bisearch. enzim.hu/, [29]) to cover the 59-flanking region spanning from 21226 to +844 of the murine Nrf2 gene (Table S1). Bisulfite- converted genomic DNA derived from 12 palpable prostate tumors of 24 weeks old TRAMP mice and 10 apparently normal prostate tissues of C57BL/6J mice was used as templates. As shown in Figure 1B, although the majority of the CpG island is barely methylated, the first 5 CpG sites were found to be hypermethylated (96%) in prostate tumors compared to apparently normal prostate tissues (4%). Representative sequencing chromatographs showing the first 5 CpGs in prostate tumor and normal prostate are shown in Figure S1. The following 10 CpGs that are separated by two CpG- free gaps (21131 to 21060 and 2886 to 2798) also displayed differential methylation status in tumors (34%) compared to normal tissues (2%). In addition, another region located in the first intron appears to be sparsely CpG-methylated in prostate tumor (4.5%). g p [ ] The transcription of Nrf2 has recently been shown to be regulated by the aryl hydrocarbon receptor (AhR) and Nrf2 itself [16,17]. However, the currently accepted paradigm of Nrf2 regulation appears to be mainly achieved via post-translational mechanisms. Hypermethylation of Specific CpG Sites in the CpG Island in Murine Nrf2 Gene As such, Nrf2 is functionally suppressed by the Kelch- like Erythroid-cell-derived protein with CNC homology (ECH)- Associated Protein 1 (Keap1), which binds to and sequesters Nrf2 in the cytoplasm leading to the degradation of Nrf2, and thus prevents Nrf2 nuclear translocation and transactivation of its target genes [18]. Upon challenges by oxidative or electrophilic stresses that could involve potential modification of critical cysteine residues in Keap1 and or Nrf2 itself coupled with phosphorylation by kinases, Nrf2 is released from Keap-1, translocates into the nucleus, dimerizes with small Maf proteins, binds to ARE and transcrip- tionally activates Nrf2-ARE target genes [4]. To date, it is not clear as to how the expression of Nrf2 in human prostate cancer or in TRAMP mouse tumor is suppressed. Epigenetic or epigenomic mechanisms, particularly DNA methylation, have been frequently implicated in the alterations of gene expression in prostate cancer [19,20]. Coordinated hypermethylation of APC and GSTP1 in early carcinogenesis has been utilized as potential diagnostic markers to detect prostate cancer [21]. In addition, alteration of DNA methylation profiles has been linked with cancer progression [20]. DNA methylation, coupled with histone modifications, would affect the interactions of the promoters of critical genes with transcriptional corepressors and coactivators leading to changes in gene expressions, which could be one of the driving forces for prostate carcinogenesis. Silencing of multiple genes by DNA methylation has been reported in TRAMP prostate tumors and cell lines derived from TRAMP prostate tumors [22–24]. Inhibition of DNA methyl- transferase activity by 5-aza-29-deoxycytidine (5-aza) has been shown to prevent prostate tumorigenesis in TRAMP mice [25]. In addition, the expression of Keap1 has been reported to be regulated by DNA methylation in lung cancer [26]. In the present study, we first identified a CpG island in the upstream 59-flanking region of the murine Nrf2 gene followed by interrogation of the DNA methylation status of the whole CpG island via bisulfite genomic sequencing. We found that certain CpG sites in the distal region of the CpG island were hypermethylated in the TRAMP tumor tissues as well as in tumorigenic TRAMP-C1 and -C2 cells, but not in normal prostatic tissue and non-tumorigenic TRAMP- C3 cells. Utilizing methylated reporter assay, chromatin immu- noprecipitation (ChIP) assay and treatments with trichostatin A TRAMP C1, C2 and C3 cell lines were originated from a heterogeneous tumor of 32-week TRAMP mouse [30]. Introduction Prostate cancer, as well as many other age-related cancers, is characterized by increased intracellular oxidative stress [1,2]. Chronic oxidative stress and its associated pathological conditions including inflammation and metabolic disorders have been postulated to drive somatic mutations and neoplastic transforma- tion, thus could play an important role in the development and progression of prostate cancer [3]. Increased oxidative stress or reactive oxygen species (ROS) levels could be a consequence of increased ROS generation and/or decreased antioxidant capacities and or ROS detoxification. Recently the impaired antioxidant defense system in carcinogenesis of prostate cancer has been gaining increased attentions. The cellular antioxidant defense system comprises a battery of antioxidant/detoxifying enzymes and proteins such as superoxide dismutase (SOD), catalase, hemeoxgenase (HO), UDP-glucurono- syltransferases (UGT), glutathione peroxidase (GPx), glutathione S- transferases (GST), and NAD(P)H:quinone oxidoreductase (NQO) Nrf2 is a helix–loop–helix basic leucine zipper transcription factor that regulates the expression of many phase II detoxifying PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 1 January 2010 | Volume 5 | Issue 1 | e8579 Epigenetic Regulation of Nrf2 and antioxidant enzymes via its binding to the antioxidant- response element (ARE) in the promoter region [4]. Knockout of Nrf2 in mice substantially abrogated the inducible expression of ARE-mediated detoxifying and antioxidant enzymes, and ren- dered these mice highly susceptible to carcinogens and/or oxidative damages [13,14]. In these context, previously we have found that the protein expression levels of Nrf2 and Nrf2-target gene heme-oxygenase-1 (HO-1) were attenuated in the skin tumors of a mouse skin carcinogenesis model [15]. Similarly, the expression of Nrf2 as well as its downstream target genes such as UGT1A1, GSTM1 and NQO1 were found to be gradually down- regulated in prostate tumors with the progression of prostate tumorigenesis in TRAMP mice [10]. Frolich et al. recently reported the expression of Nrf2 and GST mu family genes were significantly decreased in TRAMP prostate tumor, and linked this phenomenon to increased oxidative stress and DNA damage in prostate cancer. Meta-analysis of tissue expression profiling data from Oncomine database (www.oncomine.org) suggested that the expressions of Nrf2 and several GST mu genes are also gradually down-regulated in human prostate cancers [11]. (TSA)/5-aza, we provided compelling evidence that the expression of Nrf2 is epigenetically regulated during the development of prostate tumors in TRAMP mice. Introduction Nrf2 expression was suppressed by methylation of certain CpG sites and this was accompanied by the recruitment of MBD2 and trimethyl-histone H3 (Lys9) to the Nrf2 gene promoter in prostate tumor of TRAMP mice. Hypermethylation of Specific CpG Sites in the CpG Island in Murine Nrf2 Gene While TRAMP C1 and C2 cells are tumorigenic when grafted into syngenic C57BL/6J hosts, TRAMP C3 cells are not. Genomic DNA from C1 and C3 cells was bisulfite-sequenced as above to detect the methylation status of the CpG island in Nrf2 gene. Surprisingly, the methylated CpG ‘‘hot spots’’ as found above in the TRAMP prostate tumor were also methylated in tumorigenic C1 cells but not in non-tumorigenic TRAMP C3 cells (Figure 1C). PLoS ONE | www.plosone.org Methylation of the First 5 CpGs Significantly Suppressed the Transcriptional Activity of Mouse Nrf2 Promoter To investigate the functional role of methylation of specific CpG sites, particularly the first 5 CpGs in the CpG island, luciferase reporters driven by the Nrf2 promoter with or without the first 5 CpGs (designated as 21367 and 21065, respectively) were constructed as described in Materials and Methods (Figure 2A). The plasmids were methylated using M. sssI CpG methyltrans- ferase in vitro and the methylation status was confirmed by HpaI/ HhaII digestion (Figure S2). As shown in Fig. 2B, the 21065 Nrf2 promoter, which had been reported previously [17,28], substan- tially increased the luciferase activity to about 200 folds. In PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 2 Epigenetic Regulation of Nrf2 Figure 1. Hypermethylation of Nrf2 promoter in TRAMP prostate was correlated with tumorigenesis. (A) A CpG island was identified in the 59-flanking region of mouse Nrf2 gene, spanning from position 21175 to +1240 with the translation initiation site set as position 1. The sequences covered by bisulfite genomic sequencing (21226 to +844) and contain methylated CpGs are schematically presented with CpG sites indicated by vertical lines. (B) The methylation patterns and extents of CpG sites in the promoter of Nrf2 gene in TRAMP prostate tumor and apparently normal prostate were determined by bisulfite genomic sequencing as described in Material & Methods. Black dots indicate methylated CpGs and open circles indicate non-methylated CpGs. (C) The methylation patterns and extents of CpG sites in the promoter of Nrf2 gene in TRAMP C1 and C3 cells were determined. The CpGs in the sequence between +296 to +594 are not displayed because methylation is insignificant. doi:10.1371/journal.pone.0008579.g001 Figure 1. Hypermethylation of Nrf2 promoter in TRAMP prostate was correlated with tumorigenesis. (A) A CpG island was identified in the 59-flanking region of mouse Nrf2 gene, spanning from position 21175 to +1240 with the translation initiation site set as position 1. The sequences covered by bisulfite genomic sequencing (21226 to +844) and contain methylated CpGs are schematically presented with CpG sites indicated by vertical lines. (B) The methylation patterns and extents of CpG sites in the promoter of Nrf2 gene in TRAMP prostate tumor and apparently normal prostate were determined by bisulfite genomic sequencing as described in Material & Methods. Black dots indicate methylated CpGs and open circles indicate non-methylated CpGs. Methylation of the First 5 CpGs Significantly Suppressed the Transcriptional Activity of Mouse Nrf2 Promoter (C) The methylation patterns and extents of CpG sites in the promoter of Nrf2 gene in TRAMP C1 and C3 cells were determined. The CpGs in the sequence between +296 to +594 are not displayed because methylation is insignificant. doi:10.1371/journal.pone.0008579.g001 contrast, the 21367 Nrf2 promoter showed a less potent transcriptional activity (,67 folds). Importantly, in vitro CpG- methylation of the reporter construct resulted in a dramatic decrease of the transcriptional activity of the 21367 Nrf2 promoter (84% decrease); while, the activity of the 21065 promoter was decreased by only 23.4% by CpG-methylation. Similar results were obtained in Hep G2 hepatoma cells and human embryonic kidney HEK 293 cells (Figure S3). positive control, b-actin promoter is equally associated with Pol II in C1 and C3 (Figure 3A). The binding of Pol II to the Nrf2 gene TSS was significantly decreased in C1 cells as compared to in C3 cells, indicating a suppressed transcription of Nrf2 in C1 cells (Figure 3A & B). In agreement with this observation, the binding of MBD2 and tri-methylated histone 3-lys9, (H3K9me3) to the methylated CpGs in Nrf2 promoter was higher in C1 cells than in C3 cells, whereas the association of acetylated histone 3 (H3Ac) displayed a reversed pattern (Figure 3A & B). Methyl CpG binding protein 2 (MeCP2) and mammalian Sin3A (mSin3A) were also tested for their binding with this Nrf2 promoter; however no detectable binding was observed (data not shown). Hypermethylated CpG Island Was Associated with Methyl-CpG-Binding Domain (MBD2) and Histone Modifications, Which Is Reversible by 5-aza/TSA Treatment Hypermethylated CpG Island Was Associated with Methyl-CpG-Binding Domain (MBD2) and Histone Modifications, Which Is Reversible by 5-aza/TSA Treatment Hypermethylated CpG Island Was Associated with Methyl-CpG-Binding Domain (MBD2) and Histone Modifications, Which Is Reversible by 5-aza/TSA Treatment The methylation status of DNA is regulated by DNA methyl- transferases (DNMTs) and 5-aza is a DNMT inhibitor, is often used to examine the effects of DNA methylation [32]. As shown in Figure 3C & D, 5-aza treatment of C1 cells decreases the binding of MBD2 and H3K9me3 to the Nrf2 promoter, while the H3Ac exhibits no observable effect. However, combined treatment of C1 cells with 5-aza and a histone deactylase (HDAC) inhibitor, TSA, substantially increases the binding of H3Ac to the Nrf2 promoter, and further decreases the bindings of MBD2 and H3K9me3 to the Nrf2 promoter. Consistent with the above results, the recruitment of Pol II to the Nrf2 promoter also increases correspondingly, whereas Pol II’s binding to b-actin promoter was not changed (Figure 3C). The repression of gene expression by CpG methylation is generally achieved by MBD proteins that bind to methylated DNA leading to recruitment of chromatin remodeling and transcrip- tional repressor complexes [31]. In the current study, ChIP assays were employed to examine the proteins that could be potentially associated with Nrf2 promoter in TRAMP C1 and C3 cells. Based on the methylation status of Nrf2 promoter, primer sets were designed to cover the first 5 CpGs and the TSS to detect the association of specified DNA-binding proteins as well as RNA polymerase II (Pol II). The specificity of ChIP assays were verified by nonspecific IgG which did not pull down anything. As a PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 January 2010 | Volume 5 | Issue 1 | e8579 3 Epigenetic Regulation of Nrf2 Figure 2. Methylation of the first 5 CpGs inhibited the transcriptional activity of Nrf2 promoter. (A) The construction of luciferase reporters is schematically presented. Nrf2 promoters with (21367–1) or without (21065–1) the extra sequence containing the first 5 CpGs were amplified from mouse genomic DNA and inserted into pGL 4.15 vector. The resulted reporters were designated as pGL-1367 and pGL-1065, respectively. (B) pGL-1367 or pGL-1065 reporters, either methylated by CpG methyltransferase or not, were co-transfected with pGL 4.75 vector which contains a Renilla reniformis luciferase gene driven by CMV promoter into TRAMP C1 cells, and the luciferase activities were measured after 24 hrs. Discussion We and others have previously reported that the expression of Nrf2 and its target genes is suppressed in TRAMP prostate tumors [10,11]. Marvis et al., had reported that the expression of GST family genes was suppressed in TRAMP C2 cells and 5-aza and TSA treatment could restore the expression [9]. Here we examined the expression of Nrf2 and one of its downstream target genes NQO1 in TRAMP C1 and C3 cells. As shown in Figure 4A, the mRNA level of Nrf2 is significantly lower in C1 cells than in C3 cells. The expression of NQO1 was readily induced by tBHQ, a classic Nrf2 agonist, in C3 cells, while such induction was blunted in C1 cells (Figure 4A & C). Treatment of C1 cells with 5-aza and TSA modestly restored Nrf2 expression and significantly enhanced the induction of NQO1 by tBHQ. However, the treatment of C3 cells under the same conditions exhibited no effect on the expression of Nrf2 or NQO1 (Figure 4A, B & C). Western blotting was performed to examine the protein expression levels of Nrf2, NQO1, MBD2, H3Ac and H3K9me3 (Figure 4D). The protein levels of Nrf2 and NQO1 were lower in C1 cells than in C3 cells, and 5-aza and TSA treatment enhanced the induction of NQO1 protein by tBHQ. Although 5-aza and TSA treatment did not increase the basal level of Nrf2 protein the treatment significantly augmented tBHQ-induced Nrf2 protein expression. TSA treatment strongly increased acetylated histone 3 protein while had no effect on histone 3 methylation status. Previous findings from several laboratories including our laboratory have demonstrated that Nrf2 plays an essential role in the development of various cancers [33]. Nrf2 regulates the expression of antioxidant and phase II detoxifying enzymes including NQO1, HO-1 and GST [33]. Therefore, the control of transcriptional activation of Nrf2 and Nrf2-target genes would appear to be an important homeostatic mechanism that protect cellular injuries or damages resulted from oxidative stress [33]. Nrf2 deficiency could lead to defect in the cellular defense system against oxidative stress, potentially resulting in cancer initiation, promotion and progression [34]. The repressed expression of antioxidant and detoxifying enzymes such as GSTP1 in prostate cancer has extensively been studied [2,5]. However the role of Nrf2 in prostate cancer have not received enough attention until recently [10,11]. Frolich et al. The Transcriptional Induction of Nrf2 and NQO-1 in TRAMP Cell Lines Was Correlated with CpG Islands Methylation Status and Could Be Restored by 5-aza/TSA Treatment The Transcriptional Induction of Nrf2 and NQO-1 in TRAMP Cell Lines Was Correlated with CpG Islands Methylation Status and Could Be Restored by 5-aza/TSA Treatment The Transcriptional Induction of Nrf2 and NQO-1 in TRAMP Cell Lines Was Correlated with CpG Islands Methylation Status and Could Be Restored by 5-aza/TSA Treatment Hypermethylated CpG Island Was Associated with Methyl-CpG-Binding Domain (MBD2) and Histone Modifications, Which Is Reversible by 5-aza/TSA Treatment The transcriptional activities of each constructs were calculated by normalizing the firefly luciferase activities with corresponding Renilla luciferase activities, and are represented as folds of induction compared with the activity of empty pGL 4.15 vector. The values are mean6SD of four separate samples. doi:10.1371/journal.pone.0008579.g002 Figure 2. Methylation of the first 5 CpGs inhibited the transcriptional activity of Nrf2 promoter. (A) The construction of luciferase reporters is schematically presented. Nrf2 promoters with (21367–1) or without (21065–1) the extra sequence containing the first 5 CpGs were amplified from mouse genomic DNA and inserted into pGL 4.15 vector. The resulted reporters were designated as pGL-1367 and pGL-1065, respectively. (B) pGL-1367 or pGL-1065 reporters, either methylated by CpG methyltransferase or not, were co-transfected with pGL 4.75 vector which contains a Renilla reniformis luciferase gene driven by CMV promoter into TRAMP C1 cells, and the luciferase activities were measured after 24 hrs. The transcriptional activities of each constructs were calculated by normalizing the firefly luciferase activities with corresponding Renilla luciferase activities, and are represented as folds of induction compared with the activity of empty pGL 4.15 vector. The values are mean6SD of four separate samples. doi:10.1371/journal.pone.0008579.g002 doi:10.1371/journal.pone.0008579.g002 Interestingly, although the protein level of MBD2 was not affected by 5-aza and TSA treatment, it was much higher in C1 cells than in C3 cells (Figure 4D). Interestingly, although the protein level of MBD2 was not affected by 5-aza and TSA treatment, it was much higher in C1 cells than in C3 cells (Figure 4D). Discussion reported that the down-regulation of Nrf2 appears to be responsible for the reduced GST expression, elevated oxidative stress and DNA damage in prostate tumorigenesis in TRAMP mice [11]. We have recently found that the expression of Nrf2 as well as Nrf2-target genes is gradually down-regulated during the progression of prostate cancer in TRAMP mice [10]. Previous analysis of the online human prostate gene expression data sets demonstrated that the expression of Nrf2 and GST [11] PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 January 2010 | Volume 5 | Issue 1 | e8579 4 Epigenetic Regulation of Nrf2 Figure 3. Hypermethylated CpG island was associated with MBD2 binding and histone modifications and 5-aza/TSA treatment reversed the association. (A) ChIP assay was performed to detect the binding of indicated proteins to specific regions of Nrf2 gene cross-linked and immunoprecipitated from TRAMP C1 and C3 cells. The results from 3 independent experiments were quantified by densitometry as shown in (B). (C) TRAMP C1 cells were treated with vehicle, 5-aza or 5-aza+TSA as described, then the cells were subjected to ChIP assay. The results from 2 independent experiments were quantified by densitometry as shown in (D). ChIP assays were performed as described in Material & Methods using antibodies against Pol II, MBD2, H3K9m3 and AcH3. 3 sets of ChIP primers were used (Table S2), with Nrf2P1 covers the first 5 CpGs (21190 to 21092) in the CpG island and Nrf2P2 covers a region close to TSS (262 to +20). Nonspecific IgG was employed as a negative control and binding of Pol II to b-actin promoter was used to verify the efficiency of ChIP assay. The experiments were repeated at least twice with similar results. doi:10.1371/journal.pone.0008579.g003 Figure 3. Hypermethylated CpG island was associated with MBD2 binding and histone modifications and 5-aza/TSA treatment reversed the association. (A) ChIP assay was performed to detect the binding of indicated proteins to specific regions of Nrf2 gene cross-linked and immunoprecipitated from TRAMP C1 and C3 cells. The results from 3 independent experiments were quantified by densitometry as shown in (B). (C) TRAMP C1 cells were treated with vehicle, 5-aza or 5-aza+TSA as described, then the cells were subjected to ChIP assay. The results from 2 independent experiments were quantified by densitometry as shown in (D). Discussion ChIP assays were performed as described in Material & Methods using antibodies against Pol II, MBD2, H3K9m3 and AcH3. 3 sets of ChIP primers were used (Table S2), with Nrf2P1 covers the first 5 CpGs (21190 to 21092) in the CpG island and Nrf2P2 covers a region close to TSS (262 to +20). Nonspecific IgG was employed as a negative control and binding of Pol II to b-actin promoter was used to verify the efficiency of ChIP assay. The experiments were repeated at least twice with similar results. doi:10.1371/journal.pone.0008579.g003 as well as NQO1 was gradually decrease during human prostate carcinogenesis (Figure S4). Furthermore, it has been reported that several GST genes are down-regulated in primary but not in metastatic TRAMP tumors [9]. In current study, we found that the expression of Nrf2 and the induction of NQO1 was compromised in tumorigenic TRAMP C1 cells but not in non- tumorigenic TRAMP C3 cells (Figure 4A). This suppression of Nrf2 expression and Nrf2-target gene NQO1 in both of these TRAMP cell lines would exclude the possibility that Nrf2 expression would be affected by the SV40 transgene, since these TRAMP cell lines do not express the SV40 transgene [30]. DNA methylation has been implicated in the silencing of the GSTP1 gene in human prostate cancer, and similarly DNA- methylation silencing of several other genes are also implicated in TRAMP prostate tumor [5,23,24]. However, interestingly Mas- sARRAY Quantitative DNA Methylation Analyses (MAQMA) analysis of the 59 region of several GST genes displayed no significant differences between normal prostatic epithelial cells and prostate tumor from the TRAMP mice [9]. Recently, several reports show that in both TRAMP and Rb2/2 prostate tumors, an Rb/ E2F-dependent increase of DNMT1 expression and methylation activity [25,35]. Hypermethylation Nrf2 promoter was ruled out using MSP and that 5-aza treatment had no effect on Nrf2 expression (with data not shown) [11]. We analyzed the 59-flanking region of Nrf2 gene and identified a CpG island that extends to position -1175 (Figure 1A). Using bisulfite sequencing, which would be more specific in identifying CpG methylation and would reveal more details about DNA methylation than MSP, we found that the first 5 CpGs in the CpG island are hypermethylated in TRAMP prostate tumors and in the tumorigenic TRAMP C1 cells but not in normal prostate tissues and non-tumorigenic TRAMP C3 cells (Figure 1B). Discussion Remarkably, these 5 CpGs are located adjacent to the previously reported Nrf2 promoter [17]. Thus, the methylation status of these specific CpGs appears to be correlated with the tumorigenicity as well as Nrf2 expression and NQO1 induction (Figures 1 and 4). Notably, similar pattern of specific methylation of the distal CpG island has also been observed in the Keap1 gene in lung cancer cells [26]. It is important to note that some of our current findings appear to be somewhat contradictory with the results reported previously [11], however previous findings of extensive down-regulation of Nrf2 and GST during prostate tumor progres- sion in TRAMP mice [11], are consistent with our current results. as well as NQO1 was gradually decrease during human prostate carcinogenesis (Figure S4). Furthermore, it has been reported that several GST genes are down-regulated in primary but not in metastatic TRAMP tumors [9]. In current study, we found that the expression of Nrf2 and the induction of NQO1 was compromised in tumorigenic TRAMP C1 cells but not in non- tumorigenic TRAMP C3 cells (Figure 4A). This suppression of Nrf2 expression and Nrf2-target gene NQO1 in both of these TRAMP cell lines would exclude the possibility that Nrf2 expression would be affected by the SV40 transgene, since these TRAMP cell lines do not express the SV40 transgene [30]. DNA methylation has been implicated in the silencing of the GSTP1 gene in human prostate cancer, and similarly DNA- methylation silencing of several other genes are also implicated in TRAMP prostate tumor [5,23,24]. However, interestingly Mas- sARRAY Quantitative DNA Methylation Analyses (MAQMA) analysis of the 59 region of several GST genes displayed no significant differences between normal prostatic epithelial cells and prostate tumor from the TRAMP mice [9]. Recently, several reports show that in both TRAMP and Rb2/2 prostate tumors, an Rb/ E2F-dependent increase of DNMT1 expression and methylation activity [25,35]. Hypermethylation Nrf2 promoter was ruled out as well as NQO1 was gradually decrease during human prostate carcinogenesis (Figure S4). Furthermore, it has been reported that several GST genes are down-regulated in primary but not in metastatic TRAMP tumors [9]. In current study, we found that the expression of Nrf2 and the induction of NQO1 was compromised in tumorigenic TRAMP C1 cells but not in non- tumorigenic TRAMP C3 cells (Figure 4A). Discussion It is highly likely that since Nrf2 signaling is primarily regulated via post-translational mechanisms, without challenges with Nrf2-activators such as tBHQ, Nrf2 protein would be rapidly turned over by proteosome-dependent degradation [18]. The precise reason as to why tBHQ is needed for NQO1 induction would require further study. Nrf2 promoter with or without these 5 CpGs were constructed (Figure 2A). The Nrf2 promoter possesses very GC-rich non- canonical promoter which contains neither TATA box nor a CCAAT box [28], however, this Nrf2-promoter potently activated the transcription of luciferase reporter gene (Figure 2B). Interest- ingly, the addition of sequences from 21065 to 21367 appears to be repressive to the transcriptional activity of the Nrf2 promoter (Figure 2B). Such repressive sequence could function by recruiting specific repressing factors [36], but the exact mechanism accounting for this repressive function of this sequence even in the absence of methylation would require further investigation. Nevertheless, when the reporters were methylated in vitro by CpG methyltransferase, the luciferase reporter activity of the Nrf2 promoter with the additional sequence containing the 5 CpGs (pGL-1367) was reduced by about 84%. In contrast, methylation of the reporter without the additional sequence resulted in about 23% reduction (Figure 2B). This is probably due to the heavy methylation of the whole construct including the luciferase gene (but further study would be needed to prove this). Altogether, these results suggest that the extra 5 CpGs (21065 to 21367) could play a critical role in methylation- dependent suppression of Nrf2 promoter activity. disease progression, delay androgen-independent disease and improve survival of TRAMP mice [25,37]. In addition, stage and phenotype-specific CpG island methylation and DNA methyltransferase expression have been well documented during prostate cancer progression in TRAMP mice [38,39]. These published findings suggest the relevance of using this TRAMP system to interrogate the possible role of epigenetic alterations in prostate carcinogenesis. 5-aza treatment of TRAMP C1 cells modestly increased the mRNA level of Nrf2, while combined treatments of 5-aza and TSA induced a more prominent increase of Nrf2 (Figure 4A). This result is consistent with the report by Mavis et al., in which combined 5-aza and TSA treatments significantly enhanced the expression of GST genes [9]. The protein level of Nrf2 in TRAMP C1 cells remained unaffected by either 5-aza or 5-aza/TSA treatments, however, addition of a potent Nrf2-activator tBHQ would enhance the accumulation of Nrf2 protein (Figure 4B). PLoS ONE | www.plosone.org Discussion TRAMP C1 and C3 cells were treated with 2 mM 5-aza, 200 nM TSA, or 1 mM 5-aza plus 100 nM TSA for 60 hrs or 48 hrs followed by incubation in the presence of 5 mM tBHQ for further 12 hrs. After treatments, the cells were harvested for total protein or RNA Figure 4. The expression of Nrf2 and NQO-1 in TRAMP cell lines was correlated with CpG islands methylation status and could be restored by 5-aza/TSA treatment. TRAMP C1 and C3 cells were treated with 2 mM 5-aza, 200 nM TSA, or 1 mM 5-aza plus 100 nM TSA for 60 hrs or 48 hrs followed by incubation in the presence of 5 mM tBHQ for further 12 hrs. After treatments, the cells were harvested for total protein or RNA extractions. (A) the mRNA levels of Nrf2 and NQO1 were determined by RT-PCR, with GAPDH serving as internal control, and the results from 3 independent experiments were quantified by densitometry and the results are shown in B and C. (D) the protein levels of Nrf2, NQO1, MBD2, H3K9me3 and H3Ac were determined by Western blotting, with actin as a loading control. Each experiment was repeated at least twice with similar results. d i /j l doi:10.1371/journal.pone.0008579.g004 disease progression, delay androgen-independent disease and improve survival of TRAMP mice [25,37]. In addition, stage and phenotype-specific CpG island methylation and DNA methyltransferase expression have been well documented during prostate cancer progression in TRAMP mice [38,39]. These published findings suggest the relevance of using this TRAMP system to interrogate the possible role of epigenetic alterations in prostate carcinogenesis. 5-aza treatment of TRAMP C1 cells modestly increased the mRNA level of Nrf2, while combined treatments of 5-aza and TSA induced a more prominent increase of Nrf2 (Figure 4A). This result is consistent with the report by Mavis et al., in which combined 5-aza and TSA treatments significantly enhanced the expression of GST genes [9]. The protein level of Nrf2 in TRAMP C1 cells remained unaffected by either 5-aza or 5-aza/TSA treatments, however, addition of a potent Nrf2-activator tBHQ would enhance the accumulation of Nrf2 protein (Figure 4B). As expected, the induction of NQO1 mRNA and protein levels displayed a similar trend with that of the Nrf2 protein. Discussion This suppression of Nrf2 expression and Nrf2-target gene NQO1 in both of these TRAMP cell lines would exclude the possibility that Nrf2 expression would be affected by the SV40 transgene, since these TRAMP cell lines do not express the SV40 transgene [30]. DNA methylation has been implicated in the silencing of the GSTP1 gene in human prostate cancer, and similarly DNA- methylation silencing of several other genes are also implicated in TRAMP prostate tumor [5,23,24]. However, interestingly Mas- sARRAY Quantitative DNA Methylation Analyses (MAQMA) analysis of the 59 region of several GST genes displayed no significant differences between normal prostatic epithelial cells and prostate tumor from the TRAMP mice [9]. Recently, several reports show that in both TRAMP and Rb2/2 prostate tumors, an Rb/ E2F-dependent increase of DNMT1 expression and methylation activity [25,35]. Hypermethylation Nrf2 promoter was ruled out To determine the functional role of methylation of these 5 CpGs in the suppression of Nrf2 expession, luciferase reporters of the PLoS ONE | www.plosone.org PLoS ONE PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 January 2010 | Volume 5 | Issue 1 | e8579 5 Epigenetic Regulation of Nrf2 Figure 4. The expression of Nrf2 and NQO-1 in TRAMP cell lines was correlated with CpG islands methylation status and could be restored by 5-aza/TSA treatment. TRAMP C1 and C3 cells were treated with 2 mM 5-aza, 200 nM TSA, or 1 mM 5-aza plus 100 nM TSA for 60 hrs or 48 hrs followed by incubation in the presence of 5 mM tBHQ for further 12 hrs. After treatments, the cells were harvested for total protein or RNA extractions. (A) the mRNA levels of Nrf2 and NQO1 were determined by RT-PCR, with GAPDH serving as internal control, and the results from 3 independent experiments were quantified by densitometry and the results are shown in B and C. (D) the protein levels of Nrf2, NQO1, MBD2, H3K9me3 and H3Ac were determined by Western blotting, with actin as a loading control. Each experiment was repeated at least twice with similar results. doi:10.1371/journal.pone.0008579.g004 Figure 4. The expression of Nrf2 and NQO-1 in TRAMP cell lines was correlated with CpG islands methylation status and could be restored by 5-aza/TSA treatment. Discussion As expected, the induction of NQO1 mRNA and protein levels displayed a similar trend with that of the Nrf2 protein. It is highly likely that since Nrf2 signaling is primarily regulated via post-translational mechanisms, without challenges with Nrf2-activators such as tBHQ, Nrf2 protein would be rapidly turned over by proteosome-dependent degradation [18]. The precise reason as to why tBHQ is needed for NQO1 induction would require further study. The role of CpG methylation in suppressing Nrf2 expression and activation was tested by treatment with 5-aza in TRAMP cells. 5-Aza has previously been shown to be able to prevent early PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 6 Epigenetic Regulation of Nrf2 To further delineate the molecular mechanism by which the specific CpG-methylation suppresses Nrf2 expression, ChIP assays were performed. The result reveals that the binding of MBD2 and H3K9m3 to the specific CpGs was substantially higher in TRAMP C1 cells than in TRAMP C3 cells correlating with the fact that the CpGs were minimally methylated in TRAMP C3 cells (Figure 3A). In contrast, AcH3 displayed an opposite binding pattern to the same CpGs sequence in TRAMP C1 cells as compared to TRAMP C3 cells, and similarly the binding of Pol II to the transcription start site showed similar pattern as AcH3 (Figure 3A). These methylation- dependent associations of corepressors could be modulated by 5-aza and TSA treatments and our results (Figure 3B) correlated very well with the transcription level of Nrf2 (mRNA level) in these two cell lines (Figure 4A). MBD2 has been reported to mediate epigenetic silencing of 14-3-3s in TRAMP C1 cells and human LNCaP prostate cells [24], and has been shown to be involved in the transcriptional repression of GSTP1 in MCF-7 breast cancer cells [40]. MBD2 and other MBD proteins bind to methylated CpGs and recruit corepressor complexes which contain HDACs, chromatin remodeling proteins as well as other proteins leading to the repression of the expression of hypermethylated genes [31]. Interestingly, the protein level of MBD2 was much higher in TRAMP C1 cells than in TRAMP C3 cells (Figure 4A), suggesting a possible common MBD2-mediated epigenetic suppression of Nrf2 in TRAMP C1 cells. In contrast, the protein level of AcH3 was apparently lower in TRAMP C1 cells than in TRAMP C3 cells but was increased tremendously by TSA treatment (Figure 4D). Discussion Since the expression of MBD2 would be dependent on HDAC activities, our above results would potentially explain the requirement of HDAC inhibitors in order to effectively modulate corepressors binding and to maximally restore the reexpression of Nrf2 as well as induction of NQO1 in TRAMP C1 cells. In addition, when this suppressive sequence containing the extra 5 CpGs was further analyzed using the Transcriptional Elements Search System (TESS, http://www.cbil.upenn.edu/tess) based on the TRANSFAC V6.0 database, several transcription factor binding sites were identified, including the binding sites of E2F1-p107 and NF-E2 (data not shown). The exact binding proteins and their functions leading to the suppression of Nrf2 expression would require further investigation. 17b-ol-3-one, and antibiotics. The cells were grown at 37uC in a humidified 5% CO2 atmosphere. 17b-ol-3-one, and antibiotics. The cells were grown at 37uC in a humidified 5% CO2 atmosphere. Plasmids The genomic sequence of murine Nrf2 containing the promoter region was retrieved from NCBI mouse genome data base. Two murine Nrf2 promoter segments, 21065–1 and 21367–1 with the translation initiation site (TSS) referred to as position 1, were amplified from mouse genomic DNA isolated from normal mouse prostate using the following primers: 1065 forward, 59-GGTACC- TAAGTACGTGTAAAGGAACCCTGAGA-39; 1367 forward, 59-GGTACCAACAGTCACTACCACCACCA-39; and a com- mon reverse primer, 59-CTCGAGGCTGAGGGCGGACGC- TGT-39. The PCR products were cloned into pCR4 TOPO vector using a TOPO TA Cloning kit (Invitrogen, Grand Island, NY) then digested with KpnI and XhoI and inserted into pGL4.15 luc2P/Hygro vector. All the sequences of recombinant plasmids were verified by sequencing (DNA Core Facility, Rutgers/ UMDNJ, Piscataway, NJ). Bisulfite Genomic Sequencing (BGS) Genomic DNA was isolated from the palpable prostate tumors of 24 weeks old TRAMP mice (n = 12), apparently normal prostate tissue of 24 weeks old C57BL/6J mice (n = 10), TRAMP-C1 and C3 cells using the DNeasy tissue kit (Qiagen, Valencia, CA). The bisulfite conversion was carried out using 500 ng of genomic DNA with the use of EZ DNA Methylation Gold Kits following manufacturer’s instructions (Zymo Research Corp., Orange, CA). The converted DNA was amplified by PCR using Platinum Blue PCR SuperMix (Invitrogen, Grand Island, NY) with specific primer sets (Table S1), with the translation initiation site (TIS) defined as position 1. The PCR products were purified by gel extraction using the QiaquickTM gel extraction kit (Qiagen, Valencia, CA), then cloned into pCR4 TOPO vector using a TOPOTM TA Cloning kit (Invitrogen, Grand Island, NY). Plasmids DNA from at least 10 colonies per each group were prepared using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) and sequenced (DNA Core Facility, Rutgers/UMDNJ, Piscataway, NJ). In summary, our present results clearly demonstrate that the expression of Nrf2 is suppressed by promoter CpG methylation in TRAMP prostate tumors. The existence and possible biological consequences of such epigenetic mechanism in the regulation of Nrf2 expression in human prostate cancer is currently under investigation in our laboratory. To the best of our knowledge, this is the first report revealing the epigenetic regulation of Nrf2 in prostate tumorigenesis. These findings would certainly open the door for further study on the role of Nrf2 as a plausible target for cancer chemoprevention and a possible diagnostic marker for detection of human prostate cancer. Animals Female hemizygous C57BL/TGN TRAMP mice, line PB Tag 8247NG, and male C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The animals were bred on same genetic background and maintained in the Animal Care Facility of Rutgers University. Housing and care of the animals were in accordance with the guidelines established by the University’s Animal Research Committee consistent with the NIH Guidelines for the Care and Use of Laboratory Animals. Transgenic males used in the current study were routinely obtained as [TRAMP 6 C57BL/6] F1 or as [TRAMP 6 C57BL/6] F2 offspring. Identities of transgenic mice were confirmed by the PCR-based genotyping. Throughout the experiment the animals were housed in cages with wood chip bedding in a temperature-controlled room (68–72uF) with a 12-h light dark cycle, at a relative humidity of 45–55%, and fed with irradiated AIN-76A diet (DYETS Inc, Bethlehem, PA). Materials and Methods Reagents and Cell Culture Cell Treatments and Western Blotting Cells were plated in 6-well or 12-well plates for 24 hrs, then treated with 2 mM 5-aza, 200 nM TSA, or 1 mM 5-aza plus 100 nM TSA in media containing 0.5% FBS for 60 hrs, or 48 hrs followed by incubation in the presence of 5 mM tert-butylhydro- quinone (tBHQ) for additional 12 hrs. After treatments, the cells were harvested in radioimmunoprecipitation assay (RIPA) buffer (Sigma, St Louis, MO). The protein concentrations of the cleared lysates were determined by using the bicinchoninic acid method (Pierce, Rockford, IL), and aliquots each containing 20 mg of total protein were resolved by 4%–15% SDS-polyacrylamide gel electrophoresis (Bio-rad, Hercules, CA). After electrophoresis, the proteins were electro-transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF mem- brane was blocked with 5% fat-free milk in phosphate-buffered saline-0.1% Tween 20 (PBST), then sequentially incubated with specified primary antibodies and HRP-conjugated secondary antibodies. The blots were visualized by SuperSignal enhanced chemiluminiscence (ECL) detection system and documented using a Gel Documentation 2000 system (Bio-Rad, Hercules, CA). RNA Isolation and Reverse Transcription-PCR Figure S2 pGL-1065 and pGL-1367 reporters were methylated in vitro by CpG methyltransferase M. sssI, then digested by HpaI or HhaII. HpaI and HhaII are CpG-methylation-sensitive restriction endonucleases whose activity is blocked by CpG methylation. Found at: doi:10.1371/journal.pone.0008579.s002 (0.09 MB PDF) Total RNA was extracted from the treated cells using the Trizol (Invitrogen, Carlsbad, CA). Steady-state mRNA levels of Nrf2 and NQO1 were determined by semi-quantitative reverse transcrip- tion-PCR (RT-PCR). First-strand cDNA was synthesized from 2 mg of total RNA using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. The cDNA was used as the template for PCR reactions performed using Platinum Blue PCR SuperMix (Invitrogen, Grand Island, NY). The PCR products were isolated by agarose gel electrophoresis and visualized by EB staining using a Gel Documentation 2000 system (Bio-Rad, Hercules, CA). Primers to specifically amplify the genes involved were shown in Table S2. Figure S3 pGL-1367 or pGL-1065 reporters, either methylated by CpG methyltransferase or not, were co-transfected with pGL 4.75 vector which contains a Renilla reniformis luciferase gene driven by CMV promoter into Hep G2 cells (A) or HEK293 cells (B), and the luciferase activities were measured after 24 hrs. The transcriptional activities of each constructs were calculated by normalizing the firefly luciferase activities with corresponding Renilla luciferase activities, and are represented as folds of induction compared with the activity of empty pGL 4.15 vector. The values are mean Aˆ 6SD of four separate samples. Found at: doi:10.1371/journal.pone.0008579.s003 (0.08 MB PDF) Transfection and Luciferase Reporter Assay TRAMP C1 cells were plated in 24-well plates for 24 hrs, then transfected with 100 ng of the indicated reporter plasmids by using GeneJuice (Novagen, Madison, WI) according to the manufac- turer’s instructions. 25 ng of pGL 4.75, which contains a Renilla reniformis luciferase gene driven by CMV promoter, was co- transfected as internal control. 24 hrs after transfection, the cells were lysed in dual luciferase lysis buffer, and 10 ml aliquots of the cell lysate were assayed using a dual luciferase assay kit with a Sirius luminometer (Berthold Technologies, Pforzheim, Germany). The transcriptional activities of each constructs were calculated by normalizing the firefly luciferase activities with corresponding Renilla luciferase activities, and were reported as folds of induction compared with the activity of empty pGL 4.15 vector. The values are mean6SD of four separate samples. Reagents and Cell Culture To determine the association of RNA polymerase complex II to the Nrf2 promoter, another primer set was designed to cover the sequence closer to the transcription start site (TSS, 262 to +20, mNrf2P2). Primers covering b-actin promoter region was used as a control to verify the efficacy of ChIP assays. the concentrations of all plasmids were determined by agarose gel electrophoresis. The efficiency of methylation reactions was confirmed by digestion using the methylation-dependent HhaI and HpaII restriction endonucleases (Figure S2). Reagents and Cell Culture 1 ml of each of the purified DNA was used as template for 30 cycles of PCR amplification using designated primers (Table S2). The PCR products were then analyzed by agarose gel electrophoresis and visualized using EB staining. Primer set was designed to cover the DNA sequence from position 21190 to 21092 (mNrf2P1) in which the first 5 CpGs locate. To determine the association of RNA polymerase complex II to the Nrf2 promoter, another primer set was designed to cover the sequence closer to the transcription start site (TSS, 262 to +20, mNrf2P2). Primers covering b-actin promoter region was used as a control to verify the efficacy of ChIP assays. 10 min, then excess formaldehyde was quenched by addition of 5M glycine. After washing twice, the cells were scraped into 2 ml cold PBS containing 16 protease inhibitor cocktail II. The cells were pelleted and then resuspended in Cell Lysis Buffer containing 16 protease inhibitor cocktail II. Nuclei were isolated after Dounce homogenization and resuspended in Nuclear Lysis Buffer containing 16protease inhibitor cocktail II. The samples were sonicated on ice using a Bioruptor sonicator (Diagenode Inc., Sparta, NJ) to shear the cross-linked DNA to an average length of 200–1000 bp and centrifuged at 12,000 rpm to remove insoluble material. The chromatin solutions were diluted 10-folds using dilution buffer, and 10 ml of each was reserved as total input control. Diluted chromatin solutions were precleared with salmon sperm DNA- protein A magnetic beads for 1 hr, and then incubated with protein A magnetic beads and antibodies specific for MBD2, Pol II, H3Ac and H3K9me3 (Millipore, Lake Placid, NY) or nonspecific IgG overnight at 4uC. The immunoprecipitated complex-magnetic beads were collected using magnetic separator and washed according to manufacturer’s instruction. The pellets were then incubated with proteinase K in ChIP Elution buffer for 2 hrs at 62uC with shaking to elute immunocomplex and reverse cross-link. The samples were incubated at 95uC for 10 min and DNA was purified according to manufacturer’s instruction. 1 ml of each of the purified DNA was used as template for 30 cycles of PCR amplification using designated primers (Table S2). The PCR products were then analyzed by agarose gel electrophoresis and visualized using EB staining. Primer set was designed to cover the DNA sequence from position 21190 to 21092 (mNrf2P1) in which the first 5 CpGs locate. Supporting Information Figure S1 Typical bisulfite genomic sequencing chromatographs show that the first 5 CpGs (indicated by arrows) are methylated in TRAMP prostate tumor but not in normal prostate. All the non- methylated cytosines were converted into thymidine by bisulfite treatment, while the methylated cytosines remained unchanged. Found at: doi:10.1371/journal.pone.0008579.s001 (0.22 MB PDF) RNA Isolation and Reverse Transcription-PCR Reagents and Cell Culture All the enzymes used in the present study were obtained from New England Biolabs Inc. (Ipswich, MA) unless specified. Human recombinant insulin was purchased from Invitrogen. Dual luciferase assay system, luciferase reporter vectors pGL 4.75 with CMV promoter and pGL 4.15 were obtained from Promega (Madison, WI). All other chemicals were purchased from Sigma (St Louis, MO, USA). The CpG-methylated reporters were generated by treating the reporter plasmids with methyl-transferase M. SssI according to the instruction provided by manufacturer. Briefly, 5 mg reporter constructs were incubated with 5 units of M. SssI for 1 hr in NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM MgCl2, and 1 mM dithiothreitol) supplemented with 160 mM S- adenosylmethionine at 37uC. Methylated plasmids were purified using QIAquick PCR purification kit (Qiagen, Valencia, CA) and TRAMP C1 and C3 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 5 mg/ml human recombinant insulin, 1028 mol/L 5-androstan- PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 7 Epigenetic Regulation of Nrf2 10 min, then excess formaldehyde was quenched by addition of 5M glycine. After washing twice, the cells were scraped into 2 ml cold PBS containing 16 protease inhibitor cocktail II. The cells were pelleted and then resuspended in Cell Lysis Buffer containing 16 protease inhibitor cocktail II. Nuclei were isolated after Dounce homogenization and resuspended in Nuclear Lysis Buffer containing 16protease inhibitor cocktail II. The samples were sonicated on ice using a Bioruptor sonicator (Diagenode Inc., Sparta, NJ) to shear the cross-linked DNA to an average length of 200–1000 bp and centrifuged at 12,000 rpm to remove insoluble material. The chromatin solutions were diluted 10-folds using dilution buffer, and 10 ml of each was reserved as total input control. Diluted chromatin solutions were precleared with salmon sperm DNA- protein A magnetic beads for 1 hr, and then incubated with protein A magnetic beads and antibodies specific for MBD2, Pol II, H3Ac and H3K9me3 (Millipore, Lake Placid, NY) or nonspecific IgG overnight at 4uC. The immunoprecipitated complex-magnetic beads were collected using magnetic separator and washed according to manufacturer’s instruction. The pellets were then incubated with proteinase K in ChIP Elution buffer for 2 hrs at 62uC with shaking to elute immunocomplex and reverse cross-link. The samples were incubated at 95uC for 10 min and DNA was purified according to manufacturer’s instruction. References Kotrikadze N, Alibegashvili M, Zibzibadze M, Abashidze N, Chigogidze T, et al. (2008) Activity and content of antioxidant enzymes in prostate tumors. Exp Oncol 30: 244–247. 27. Kwak MK, Egner PA, Dolan PM, Ramos-Gomez M, Groopman JD, et al. (2001) Role of Phase 2 enzyme induction in chemoprotection by dithiole- nethiones. Mutat Res 480: 305–315. 8. Bostwick DG, Alexander EE, Singh R, Shan A, Qian J, et al. (2000) Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraep- ithelial neoplasia and cancer. Cancer 89: 123–134. 28. Chan K, Lu R, Chang JC, Kan YW (1996) NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci U S A 93: 13943–13948. 9. 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Minelli A, Bellezza I, Conte C, Culig Z (2009) Oxidative stress-related aging: A role for prostate cancer? Biochim Biophys Acta 1795: 83–91. 3. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C (2007) Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 121: 2381–2386. 23. Rauhala HE, Porkka KP, Saramaki OR, Tammela TL, Visakorpi T (2008) Clusterin is epigenetically regulated in prostate cancer. Int J Cancer 123: 1601–1609. 4. Yu S, Kong AN (2007) Targeting carcinogen metabolism by dietary cancer preventive compounds. Curr Cancer Drug Targets 7: 416–424. 24. Pulukuri SM, Rao JS (2006) CpG island promoter methylation and silencing of 14-3-3sigma gene expression in LNCaP and Tramp-C1 prostate cancer cell lines is associated with methyl-CpG-binding protein MBD2. Oncogene 25: 4559–4572. 5. Nakayama M, Gonzalgo ML, Yegnasubramanian S, Lin X, De Marzo AM, et al. (2004) GSTP1 CpG island hypermethylation as a molecular biomarker for prostate cancer. 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Trends Pharmacol Sci 26: 318–326. 14. Khor TO, Huang MT, Prawan A, Liu Y, Hao X, et al. (2008) Increased susceptibility of Nrf2 knockout mice to colitis-associated colorectal cancer. Cancer Prev Res (Phila Pa) 1: 187–191. 35. McCabe MT, Davis JN, Day ML (2005) Regulation of DNA methyltransferase 1 by the pRb/E2F1 pathway. Cancer Res 65: 3624–3632. 36. Nourbakhsh M, Kalble S, Dorrie A, Hauser H, Resch K, et al. (2001) The NF- kappa b repressing factor is involved in basal repression and interleukin (IL)-1- induced activation of IL-8 transcription by binding to a conserved NF-kappa b-flanking sequence element. J Biol Chem 276: 4501–4508. ( ) 15. Xu C, Huang MT, Shen G, Yuan X, Lin W, et al. (2006) Inhibition of 7,12- dimethylbenz(a)anthracene-induced skin tumorigenesis in C57BL/6 mice by sulforaphane is mediated by nuclear factor E2-related factor 2. Cancer Res 66: 8293–8296. b-flanking sequence element. J Biol Chem 276: 4501–4508. 37. Zorn CS, Wojno KJ, McCabe MT, Kuefer R, Gschwend JE, et al. (2007) 5-aza- 29-deoxycytidine delays androgen-independent disease and improves survival in the transgenic adenocarcinoma of the mouse prostate mouse model of prostate cancer. Clin Cancer Res 13: 2136–2143. 16. Miao W, Hu L, Scrivens PJ, Batist G (2005) Transcriptional regulation of NF-E2 p45-related factor (NRF2) expression by the aryl hydrocarbon receptor- xenobiotic response element signaling pathway: direct cross-talk between phase I and II drug-metabolizing enzymes. J Biol Chem 280: 20340–20348. 38. Camoriano M, Kinney SR, Moser MT, Foster BA, Mohler JL, et al. (2008) Phenotype-specific CpG island methylation events in a murine model of prostate cancer. Cancer Res 68: 4173–4182. 17. Kwak MK, Itoh K, Yamamoto M, Kensler TW (2002) Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive agents: role of antioxidant response element-like sequences in the nrf2 promoter. Mol Cell Biol 22: 2883–2892. 39. Morey Kinney SR, Smiraglia DJ, James SR, Moser MT, Foster BA, et al. (2008) Stage-specific alterations of DNA methyltransferase expression, DNA hyper- methylation, and DNA hypomethylation during prostate cancer progression in the transgenic adenocarcinoma of mouse prostate model. Mol Cancer Res 6: 1365–1374. 18. Chromatin Immunoprecipitation (ChIP) Assay Chromoatin immunoprecipitation (ChIP) assay was carried out using Millipore’s Magna-ChIP A kit (Millipore, Lake Placid, NY) following manufacturer’s protocol. In brief, freshly prepared 18.5% formaldehyde was added to the cells (,16107 cells in 150 mm dish) at a final concentration of 1%. Cells were incubated at 37uC for Found at: doi:10.1371/journal.pone.0008579.s003 (0.08 MB PDF) Figure S4 The normalized expression of NQO1 in human prostate specimens is decreasing along with the progression of PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 January 2010 | Volume 5 | Issue 1 | e8579 8 Epigenetic Regulation of Nrf2 prostate cancer. Gene expression profiling data sets were retrieved from online database (www.oncomine.org) and analyzed for the expression of NQO1 in normal benign prostate, prostate carcinoma and metastatic prostate tumor. Found at: doi:10.1371/journal.pone.0008579.s004 (0.09 MB PDF) prostate cancer. Gene expression profiling data sets were retrieved from online database (www.oncomine.org) and analyzed for the expression of NQO1 in normal benign prostate, prostate carcinoma and metastatic prostate tumor. Found at: doi:10.1371/journal.pone.0008579.s004 (0.09 MB PDF) prostate cancer. Gene expression profiling data sets were retrieved from online database (www.oncomine.org) and analyzed for the expression of NQO1 in normal benign prostate, prostate carcinoma and metastatic prostate tumor. Table S2 Table S2 Found at: doi:10.1371/journal.pone.0008579.s006 (0.03 MB DOC) Found at: doi:10.1371/journal.pone.0008579.s006 (0.03 MB DOC) Table S1 Conceived and designed the experiments: SY TOK ANTK. Performed the experiments: SY TOK KLC WL TYW YH. Analyzed the data: SY TOK WL ANTK. Contributed reagents/materials/analysis tools: WL BAF YWK. Wrote the paper: SY TOK ANTK. Found at: doi:10.1371/journal.pone.0008579.s005 (0.03 MB DOC) References Li W, Kong AN (2009) Molecular mechanisms of Nrf2-mediated antioxidant response. Mol Carcinog 48: 91–104. 19. Schulz WA, Hatina J (2006) Epigenetics of prostate cancer: beyond DNA methylation. J Cell Mol Med 10: 100–125. 40. Lin X, Nelson WG (2003) Methyl-CpG-binding domain protein-2 mediates transcriptional repression associated with hypermethylated GSTP1 CpG islands in MCF-7 breast cancer cells. Cancer Res 63: 498–504. 20. Nelson WG, Yegnasubramanian S, Agoston AT, Bastian PJ, Lee BH, et al. (2007) Abnormal DNA methylation, epigenetics, and prostate cancer. Front Biosci 12: 4254–4266. PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8579 PLoS ONE | www.plosone.org 9

https://openalex.org/W2028214808

https://europepmc.org/articles/pmc2898798?pdf=render

English

A boosting method for maximizing the partial area under the ROC curve

BMC bioinformatics

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Abstract Background: The receiver operating characteristic (ROC) curve is a fundamental tool to assess the discriminant performance for not only a single marker but also a score function combining multiple markers. The area under the ROC curve (AUC) for a score function measures the intrinsic ability for the score function to discriminate between the controls and cases. Recently, the partial AUC (pAUC) has been paid more attention than the AUC, because a suitable range of the false positive rate can be focused according to various clinical situations. However, existing pAUC-based methods only handle a few markers and do not take nonlinear combination of markers into consideration. Results: We have developed a new statistical method that focuses on the pAUC based on a boosting technique. The markers are combined componentially for maximizing the pAUC in the boosting algorithm using natural cubic splines or decision stumps (single-level decision trees), according to the values of markers (continuous or discrete). We show that the resulting score plots are useful for understanding how each marker is associated with the outcome variable. We compare the performance of the proposed boosting method with those of other existing methods, and demonstrate the utility using real data sets. As a result, we have much better discrimination performances in the sense of the pAUC in both simulation studies and real data analysis. Conclusions: The proposed method addresses how to combine the markers after a pAUC-based filtering procedure in high dimensional setting. Hence, it provides a consistent way of analyzing data based on the pAUC from maker selection to marker combination for discrimination problems. The method can capture not only linear but also nonlinear association between the outcome variable and the markers, about which the nonlinearity is known to be necessary in general for the maximization of the pAUC. The method also puts importance on the accuracy of classification performance as well as interpretability of the association, by offering simple and smooth resultant score plots for each marker. subject being correctly judged as positive; the latter is that of an unaffected subject being improperly judged as positive. These two rates are shown to be more adequate to evaluate the classification accuracy than the odds ratio or relative risk [2]. However, the AUC has been severely criticized for inconsistency arising between statistical sig- nificance and the corresponding clinical significance when the usefulness of a new marker is evaluated [3]. Recently, Pencina et al. METHODOLOGY ARTICLE Open Access Open Access © 2010 Komori and Eguchi; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com- mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc- tion in any medium, provided the original work is properly cited. Abstract [4] propose a criterion termed integrated discriminant improvement and show the advantage over the AUC in the assessment of a new marker. In this context, the partial AUC (pAUC) is paid more attention than the AUC, especially in clinical set- tings where a low FPR or a high TPR is required [5-7]. * Correspondence: [email protected] 1 Prediction and Knowledge Discovery Research Center, The Institute of Statistical Mathematics, Midori-cho, Tachikawa, Tokyo 190-8562, Japan 2 The Institute of Statistical Mathematics and Department of Statistical Science, The Graduate University for Advanced Studies Midori-cho, Tachikawa, Tokyo 190-8562, Japan Full list of author information is available at the end of the article Methodology article A boosting method for maximizing the partial area under the ROC curve Osamu Komori*1 and Shinto Eguchi1,2 Osamu Komori*1 and Shinto Eguchi1,2 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Background However, for the sake of simplicity, we abbreviate it when the abbreviation does not cause ambiguity. Then, the ROC curve is defined as a plot of TPR against FPR when the threshold c moves on a real number line: ROC FPR TPR R F c c c ( ) = ( ) ( ) ( ) ∈ { } , , In this paper, we propose a new statistical method designed to maximize the pAUC, as an extension of AUCBoost [12], using a boosting technique and the approximate pAUC. The approximation-based method makes it possible to nonlinearly combine more than two markers, based on basis functions of natural cubic splines as well as decision stumps. The resultant score plots for each marker enable us to observe how the markers are associated with the outcome variable in a visually appar- ent way. Hence, our boosting method attaches impor- tance not only to the classification performance but also to the interpretation of the results, which is essential in clinical and medical fields. and the area under the ROC curve (AUC) is given as and the area under the ROC curve (AUC) is given as AUC TPR FPR F c d c ( ) = ( ) ( ) ∞ −∞∫ . In this setting, we consider a part of the AUC by limit- ing the value of FPR between α1 and α2, with correspond- ing thresholds c1 and c2, respectively: a a 1 1 0 2 2 0 = ( ) − ( ) ( ) = ( ) − ( ) ( ) ∫ ∫ H H F c g d F c g d x x x x x x , , (1) This paper is organized as follows. In the Methods sec- tion, we present a new boosting method for the maximi- zation of the pAUC after giving a brief review of the pAUC and the approximate pAUC. Then, we show a rela- tionship between the pAUC and the approximate pAUC in Theorem 1, which justifies the use of the approximate pAUC in the boosting algorithm. In the Results and Dis- cussion section, we compare the proposed method with other existing ones such as SDF [10], AdaBoost [15], Log- itBoost [16] and GAMBoost [17]. Background The receiver operating characteristic (ROC) curve has been widely used in various scientific fields, in situations where the evaluation of discrimination performance is of great concern for the researchers. The area under the ROC curve (AUC) is the most popular metric because it has a simple probabilistic interpretation [1] and consists of two important rates used to assess classification per- formance: the true positive rate (TPR) and the false posi- tive rate (FPR). The former is a probability of an affected * Correspondence: [email protected] 1 Prediction and Knowledge Discovery Research Center, The Institute of Statistical Mathematics, Midori-cho, Tachikawa, Tokyo 190-8562, Japan 2 The Institute of Statistical Mathematics and Department of Statistical Science, The Graduate University for Advanced Studies Midori-cho, Tachikawa, Tokyo 190-8562, Japan Full list of author information is available at the end of the article Dodd and Pepe [8] propose a regression modeling framework based on the pAUC, and apply this framework Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 2 of 17 Page 2 of 17 FPR H TPR H c F c g dx c F c g dx ( ) = ( ) − ( ) ( ) ( ) = ( ) − ( ) ( ) ∫ ∫ x x x x 0 1 , , to investigation of a relationship between a test result and the patient characteristics. Cai and Dodd [9] make some modifications to improve the efficiency of the estimation for parameters, and provide graphical tools for the model checking. In regard to classification problems, Pepe and Thompson [10] propose a method for deriving a linear combination of two markers that optimizes the AUC as well as the pAUC. However, as recognized by Pepe et al. [11], more general approaches are required when the number of markers is large. Moreover, the nonlinear combination of markers is necessary to maximize the AUC as well as the pAUC even in a simple setting such that normality is assumed to the distribution of markers [12]. However, the existing methods [10,13,14] only deal with the linear combination of markers. where H is the Heaviside function: H(z) = 1 if z ≥ 0 and 0 otherwise, and g0(x) and g1(x) are probability density functions given class 0 and class 1, respectively. Note that FPR and TPR are also dependent on the score function F. Background In addition, we demon- strate the utility of the proposed method using real data sets; one of them is breast cancer data, in which we use both clinical and genomic data. In the last section, we summarize and make concluding remarks. (1) where 0 ≤ α1 <α2 ≤ 1 (c2 <c1). In this paper, we set the val- ues to be 0 and 0.1, respectively. However, it is also worth considering to take α1>0 and choose α2- α1 to be small enough, so that we essentially maximize TPR for the fixed range of FPR. Then, the pAUC can be divided into a fan- shaped part and a rectangular part: pAUC F, , a a 1 2 ( ) TPR FPR c d c c c 1 2 = ( ) ( ) ∫ TPR FPR c d c c c 1 2 = ( ) ( ) ∫ H FPR F c g d d c c 1 2 = ( ) − ( ) ( ) ( ) x x x ≤( )≤ ∫ ∫ + ( ) − ( ) F c c c c x 1 1 2 1 2 1 TPR a a . pAUC and approximate pAUC Partial area under the ROC curve Partial area under the ROC curve Let y denote a class label for cases (y = 1) and controls (y = 0), and x be a vector of p markers as x = (x1, x2, ..., xp). Given a score function F (x) and a threshold c, we judge the subject as positive if F (x) ≥ c, and as negative if F(x) <c. The corresponding false positive rate (FPR) and true positive rate (TPR) are given as Its probabilistic interpretation is offered by Dodd [18] and Pepe [19] as pAUC F P F F c F c P F F , , , a a a a 1 2 1 0 2 0 1 2 1 1 0 ( ) = ( ) ≥ ( ) ≤ ( ) ≤ ( ) = − ( ) ( ) ≥ X X X X X ( ) ≤ ( ) ≤ ( ) c F c 2 0 1 X . pAUC F, , a a 1 2 ( ) P F F c F c P F F , a a 1 0 2 0 1 2 1 1 0 = ( ) ≥ ( ) ≤ ( ) ≤ ( ) = − ( ) ( ) ≥ X X X X X ( ) ≤ ( ) ≤ ( ) c F c 2 0 1 X . Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 3 of 17 Approximate pAUC As seen in Equation (2), the empirical pAUC is non-dif- ferentiable. Eguchi and Copas [20] use the standard nor- mal distribution function to approximate the AUC, and applies an algorithms in order to maximize the AUC. Ma and Huang [13] and Wang et al. [14] employ the similar approximation to the AUC by a sigmoid function for mul- tiple marker combination. Since there is no essential dif- ference between the two approximations, we use the standard normal distribution for the approximation of the pAUC: (b) For err I 2 t t i n i i f w i y f ( ) = ( ) − ≠ ( ) ( ) = ∑ 1 1 x (b) For , find the best weak classifier ft err I 2 t t i n i i f w i y f ( ) = ( ) − ≠ ( ) ( ) = ∑ 1 1 x t i n w i( ) = ∑ 1 , find the best weak classifier ft t i n w i( ) = ∑ 1 , find the best weak classifier ft t i n w i( ) = ∑ 1 f f t f t Ada = ( ) ∈ arg min , F err (3) (3) where Ada is a set of weak classifiers taking values 1 or -1, and I(·) is the indicator function.  where Ada is a set of weak classifiers taking values 1 or -1, and I(·) is the indicator function.  pAUC H FPR TP s s a a F F c g d d c c c c F c , , 1 2 1 2 1 1 2 ( ) = ( ) − ( ) ( ) ( ) + ≤( )≤ ∫ ∫ x x x x R c1 2 1 ( ) − ( ) a a , (c) Calculate the coefficient βt (c) Calculate the coefficient βt bt t ft t ft = − ( ) ( ) 1 2 1 log . err err (4) (4) where α1 and α2 are defined in Equation (1), and Hσ (z) is an approximation of H(z) by the standard normal distri- bution function, that is, Hσ (z) = Φ(z/σ). pAUCBoost with natural cubic splines Boosting Given samples from class 0 and class 1 , the empirical form is expressed as x i n i 0 0 1 2 : , , , = { } … x j n j 1 1 1 2 : , , , = { } … Boosting is one of the most popular method for classifica- tion in machine learning community. The main concept is that the score function F is constructed based on vari- ous simple functions, termed weak classifiers. There exist many boosting methods according to the objective func- tions [15-17,21,22]. The seminal and important one is AdaBoost, whose objective function is the exponential loss and its algorithm with the iteration number T is as follows. pAUC H F n n F x F x j i j n i I , , , a a 1 2 1 0 1 1 0 1 1 ( ) = ( ) − ( ) ( ) = ∈∑ ∑ (2) where and are empirical values that are the clos- est to α1 and α1 respectively; where and are thresholds determined by and . a1 a 2 I i c F c i = ≤ ( ) ≤ { } 2 0 1 x , c1 c2 a1 a 2 1. Start with a score function F0(xi) = 0, i = 1, 2, ..., n, 1. Start with a score function F0(xi) = 0, i = 1, 2, ..., n, 1. Start with a score function F0(xi) = 0, i = 1, 2, ..., n, where n = n0 + n1. 2. For t = 1, ..., T (a) Calculate the weights wt(i) where n = n0 + n1. 2. For t = 1, ..., T (a) Calculate the weights wt(i) w i n F y t t i i ( ) = − ( ) − ( ) { } − 1 2 1 1 exp x Approximate pAUC Similarly, the cor- responding empirical pAUC is defined as (d) Update the score function as (d) Update the score function as F F f t t t t x x x ( ) = ( ) + ( ) −1 b 3. Finally, output a final score function as pAUC H H fan s s a a F n n F F F x j i j J i I , , 1 2 1 0 1 1 0 1 ( ) = ( ) − ( ) ( ) ⎧ ⎨⎪ ⎩⎪ + ∈ ∈∑ ∑ x x j i j J F x ( ) − ( ) ( )} ∈∑ 0 rec , F f t t t T x x ( ) = ( ) =∑b 1 . Based on this iterative procedure, we propose the pAUCBoost algorithm after defining the object function. Objective function We construct a score function F(x) in an additive model for the maximization of the pAUC: where and J j c F c j fan = ≤( ) ≤ { } 2 1 1 x J j c rec = < { 1 where and J j c F c j fan = ≤( ) ≤ { } 2 1 1 x J j c rec = < { 1 . A smaller scale parameter σ implies a better F j ( )} 1 x . A smaller scale parameter σ implies a better approximation of H(z). F j ( )} 1 x F F x k k k p x ( ) = ( ) =∑ 1 , (5) (5) approximation of H(z). F F x k k k x ( ) = ( ) =∑ 1 , Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 4 of 17 where Fk(xk) is the k-th component of F(x), and the plot of Fk(xk) against xk is called a score plot that describes the association between xk and an outcome variable. The sub- set of weak classifiers for xk is given as pAUC pAUC l l a a a a F F , , , , , 1 2 1 1 2 ( ) ≡ ( ) without loss of generality. without loss of generality. pAUCBoost algorithm In this setting, the objective function we propose is given as p (c) Find the best weak classifier ft pAUC pAUC , s l s a a a a l F F F x dx k k k k p , , , , , ’’ 1 2 1 2 2 1 ( ) = ( ) − ( ) { } ∫ ∑ = (6) pAUC , s l a a F, , 1 2 ( ) f F f f t f F t t = + ( ) ( ) ∈ − arg , , max pAUCl b a a 1 1 1 (9) A F pAUCs a a l F F x dx k k k k p , , , ’’ 1 2 2 1 = ( ) − ( ) { } ∫ ∑ = (6) (d) Update the score function as (d) Update the score function as (6) F F f f t t t t t x x x ( ) = ( ) + ( ) ( ) −1 b (10) (10) where is the second derivative of Fk(xk) and λ is a smoothing parameter that controls the smoothness of F(x). It is rewritten as F x k k ’’ ( ) 3. Finally, output a final score function as F f f t t t t T x x ( ) = ( ) ( ) =∑b 1 . pAUC pAUC , s l ls a a s a a F F , , , , , 1 2 1 1 2 2 ( ) = ( ) (7) (7) Therefore, we have The dependency of the pAUCl b a a F f f t t −+ ( ) ( ) 1 1 2 , , The dependency of the pAUCl b a a F f f t t −+ ( ) ( ) 1 1 2 , , max , , max , , . , , , , s l s l l l a a a a F F F F pAUC pAUC , 1 2 1 1 2 ( ) = ( ) (8) on thresholds and makes it necessary to pick up the best pair (βt(ft), ft) at the same time in step 2.(c). Approximate pAUC The maximum value that is attained by a set of (F1, F2, ..., Fp) can take the larger value by replacing the functions with p sets of natural cubic splines. This can be proved in the same way as the result of generalized additive models [23], because the penalty term is the same. Hence, we find that the maximizer of the pAUCBoost objective function is the natural cubic spline. Fk k l k k l k k l k f x N x Z l m = ( ) = ( ) = { } , , , , , 1 … where Nk, l (xk) is a basis function of xk for representing a natural cubic spline with mk knots, and Zk, l is a standard- ization factor that makes the heights of Nk, l's uniform. Thus, Fk (xk) in Equation (5) has the following expression. pAUCBoost algorithm Using weak classifiers f 's  , we construct a score function F for the maximization of the pAUC. Note that the coefficient β cannot be determined independently of the weak classifier, so we denote it as β(f) in the following algorithm.  F x f x k k l k l k l ( ) = ( ) ∑b , , 1.Start with a score function F0(x) = 0 and set every where βl's are coefficients that are calculated in the pAUCBoost algorithm. Then, the set of weak classifiers that we use in pAUCBoost is defined as 1.Start with a score function F0(x) = 0 and set every coefficient β0(f) to be 1 or -1, so that the candidates of the initial score function have positive or negative derivatives. 1.Start with a score function F0(x) = 0 and set every coefficient β0(f) to be 1 or -1, so that the candidates of the initial score function have positive or negative derivatives. 2. For t = 1, ..., T 2. For t = 1, ..., T 2. For t = 1, ..., T F F = = k k p 1∪ . F F = = k k p 1∪ . (a) For all f 's  , calculate the values of thresh- olds and of Ft-1 + βt-1 (f)f.  c1 c2 (b) Update βt-1(f) to βt(f) with a one-step Newton- Raphson iteration. (b) Update βt-1(f) to βt(f) with a one-step Newton- Raphson iteration. (b) Update βt-1(f) to βt(f) with a one-step Newton- Raphson iteration. pAUCBoost algorithm This process is quite different from that of AdaBoost, in which βt and ft are determined independently in Equations (3) and (4). Because of the dependency and the difficulty of getting the exact solution of βt(ft), the one-step Newton- Raphson calculation is conducted in the boosting pro- c1 c2 We remark that the scale parameter σ in the definition of in Equation (6) can be fixed to 1 because of Equation (8). Hence, we redefine the objective function as pAUCs l , Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 5 of 17 version of the Bayes risk consistency for the nonconvex function in the limiting sense. cess. The one-step Newton-Raphson update is also employed in LogitBoost [16] and GAMBoost [17]. The details of the pAUCBoost algorithm are given in addi- tional file 1: Details of the pAUCBoost algorithm. Tuning procedure We also have a following corollary from Theorem 1. C ll 1 F f ti F l t We also have a following corollary from Theorem 1. Corollary 1. For any score function F, let We also have a following corollary from Theorem 1. Corollary 1. For any score function F, let Corollary 1. For any score function F, let F F gh g h x x x ( ) = ( ) + ( ), We conduct K-fold cross validation to determine the smoothing parameter λ and the iteration number T. We divide the whole data into K subsets, and calculate the following objective function. where η is a score function, and γ is a scalar. For a fixed FPR of Fγη, the TPR of Fγηbecomes a increasing function of γ if and only if η = m(Λ), where m is a strictly increasing function. pAUC pAUC CV l a a l , , , , T K F i i K i ( ) = ( ) ( ) = − ( ) ∑ 1 1 1 2 See additional file 2: Proof of Theorem 1 and Corollary 1 for the details. Note that the corollary holds for any FPR in the range of (0,1). Hence, we find that the score func- tion that moves every and all TPR's upward from the original positions, is nothing but the optimal score func- tion derived from likelihood. Results and Discussion Simulation studies We compare the performance of pAUCBoost with that of the smooth distribution-free (SDF) method proposed by [10] in a two-dimensional setting, and with those of other existing boosting methods: AdaBoost, LogitBoost and GAMBoost in a higher-dimensional setting. The simula- tion setting is similar to that of [27]. Suppose that there are four types of sample distributions for each class, y = 0 or y = 1, as shown in Figure 1. The first panel shows an ideal situation, where we see very little overlap between the two class-conditional distributions. The second situa- tion is of practical interest for disease screening, where FPR must be restricted to be as small as possible, in a case where invasive or costly diagnostic treatments will follow. A small portion of samples from class 1 (cases) is clearly distinguishable from the bulk of samples from class 0 (controls). On the other hand, in the third situation, cases are completely within the range of controls, and therefore not useful for disease screening. The fourth situation is similar to the second one, but some of the samples from cases deviate from controls clearly on both side of the dis- tribution, rather than only on one side. This situation could be worth consideration in a case where high TPR is required with very low FPR in the same way as in the sec- ond situation. pAUCBoost algorithm This fact is not derived from the Neyman-Pearson fundamental lemma [26], from which m(Λ) is proved to maximize the AUC as well as pAUC. This corollary characterizes another property of the optimal score function m Λ. where F(-i) denotes a score function that is generated by the data without i-th subset, and is calculated by the i-th subset only. The optimal parame- ters are obtained at the maximum value of pAUCcv(λ, T) in a set of grid points (λ, T). In the case where the values of the pAUCcv(λ, T) are unstable, we calculate the pAUCcv(λ, T) 10 times and take the average to determine the optimal parameters. In our subsequent discussion, we set K = 10 and explicitly demonstrate the procedure in the section regarding real data analysis. pAUCl i( ) pAUCl where F(-i) denotes a score function that is generated by Relationship between pAUC and approximate pAUC We investigate the relationship between the pAUC and the approximate pAUC, which gives a theoretical justifi- cation of the use of the approximate pAUC in the pAUC- Boost algorithm. Theorem 1. For a pair of fixed α1 and α2, let Ψ Λ g g a a s ( ) = + ( ) ( ) pAUC F m , , , 1 2 where γ is a scalar, Λ(x) = g1 (x)/g0(x) and m is a strictly increasing function. Then, Ψ(γ) is a strictly increasing function of γ, and we have sup , , lim , . F F pAUC pAUC , s g a a g a a 1 2 1 2 ( ) = ( ) = ( ) →∞Ψ Λ sup , , lim F F pAUCs g a a g 1 2 ( ) = ( ) →∞Ψ , . pAUC ,a a 1 2 = ( ) Λ See additional file 2: Proof of Theorem 1 and Corollary 1 for the details. Note that Theorem 1 holds for the approximate pAUC by a sigmoid function, so it also gives the justification of the AUC-based methods of Ma and Huang [13] and Wang et al. [14], as a special case where α1 = 0 and α2 = 1. As proved in Eguchi and Copas [20] and Mcintosh and Pepe [24], the likelihood ratio Λ(x) is the optimal score function that maximizes the AUC as well as the pAUC. In general, the Bayes risk consistency has been well discussed under an assumption of convexity for a variety of loss functions [25]. Theorem 1 suggests a weak In the simulation study, we apply pAUCBoost with a1 In the simulation study, we apply pAUCBoost with a1 = 0 and = 0.1. The training data set contains 50 con- trols and 50 cases, and the accuracy of the performance is evaluated based on 100 repetitions using test data sets of size 1000 (500 for each class). a 2 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 6 of 17 Figure 1 Illustration of simulation setting. Illustration of four different types of sample distributions for class 0 (black) and class 1 (gray). Comparison with SDF p We consider the second situation, where we assume nor- mality distributions such as and with mixing pro- portion π = 0.9, and the last situation: . That is, the conditional probability density function of a class label y given x is given by X20 0 1 ∼N , ( ) X21 0 1 1 3 1 ∼p p N N , , ( ) + − ( ) ( ) X X 40 41 0 1 100 0 4 100 ∼ ∼ N N , , , ( ) ( ) We consider the second situation, where we assume nor- mality distributions such as and X20 0 1 ∼N , ( ) with mixing pro- portion π = 0.9, and the last situation: . That is, the conditional probability density function of a class label y given x is given by X21 0 1 1 3 1 ∼p p N N , , ( ) + − ( ) ( ) X X 40 41 0 1 100 0 4 100 ∼ ∼ N N , , , ( ) ( ) F x x x ( ) = + g g 2 2 4 4, and the coefficient of x4 is estimated by SDF to be around 0 as shown in Figure 2 (a), under the condition that λ2 is fixed to 1. On the other hand, pAUCBoost considers the nonlinearity of F(x) as p y y x x x ( ) = ( )− ( )+ ( )+ Λ Λ 1 1 1 1 1 , (11) (11) (11) where Λ1(x) is the likelihood ratio: F F x F x x ( ) = + 2 2 4 4 ( ) ( ), Λ1 2 2 1 2 3 5 4 2 2 10 4 1 20 9 3 x ( ) = ( )+ − ( ) − ( ) { } ( ) ( ) ( ) = + pf p f f f f x x x x x x exp − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ ⎛ ⎝⎜ ⎞ ⎠⎟ ( ) 9 2 75 2 12 4 2 exp x as shown in Figure 2(b). The left panel shows the score plot of x2, and the right one shows that of x4. Relationship between pAUC and approximate pAUC -5 0 5 x1 0.0 0.1 0.2 0.3 0.4 0.5 density -5 0 5 x2 0.0 0.1 0.2 0.3 0.4 0.5 density -500 0 500 x3 0.000 0.001 0.002 0.003 0.004 0.005 density -0.05 0.00 0.05 x4 0 10 20 30 40 50 density x1 x2 x3 x4 Figure 1 Illustration of simulation setting. Illustration of four different types of sample distributions for class 0 (black) and class 1 (gray). and (z) is the standard normal density function. The resultant mean value (and the 95 percent confidence interval) of the pAUC based on pAUCBoost turns out to be 0.017 (0.012, 0.020), and the value of SDF to be 0.011 (0.005, 0.017). This large difference is because SDF assumes linearity of the score function of F(x) as Comparison with SDF The pAUC- Boost clearly captures the nonlinearity of F4 (x4), where one of the optimal score function in this setting is derived from Equation (12) as ⎞ ( ) 12 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 7 of 17 t k th li it i t id ti i th th d Figure 2 Comparison of SDF method and pAUCBoost method. (a) Illustration of the estimated value of pAUC by SDF method, where and 2-1/γ4 otherwise; (b) the resultant score plots by pAUCBoost. The rug plot along the bottom of each graph de- scribes the observations from class 0; the rug plot along the top of each graph describes those from class 0. -1 0 1 2 3 γ∗ 4 0.005 0.010 0.015 0.020 pAUC (a) -2 0 2 4 x2 0.0 0.5 1.0 1.5 2.0 2.5 score -0.02 0.00 0.02 x4 0.0 0.5 1.0 1.5 2.0 2.5 score (b) g g g 4 4 4 1 1 * = − ≤ ≤ if 0.005 0.010 0.015 0.020 pAUC 2 (a) -2 0 2 4 x2 0.0 0.5 1.0 1.5 2.0 2.5 score -0.02 0.00 0.02 x4 0.0 0.5 1.0 1.5 2.0 2.5 score (b) (b) Figure 2 Comparison of SDF method and pAUCBoost method. (a) Illustration of the estimated value of pAUC by SDF method, where and 2-1/γ4 otherwise; (b) the resultant score plots by pAUCBoost. The rug plot along the bottom of each graph de- scribes the observations from class 0; the rug plot along the top of each graph describes those from class 0. g g g 4 4 4 1 1 * = − ≤ ≤ if take the nonlinearity into consideration in the method itself in this way. m x x x 1 2 4 2 9 3 9 2 75 2 ( ) = + − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ + log exp . We have also confirmed that the performance of pAUCBoost is compatible with that of SDF, in a setting when linearity of the score function is reasonable. We have averages of 0.013 (0.007, 0.017) and 0.013 (0.011, 0.015 pAUCBoost and the SDF method, respectively, under the situation that x4 is also distributed as and . Comparison with SDF Λ 2 2 1 2 3 2 4 1 4 3 4 x ( ) = ( )+ − ( ) − ( ) { } ( ) × ( )+ − ( ) − ( ) { } ( pf p f f pf p f f x x x x x x ) = + ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ + − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ 1 10 9 3 9 2 1 10 9 3 9 2 2 4 exp exp . x x 2 1 2 3 2 4 1 4 3 4 = ( )+ − ( ) − ( ) { } ( ) × ( )+ − ( ) − ( ) { } ( pf p f f pf p f f x x x x x x ) Comparison with SDF X40 0 1 ∼N , ( ) X41 0 1 1 3 1 ∼p p N N , , ( ) + − ( ) ( ) Note that the ROC curve is invariant to a monotone transformation of the score function. Although a nonlinear transformation could be applied to the data in advance, it is not practical to examine all marginal distributions and decide the appropriate trans- formations in general situations. Hence, it is better to Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 8 of 17 Figure 3 Results of simulation study based on the values of the pAUC. (a) The results of the pAUC with FPR between 0 and 0.1 for training data (left panel) and test data (right panel) with only informative genes. The gray dashed lines indicate the 95% confidence bands. (b) the results of the pAUC with noninformative genes added. 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (training) pAUC AUC Ada Logit GAM 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (test) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (training) pAUC AUC Ada Logit GAM (a) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (test) (b) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (test) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (training) pAUC AUC Ada Logit GAM (a) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (test) (a) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (training) pAUC AUC Ada Logit GAM (a) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (test) (b) 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (training) pAUC AUC Ada Logit GAM 0 20 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (test) (b) Figure 3 Results of simulation study based on the values of the pAUC. Comparison with SDF (a) The results of the pAUC with FPR between 0 and 0.1 for training data 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (training) pAUC AUC Ada Logit GAM 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC (test) (b) (b) Figure 3 Results of simulation study based on the values of the pAUC. (a) The results of the pAUC with FPR between 0 and 0.1 for training data (left panel) and test data (right panel) with only informative genes. The gray dashed lines indicate the 95% confidence bands. (b) the results of the pAUC with noninformative genes added. That is, the conditional probability density function of y given x is given same way as Equation (11): The one of the optimal score function is given by That is, the conditional probability density function of y given x is given same way as Equation (11): The one of the optimal score function is given by m x 2 2 4 9 3 9 2 9 3 9 2 x x ( ) = + − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ + + − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ log exp log exp ⎭ . p y y x x x ( ) = ( )− ( )+ ( )+ Λ Λ 2 1 1 2 1 , m x 2 2 4 9 3 9 2 9 3 9 2 x x ( ) = + − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ + + − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ log exp log exp ⎭ . p y y x x x ( ) = ( )− ( )+ ( )+ Λ Λ 2 1 1 2 1 , where It is interesting to note that almost the same results are obtained by these quite different statistical methods. SDF uses the estimated values of pAUC to derive a score func- tion; on the other hand, pAUCBoost directly uses the empirical value of the approximate pAUC in the algo- rithm. Comparison with other boosting methods We focus on only the most practical situation in disease screening such as the second situation in Figure 1. Pepe et al. [27] show the utility of the use of the pAUC, in selec- = + ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ + − ⎛ ⎝⎜ ⎞ ⎠⎟ ⎧ ⎨ ⎩ ⎫ ⎬ ⎭ 1 10 9 3 9 2 1 10 9 3 9 2 2 4 exp exp . x x Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 9 of 17 Figure 4 Results of simulation study based on the marker selection. The mean values of percentage of false discovery (left panel) and 95% con- fidence bands (right panel) for each boosting method. The horizontal axis denotes the iteration number of T. The lower sides of the 95% confidence bands of AUCBoost are shown by the heavy black line to emphasize the difference from those of pAUCBoost. 0 20 40 60 80 100 T 0.0 0.2 0.4 0.6 0.8 1.0 percentage of false discovery (mean values) pAUC AUC Ada Logit GAM 0 20 40 60 80 100 T 0.0 0.2 0.4 0.6 0.8 1.0 percentage of false discovery (95 % confidence bands) pAUC AUC Ada Logit GAM 0 20 40 60 80 100 T 0.0 0.2 0.4 0.6 0.8 1.0 percentage of false discovery (95 % confidence bands) pAUC AUC Ada Logit GAM Figure 4 Results of simulation study based on the marker selection. The mean values of percentage of false discovery (left panel) and 95% con- fidence bands (right panel) for each boosting method. The horizontal axis denotes the iteration number of T. The lower sides of the 95% confidence bands of AUCBoost are shown by the heavy black line to emphasize the difference from those of pAUCBoost. tion of potential genes that are useful for discrimination between normal and cancer tissues. The point is that the value of pAUC reflects the overlap of two distributions of controls and cases, so that we can select genes that are suitable for the purpose of further investigation. For example, some overexpressed genes encourage us to investigate the corresponding protein products. However, the task of how to combine the selected genes for better discrimination is still pending. -0.8. Comparison with other boosting methods Then, we randomly replace 10 percent of samples from class 1 with those that are distributed as for each gene, so that each gene is informative in the sense of the pAUC as shown in the second situation of Figure 1. N 3 1 , ( ) for each gene, so that each gene is informative in the sense of the pAUC as shown in the second situation of Figure 1. Figure 3(a) shows plots of the average of the pAUC against iteration number T for five boosting methods. For all the boosting methods, the values of the pAUC based on the training data almost reach the upper bound values 0.1 after a number of iterations. However, the values based on the test data show clear differences. The pAUC- Boost properly detects the small difference of the two dis- tributions illustrated in the second panel in Figure 1, and shows the best performance. On the other hand, Ada- Boost, LogitBoost and GAMBoost cannot distinguish the two groups at all. Suppose we select 50 genes by a filtering procedure, which are closely correlated each other, such that 50- dimensional gene vectors for class 0 and class 1 are dis- tributed as and , respectively. The covariance matrices are designed as Σ0 = 0.95 × W0 + 0.05 × I and Σ1 = 0.95 × W1 + 0.05 × I, where W0 and W1 are 50 × 50 matrices that are sampled from Wishart distribution with the identity matrix and 10 degrees of freedom at every repetition of the simulation. The identity matrix I is added for avoiding the singularity of the covariance matrices. These matrices are normal- ized to have 1's on the diagonal part in the similar way to the simulation setting of Zhao and Yu [28], and the range of the correlations turns out to be about between 0.8 and X 0 0 ∼N 0,åå ( ) X1 1 ∼N 0,åå ( ) Suppose we select 50 genes by a filtering procedure, which are closely correlated each other, such that 50- dimensional gene vectors for class 0 and class 1 are dis- tributed as and , respectively. Comparison with other boosting methods The covariance matrices are designed as Σ0 = 0.95 × W0 + 0.05 × I and Σ1 = 0.95 × W1 + 0.05 × I, where W0 and W1 are 50 × 50 matrices that are sampled from Wishart distribution with the identity matrix and 10 degrees of freedom at every repetition of the simulation. h d dd d f d h l X 0 0 ∼N 0,åå ( ) X1 1 ∼N 0,åå ( ) Next for illustration of the gene selection of pAUC- Boost, we added some noninformative genes to the 50 genes above, i.e., genes that are assumed to be normally distributed with the same mean and the same covariance matrix: , where  is generated in the same way as above. The results in the left panel of Figure 3(b) are the almost the same as those in Figure X X 0 1 noise noise , , ∼N 0 åå ( ) Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 10 of 17 Figure 5 Score plots of clinical markers in breast cancer data. Score plots of clinical markers that describe the association between the markers and the outcome variable. The rug plot at the bottoms of each score plot shows the observations from patients with good prognosis; the rug plot for patients with distant metastases is described at the top of each score plot. Age score 30 35 40 45 50 55 0 1 2 3 4 Size score 10 20 30 40 0 1 2 3 4 Grade score 1 2 3 0 1 2 3 4 Angi score 0 1 0 1 2 3 4 ERp score 0 1 0 1 2 3 4 PRp score 0 1 0 1 2 3 4 Lymp score 0 1 0 1 2 3 4 Lymp score 0 1 0 1 2 3 4 Age score 30 35 40 45 50 55 0 1 2 3 4 Angi score 0 1 0 1 2 3 4 Angi Size score 10 20 30 40 0 1 2 3 4 ERp score 0 1 0 1 2 3 4 Grade score 1 2 3 0 1 2 3 4 PRp score 0 1 0 1 2 3 4 score PRp Figure 5 Score plots of clinical markers in breast cancer data. Score plots of clinical markers that describe the association between the markers and the outcome variable. Comparison with other boosting methods The rug plot at the bottoms of each score plot shows the observations from patients with good prognosis; the rug plot for patients with distant metastases is described at the top of each score plot. 3(a). However, we can find a clear difference between the right panels. The performances of all methods except for pAUCBoost go down on a relatively large scale. We can observe that the mean values of the pAUC by pAUC- Boost are above the upper sides of the 95% confidence bands of those by AUCBoost after around T = 20. This is mainly because of "false discovery", or selection of nonin- formative genes by chance. Figure 4 shows the resistance of pAUCBoost to false discovery. The mean values of per- centage of false discovery (the number of selected nonin- formative genes over the number of selected genes) are plotted in the left panel; the 95 percent confidence bands (gray lines) are plotted in the right panel, against the iter- ation number T, respectively. We see that the boosting methods other than pAUCBoost select noninformative genes from the early stage of boosting procedure. The dif- ference of performance of pAUCBoost from the others is 95% significant after around T = 15 as shown in the right panel. The upper side bands of the 95% confidence bands reached 1 at the very beginning of the iteration for AUC- 3(a). However, we can find a clear difference between the right panels. The performances of all methods except for pAUCBoost go down on a relatively large scale. We can observe that the mean values of the pAUC by pAUC- Boost are above the upper sides of the 95% confidence bands of those by AUCBoost after around T = 20. This is mainly because of "false discovery", or selection of nonin- formative genes by chance. Figure 4 shows the resistance of pAUCBoost to false discovery. The mean values of per- centage of false discovery (the number of selected nonin- formative genes over the number of selected genes) are plotted in the left panel; the 95 percent confidence bands (gray lines) are plotted in the right panel, against the iter- ation number T, respectively. We see that the boosting methods other than pAUCBoost select noninformative genes from the early stage of boosting procedure. Comparison with other boosting methods The dif- ference of performance of pAUCBoost from the others is 95% significant after around T = 15 as shown in the right panel. The upper side bands of the 95% confidence bands reached 1 at the very beginning of the iteration for AUC- Boost, AdaBoost, LogitBoost and GAMBoost. The boost- ing methods other than pAUCBoost clearly suffer from false discovery. pAUCBoost seems to have an advantage because it focuses on the essential part of the sample dis- tribution in the sense of the pAUC. Mainly, there are two types of weak classifiers: smooth- ing splines and decision stumps. Buhlmann and Yu [21] proposed to use smoothing splines in the L2 Boost algo- rithm, and Tutz and Binder [17] used B-splines in GAM- Boost. However, the way of fitting the weak classifiers in pAUCBoost is different from those methods. Our algo- rithm updates a score function with a basis function of a natural cubic spline for one marker in Equations (9) and (10). On the other hand, their algorithms update a score function with a set of basis functions for one marker. Hence, our resultant score functions have tendency to have simpler forms (See the illustrations of score plots in the next section), which also leads to simple interpreta- tion of the association between the markers and the out- come variable. Note that there exists a trade-off between Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 11 of 17 the simplicity and the number of markers necessary for good performance of discrimination. However, the sim- plicity depends on the number of basis functions used for the selected markers, so the more complicated associa- tion can be expressed by increasing the number of the basis functions. In AdaBoost and LogitBoost, decision stumps are used as weak classifiers [29,30]. The advantage of using deci- sion stumps is that we can apply the boosting methods independently of the scale of the marker values. Hence, Table 1: The top 30 genes ranked by the probability of gene selection, and the values of the pAUC and AUC. Comparison with other boosting methods No gene name Pg(100) pAUC AUC 1 Contig41613_RC 0.728 0.036 0.666 2 NM_006931 0.728 0.035 0.678 3 Contig40831_RC 0.706 0.037 0.672 4 Contig55574_RC 0.639 0.035 0.654 5 AB023173 0.636 0.034 0.684 6 Contig63649_RC 0.626 0.034 0.749 7 NM_018964 0.586 0.034 0.660 8 AL137615 0.571 0.033 0.655 9 NM_006201 0.541 0.032 0.664 10 NM_001710 0.520 0.032 0.638 11 AA555029_RC 0.519 0.032 0.708 12 NM_020386 0.490 0.030 0.699 13 Contig7558_RC 0.488 0.032 0.659 14 Contig51464_RC 0.482 0.030 0.668 15 NM_014246 0.474 0.032 0.613 16 NM_007359 0.463 0.032 0.696 17 NM_006148 0.450 0.029 0.661 18 NM_004163 0.442 0.029 0.729 19 Contig37562_RC 0.423 0.031 0.630 20 Contig55377_RC 0.416 0.029 0.726 21 Contig47405_RC 0.404 0.029 0.718 22 NM_012261 0.393 0.029 0.721 23 NM_014400 0.379 0.028 0.681 24 Contig44409 0.368 0.029 0.692 25 AL080059 0.364 0.027 0.801 26 Contig60864 RC 0.358 0.029 0.637 27 NM_003748 0.353 0.025 0.793 28 AL080110 0.349 0.026 0.652 29 AL122101 0.343 0.028 0.708 30 NM_018120 0.336 0.026 0.671 Table 1: The top 30 genes ranked by the probability of gene selection, and the values of the pAUC and AUC. ies. This causes poor performance in a setting where non- informative genes are mixed with informative ones. We have also confirmed that pAUCBoost with decision stumps for weak classifiers shows worse performance than that of pAUCBoost with natural cubic splines. Hence, we have to be much careful about which weak classifiers to be employed. It depends on the types of markers or the purpose of the analysis we are engaged in. Application of pAUCBoost to real data Breast cancer data The breast cancer data of van't Veer et al. [31] contains not only gene expression profiles but also clinical markers such as Age, age of patients; Size, diameter of breast can- cer; Grade, tumour grade; Angi, existence or nonexis- tence of angioinvasion; ERp, ER expression; PRp, PR expression; and Lymp, existence or nonexistence of lym- phocytic infiltrate. First, we apply AUCBoost to these clinical markers and investigate their utility. The weak classifiers we use are natural cubic splines for continuous markers (Age and Size), and decision stumps to discrete or categorical markers. Second, we apply pAUCBoost with = 0 and = 0.1 to the gene expression data after a pAUC-based filtering procedure proposed by Pepe et al. [27]. The training data set and the test data set are the same as those in [31], that is, 44 patients with good prognosis and 34 patients with distant metastases for training data, and 7 and 12 patients for test data, respec- tively. a1 a 2 y Figure 5 shows the results of the score plot generated by AUCBoost with λ = 0.01 and T = 20, which were deter- mined by a 10-fold cross validation. The Age and Size showed almost linear association with the prognosis, and a tendency to develop metastases increased as the value of Grade. The patients with negative ER and negative PR were estimated to have high risk of metastases, which are consistent with the result of van't Veer et al. [31]. The order of description of the score plots is in accordance with that of markers selected in the AUCBoost algorithm. Hence, Age has the largest contribution to the value of the AUC. The order is from the upper left panel to the lower right panel, so the second important marker is Size and the last one is Lymp. We have found that the values of the AUC for training and test data are 0.846 and 0.964, respectively. These results are comparable to those of van't Veer et al. [31] that were derived from the gene expression data: 0.882 and 0.869, respectively. This means that clinical markers themselves also have the ability to discriminate to some extent the patients with good prog- nosis from those with metastases. the simplicity and the number of markers necessary for good performance of discrimination. Application of pAUCBoost to real data Breast cancer data -1.0 -0.5 0.0 Contig41613_RC 0 2 4 6 8 score -0.2 0.0 0.2 0.4 NM_006931 0 2 4 6 8 score -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig40831_RC 0 2 4 6 8 score -0.4 -0.2 0.0 0.2 0.4 Contig55574_RC 0 2 4 6 8 score -2.0 -1.5 -1.0 -0.5 0.0 AB023173 0 2 4 6 8 score -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig63649_RC 0 2 4 6 8 score -0.4 -0.2 0.0 0.2 0.4 NM_018964 0 2 4 6 8 score -0.2 0.0 0.2 AL137615 0 2 4 6 8 score -0.2 0.0 0.2 0.4 0.6 NM_006201 0 2 4 6 8 score -1.0 -0.5 0.0 0.5 NM_001710 0 2 4 6 8 score -0.4 -0.2 0.0 0.2 0.4 0.6 AA555029_RC 0 2 4 6 8 score -1.0 -0.5 0.0 Contig41613_RC 0 2 4 6 8 score -2.0 -1.5 -1.0 -0.5 0.0 AB023173 0 2 4 6 8 score Contig41613_RC -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig63649_RC 0 2 4 6 8 score -0.2 0.0 0.2 0.4 NM_006931 0 2 4 6 8 score score NM_001710 -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig40831_RC 0 2 4 6 8 score -0.4 -0.2 0.0 0.2 0.4 NM_018964 0 2 4 6 8 score score score NM_018964 AA555029_RC -0.2 0.0 0.2 AL137615 0 2 4 6 8 score -0.4 -0.2 0.0 0.2 0.4 Contig55574_RC 0 2 4 6 8 score Figure 6 Score plots of gene expressions in breast cancer data. Score plots of the selected 11 genes that describe the association between the genes and the outcome variable. The rug plot at the bottoms of each score plot shows the observations from patients with good prognosis; the rug plot for patients with distant metastases is described at the top of each score plot. ria that the genes are two-fold regulated and that the p- values are less than 0.01 in more than 3 patients. Then, the approximately 5000 filtered genes were ordered based on their values of the pAUC with = 0 and = 0.1. In order to assess the variability of the top genes, we used the probability of gene selection proposed by Pepe et al. [27], that is a1 a 2 Among the 11 genes, Contig41613_RC showed a nonlin- ear and nonmonotonic association. Application of pAUCBoost to real data Breast cancer data However, the sim- plicity depends on the number of basis functions used for the selected markers, so the more complicated associa- tion can be expressed by increasing the number of the basis functions. In AdaBoost and LogitBoost, decision stumps are used as weak classifiers [29,30]. The advantage of using deci- sion stumps is that we can apply the boosting methods independently of the scale of the marker values. Hence, the decision stump-based method is resistant to outliers, which often occur in real data. However, it easily suffers from false discovery, as clearly shown in simulation stud- Next, we analyze the gene expression data as follows. The informative genes were selected, in the same way as [31], from the total of 25000 genes according to the crite- Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 12 of 17 Figure 6 Score plots of gene expressions in breast cancer data. Score plots of the selected 11 genes that describe the association between the genes and the outcome variable. The rug plot at the bottoms of each score plot shows the observations from patients with good prognosis; the rug plot for patients with distant metastases is described at the top of each score plot. Application of pAUCBoost to real data Breast cancer data That is, the gene expression of the patients with metastases had large vari- ance as shown by the rug plot, compared with that of patients with good prognosis, which had a tendency to take small absolute values and concentrate around the origin. The nonlinearity of the associations can be cap- tured by pAUCBoost in this way. The values of tuning parameter λ and T were determined to be 10-6 and 65 by 10-cross validation, as described in the left panel in Fig- ure 7. The value of A is very small, and it seems to be ignorable. However, since the value of A has an implicit role to control the accuracy of approximation of the pAUC as seen Equations (7) and (8), it should not be set to 0. The right panel in Figure 7 shows the pAUC for training (solid) and test (dashed) data, as a function of T with λ = 10-6. We saw that both of the values for training and test data are more than 3 times larger than those of van't Veer et al. [31]: 0.025 and 0.0084, respectively. Finally, we confirmed that the nonlinearity of score func- ria that the genes are two-fold regulated and that the p- values are less than 0.01 in more than 3 patients. Then, the approximately 5000 filtered genes were ordered based on their values of the pAUC with = 0 and = 0.1. In order to assess the variability of the top genes, we used the probability of gene selection proposed by Pepe et al. [27], that is a1 a 2 (13) P k P g k g ( ) = ( ) gene ranked the top in , (13) where k was set to 100 in this analysis and this probability was calculated by 1000 bootstrap resampling replications. Table 1 shows the results of the top 30 genes ranked by Pg (100), along with the values of pAUC and AUC calculated from the original data. We picked up significant genes with Pg (100) > 0.5, and applied pAUCBoost to the 11 genes. The score plots in Figure 6 describe the nonlinear association between gene expressions and the prognosis. where k was set to 100 in this analysis and this probability was calculated by 1000 bootstrap resampling replications. Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 13 of 17 Figure 7 Results of the pAUC. Application of pAUCBoost to real data Breast cancer data The results of 10-fold cross validation with different values of smoothing parameter λ and iteration number T (left pan- el); the results of the values of pAUC for training data (solid) and test data (dashed) by pAUCBoost, as a function of T with λ = 10-6 (right panel). 0 20 40 60 80 T 0.04 0.05 0.06 0.07 0.08 pAUCcv λ= 1e-006 λ= 1e-005 λ= 1e-004 λ= 1e-003 λ= 1e-002 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC training test -1.0 -0.5 0.0 Contig41613_RC 0 1 2 3 score -0.2 0.0 0.2 0.4 NM_006931 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig40831_RC 0 1 2 3 score -0.4 -0.2 0.0 0.2 0.4 Contig55574_RC 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 AB023173 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig63649_RC 0 1 2 3 score -0.4 -0.2 0.0 0.2 0.4 NM_018964 0 1 2 3 score -0.2 0.0 0.2 AL137615 0 1 2 3 score -0.2 0.0 0.2 0.4 0.6 NM_006201 0 1 2 3 score -1.0 -0.5 0.0 0.5 NM_001710 0 1 2 3 score -0.4 -0.2 0.0 0.2 0.4 0.6 AA555029_RC 0 1 2 3 score Figure 7 Results of the pAUC. The results of 10-fold cross validation with different values of smoothing parameter λ and iteration number T (left pan- el); the results of the values of pAUC for training data (solid) and test data (dashed) by pAUCBoost, as a function of T with λ = 10-6 (right panel). 0 20 40 60 80 T 0.04 0.05 0.06 0.07 0.08 pAUCcv λ= 1e-006 λ= 1e-005 λ= 1e-004 λ= 1e-003 λ= 1e-002 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC training test 0 20 40 60 80 T 0.04 0.05 0.06 0.07 0.08 pAUCcv λ= 1e-006 λ= 1e-005 λ= 1e-004 λ= 1e-003 λ= 1e-002 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC training test Figure 7 Results of the pAUC. The results of 10-fold cross validation with different values of smoothing parameter λ and iteration number T (left pan- el); the results of the values of pAUC for training data (solid) and test data (dashed) by pAUCBoost, as a function of T with λ = 10-6 (right panel). Application of pAUCBoost to real data Breast cancer data -0.2 0.0 0.2 0.4 0.6 NM_006201 0 1 2 3 score -1.0 -0.5 0.0 0.5 NM_001710 0 1 2 3 score Figure 8 Linear score plots of gene expressions in breast cancer data. Score plots of the selected 11 genes generated by pAUCBoost using only linear weak classifiers. -1.0 -0.5 0.0 Contig41613_RC 0 1 2 3 score -0.2 0.0 0.2 0.4 NM_006931 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig40831_RC 0 1 2 3 score -0.4 -0.2 0.0 0.2 0.4 Contig55574_RC 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 AB023173 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig63649_RC 0 1 2 3 score -0.4 -0.2 0.0 0.2 0.4 NM_018964 0 1 2 3 score -0.2 0.0 0.2 AL137615 0 1 2 3 score -0.2 0.0 0.2 0.4 0.6 NM_006201 0 1 2 3 score -1.0 -0.5 0.0 0.5 NM_001710 0 1 2 3 score -0.4 -0.2 0.0 0.2 0.4 0.6 AA555029_RC 0 1 2 3 score -1.0 -0.5 0.0 Contig41613_RC 0 1 2 3 score -0.2 0.0 0.2 0.4 NM_006931 0 1 2 3 score -1.0 -0.5 0.0 Contig41613_RC 0 1 2 3 score -0.2 0.0 0.2 0.4 0.6 NM_006201 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 AB023173 0 1 2 3 score -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig63649_RC 0 1 2 3 score NM_001710 NM_006931 Contig63649_RC Contig63649_RC -2.0 -1.5 -1.0 -0.5 0.0 0.5 Contig40831_RC 0 1 2 3 score AA555029_RC Figure 8 Linear score plots of gene expressions in breast cancer data. Score plots of the selected 11 genes generated by pAUCBoost using only linear weak classifiers. Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 14 of 17 Figure 9 Comparison between linear and nonlinear score functions. Comparison based on the values of the pAUC between linear and nolinear score functions generated by pAUCBoost. 0 20 40 60 80 100 T 0.00 0.02 0.04 0.06 0.08 0.10 pAUC training test training (linear) test (linear) 0.08 0.04 0.06 pAUC 0.06 T Figure 9 Comparison between linear and nonlinear score functions. Comparison based on the values of the pAUC between linear and nolinear score functions generated by pAUCBoost. tion F as shown in Figure 6 played an important role for the classification performance. Ovarian cancer data This dataset was analyzed by Pepe et al. [27] for illustra- tion of their pAUC-based filtering procedure in Equation (13). It consists of 1536 genes spotted on the glass arrays, and is available from the website of a textbook by Pepe [19]. It includes 23 controls with normal ovarian tissues and 30 cases with ovarian cancers. We divided the whole data into training data and test data in the ratio of 2 to 1. That is, the first 15 controls and 20 cases in the original data are used for training data; the others are for test data. Application of pAUCBoost to real data Breast cancer data See the score plots that are generated by only linear basis functions of natural cubic splines in Figure 8, and resultant values of the pAUC in Figure 9. Both the values of the pAUC based on training data and those on test data changed for the worse. The results of other boosting methods, and the results for less stringent bounds on the values of are presented in additional file 3: Supplementary results of breast cancer data analysis. a 2 Using the training data only, we ranked the genes accord- ing to the value of the pAUC with = 0 and = 0.1, and assessed the variability as in the breast cancer analy- sis above. Then, we picked up 20 genes that satisfy Pg(100) > 0.9 in the same way as Pepe et al. [27]. We found that there are 12 common genes to theirs, including g1483 that ranked best in their analysis. For these 20 fil- tered genes, we applied pAUCBoost and had the resul- tant score plots in Figure 10. As seen is Figure 10, the pAUCBoost selected 11 genes and attained the maximum value of the pAUC (0.1). Finally, we assessed the classifi- cation performance based on the independent test data, and had a high value of the pAUC (0.08). The classifica- tion is relatively easy, so the results of other boosting methods also reached the same values of the pAUC based on the independent test data. a1 a 2 Leukemia data The third data we analyzed is leukemia data [32]. It con- tains 38 training samples and 34 test samples with 7129 genes for acute myeloid leukemia (AML) and acute lym- Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 15 of 17 Figure 10 Score plots of gene expressions in ovarian cancer data. Score plots of the selected 11 genes by pAUCBoost based on ovarian cancer data. The rug plot at the bottoms of each score plot shows the observations from normal controls; the rug plot for ovarian cancer cases is described at the top of each score plot. Leukemia data We repeated the same proce- dure as the previous two analyses above using the 8 filtered genes that satisfy Pg(100) > 0.9. We achieved the perfect classification performance regarding both train- ing and test data sets, and had the score plots in Figure 11. The results of other boosting methods produced simi- lar but a little worse values of the pAUC. That is, the val- ues are more than 0.08 but less than 0.1 on the basis of test data. phoblastic leukemia (ALL). We repeated the same proce- dure as the previous two analyses above using the 8 filtered genes that satisfy Pg(100) > 0.9. We achieved the perfect classification performance regarding both train- ing and test data sets, and had the score plots in Figure 11. The results of other boosting methods produced simi- lar but a little worse values of the pAUC. That is, the val- ues are more than 0.08 but less than 0.1 on the basis of test data. able, as shown in real data analysis. Natural cubic splines that give the maximum of the pAUCBoost objective func- tion are used for markers taking continuous values. In addition, using decision stumps for markers that take dis- crete or categorical values the proposed method enables us to treat various kinds of markers together. We have also provided a consistent way to analyze gene expression data in the sense of the pAUC, as shown in the analysis of the breast cancer data, ovarian cancer data and leukemia data. The pAUC is shown to be useful by Pepe et al. [27] for selection of informative genes, some of which are overexpressed or underexpressed in cancer tis- sues. However, how to combine the selected genes and how to discriminate the cancer tissues from normal tis- sues, have not been addressed. We nonlinearly combined the genes ranked by the pAUC in order to produce a score function, by which the classification of controls and cases is done. Interestingly, we have found 4 genes in common with the 70 genes of van't Veer et al. [31]: Contig63649_RC, AA555029_RC, Contig40831_RC, Leukemia data 0.5 1.0 1.5 2.0 2.5 3.0 g1441 0 2 4 6 8 10 12 score 0.5 1.0 1.5 2.0 2.5 g1117 0 2 4 6 8 10 12 score 1 2 3 4 g9 0 2 4 6 8 10 12 score 0.8 1.0 1.2 1.4 1.6 g1238 0 2 4 6 8 10 12 score 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 g1483 0 2 4 6 8 10 12 score 0.8 1.0 1.2 1.4 1.6 1.8 2.0 g1409 0 2 4 6 8 10 12 score 0.0 0.5 1.0 1.5 2.0 2.5 g1491 0 2 4 6 8 10 12 score 0.6 0.8 1.0 1.2 1.4 1.6 1.8 g49 0 2 4 6 8 10 12 score 0.8 1.0 1.2 1.4 1.6 g1019 0 2 4 6 8 10 12 score 0.6 0.8 1.0 1.2 1.4 g176 0 2 4 6 8 10 12 score 1.0 1.5 2.0 g1277 0 2 4 6 8 10 12 score 0.5 1.0 1.5 2.0 2.5 3.0 g1441 0 2 4 6 8 10 12 score 0.6 0.8 1.0 1.2 1.4 g176 0 2 4 6 8 10 12 score 0.8 1.0 1.2 1.4 1.6 g1238 0 2 4 6 8 10 12 score 0.0 0.5 1.0 1.5 2.0 2.5 g1491 0 2 4 6 8 10 12 score 0.5 1.0 1.5 2.0 2.5 g1117 0 2 4 6 8 10 12 score 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 g1483 0 2 4 6 8 10 12 score 1.0 1.5 2.0 g1277 0 2 4 6 8 10 12 score 0.6 0.8 1.0 1.2 1.4 1.6 1.8 g49 0 2 4 6 8 10 12 score g49 1 2 3 4 g9 0 2 4 6 8 10 12 score 0.8 1.0 1.2 1.4 1.6 g1019 0 2 4 6 8 10 12 score 0.8 1.0 1.2 1.4 1.6 1.8 2.0 g1409 0 2 4 6 8 10 12 score score g9 Figure 10 Score plots of gene expressions in ovarian cancer data. Score plots of the selected 11 genes by pAUCBoost based on ovarian cancer data. The rug plot at the bottoms of each score plot shows the observations from normal controls; the rug plot for ovarian cancer cases is described at the top of each score plot. phoblastic leukemia (ALL). Acknowledgements The authors would like to express acknowledgement to Professor John Copas who kindly gave us some useful comments and suggestions to this paper. We also note that this study is supported by the Program for Promotion of Funda- mental Studies in Health Sciences of the National Institute of Biomedical Inno- vation (NIBIO). Authors' contributions OK carried out the simulation study and the real data analysis. OK and SE are responsible for the algorithm of proposed method, the proof of Theorem 1 and Corollary 1 in Additional File 2. They drafted the manuscript and approved the final manuscript. Conclusions We have developed the pAUCBoost algorithm to maxi- mize the pAUC based on the approximate pAUC in the additive model. The use of the approximate pAUC is jus- tified by showing a relationship with the pAUC in Theorem 1. A resultant score function is decomposed component- wise into functions that are useful for understanding the associations between each marker and the outcome vari- Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Page 16 of 17 -5 -4 -3 -2 -1 0 1 X95735.at 0 2 4 6 8 10 12 score Figure 11 Score plots of gene expressions in leukemia data. Score plots of the selected 5 genes by pAUCBoost based on leukemia data. The rug plot at the bottoms of each score plot shows the observations from ALL; the rug plot for AML is described at the top of each score plot. -5 -4 -3 -2 -1 0 1 X95735.at 0 2 4 6 8 10 12 score -1 0 1 2 M27891.at 0 2 4 6 8 10 12 score 0 1 2 3 4 M31166.at 0 2 4 6 8 10 12 score -1 0 1 2 M23197.at 0 2 4 6 8 10 12 score -1 0 1 2 Y12670.at 0 2 4 6 8 10 12 score -1 0 1 2 Y12670.at 0 2 4 6 8 10 12 score 0 1 2 3 4 M31166.at 0 2 4 6 8 10 12 score score score Y12670.at M31166.at X95735.at -1 0 1 2 M27891.at 0 2 4 6 8 10 12 score -1 0 1 2 M23197.at 0 2 4 6 8 10 12 score score score score M23197.at Figure 11 Score plots of gene expressions in leukemia data. Score plots of the selected 5 genes by pAUCBoost based on leukemia data. The rug plot at the bottoms of each score plot shows the observations from ALL; the rug plot for AML is described at the top of each score plot. NM_L006931. 6 genes among the selected 11 genes are related to protein coding. We also applied pAUCBoost to the 70 genes for comparison with the result from the 11 genes. We found that it yielded a poor result, especially about the value of pAUC for test data. Hence, pAUC- Boost with FPR restricted to be small should be applied to the genes or markers that have gone through a pAUC- based filtering procedure beforehand. Conclusions In the usual analy- sis setting, in which markers do not have especially high values of the pAUC, AUCBoost is preferable because of the stable performance due to the comprehensive infor- mation it can take into the algorithm. NM_L006931. 6 genes among the selected 11 genes are related to protein coding. We also applied pAUCBoost to the 70 genes for comparison with the result from the 11 genes. We found that it yielded a poor result, especially about the value of pAUC for test data. Hence, pAUC- Boost with FPR restricted to be small should be applied to the genes or markers that have gone through a pAUC- based filtering procedure beforehand. In the usual analy- sis setting, in which markers do not have especially high values of the pAUC, AUCBoost is preferable because of the stable performance due to the comprehensive infor- mation it can take into the algorithm. Additional file 3 Supplementary results of breast cancer data analy- sis. describes the supplementary results of breast cancer data, where the range of FPR is more relaxed. Additional file 3 Supplementary results of breast cancer data analy- sis. describes the supplementary results of breast cancer data, where the range of FPR is more relaxed. References 27. Pepe MS, Longton G, Anderson GL, Schummer M: Selecting differentially expressed genes from microarray experiments. Biometrics 2003, 59:133-142. 1. 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Circulation 2007, 115:928-935. 31. van't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AAM, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH: Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002, 415:530-536. 4. Pencina MJ, D'Agostino RB Sr, D'Agostino RB Jr, Vasan RS: Evaluating the added predictive ability of a new marker: From area under the ROC curve to reclassification and beyond. Statistics in Medicine 2008, 27:157-172. 4. Pencina MJ, D'Agostino RB Sr, D'Agostino RB Jr, Vasan RS: Evaluating the added predictive ability of a new marker: From area under the ROC curve to reclassification and beyond. Statistics in Medicine 2008, 27:157-172. 5. Baker SG: The central role of receiver operating characteristic (ROC) curves in evaluating tests for the early detection of cancer. Journal of the National Cancer Institute 2003, 95:511-515. 5. Baker SG: The central role of receiver operating characteristic (ROC) curves in evaluating tests for the early detection of cancer. Journal of the National Cancer Institute 2003, 95:511-515. 32. Golub TT, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, Coller H, Loh ML, Downing JR, Caligiuri MA, Bloomfield CD, Lander ES: Molecular classification of cancer: Class discovery and class prediction by gene expression monitoring. Science 1999, 286:531-537. 6. Author Details 1Prediction and Knowledge Discovery Research Center, The Institute of Statistical Mathematics, Midori-cho, Tachikawa, Tokyo 190-8562, Japan and 2The Institute of Statistical Mathematics and Department of Statistical Science, The Graduate University for Advanced Studies Midori-cho, Tachikawa, Tokyo 190-8562, Japan Additional file 1 Details of the pAUCBoost algorithm. gives the details of the pAUCBoost algorithm. Additional file 1 Details of the pAUCBoost algorithm. gives the details of the pAUCBoost algorithm. Additional file 2 Proof of Theorem 1 and Corollary 1. contains the details of the proof of Theorem 1 and Corollary 1. Additional file 2 Proof of Theorem 1 and Corollary 1. contains the details of the proof of Theorem 1 and Corollary 1. Page 17 of 17 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 Komori and Eguchi BMC Bioinformatics 2010, 11:314 http://www.biomedcentral.com/1471-2105/11/314 References Qi Y, Joseph ZB, Seetharaman JK: Evaluation of different biological data and computational classification methods for use in protein interaction prediction. Proteins: Structure, Function, and Bioinformatics 2006, 63:490-500. doi: 10.1186/1471-2105-11-314 Cite this article as: Komori and Eguchi, A boosting method for maximizing the partial area under the ROC curve BMC Bioinformatics 2010, 11:314 7. Hadjiiski L, Sahiner B, Chan HP, Petrick N, Helvie M: Classification of malignant and benign masses based on hybrid ART2LDA approach. IEEE Transactions on Medical Imaging 1999, 18:1178-1187. 8. Dodd LE, Pepe MS: Partial AUC estimation and regression. Biometrics 2003, 59:614-623. 9. Cai T, Dodd LE: Regression analysis for the partial area under the ROC curve. Statistica Sinica 2008, 18:817-836. 10. Pepe MS, Thompson ML: Combining diagnostic test results to increase accuracy. Biostatistics 2000, 1:123-140. 11. Pepe MS, Cai T, Longton G: Combining predictors for classification using the area under the Receiver Operating Characteristic curve. Biometrics 2006, 62:221-229. 12. Komori O: A boosting method for maximization of the area under the ROC curve. Annals of the Institute of Statistical Mathematics 2009. 13. Ma S, Huang J: Regularized ROC method for disease classification and biomarker selection with microarray data. Bioinformatics 2005, 21:4356-4362. 14. 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Eguchi S, Copas J: A class of logistic-type discriminant functions. Biometrika 2002, 89:1-22. 21. Bühlmann P, Yu B: Boosting with the L2 loss: regression and classification. Journal of the American Statistical Association 2003, 98:324-339. 22. Murata N, Takenouchi T, Kanamori T, Eguchi S: Information geometry of U-boost and Bregman divergence. Neural Computation 2004, 16:1437-1481. 23. Hastie T, Tibshirani R, (Eds): Generalized Additive Models. Chapman & Hall; 1990. 24. Received: 24 February 2010 Accepted: 10 June 2010 Published: 10 June 2010 This article is available from: http://www.biomedcentral.com/1471-2105/11/314 © 2010 Komori and Eguchi; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and re BMC Bioinformatics 2010, 11:314 Received: 24 February 2010 Accepted: 10 June 2010 Published: 10 June 2010 This article is available from: http://www.biomedcentral.com/1471-2105/11/314 © 2010 Komori and Eguchi; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and re BMC Bioinformatics 2010, 11:314 26. Neyman J, Pearson ES: On the problem of the most efficient tests of statistical hypotheses. Philosophical Transaction of the Royal Society of London. Series A 1933, 231:289-337. References McIntosh MW, Pepe MS: Combining several screening tests: Optimality of the risk score. Biometrics 2002, 58:657-664. 25. 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https://openalex.org/W3134448299

https://link.springer.com/content/pdf/10.1007/s10798-021-09659-5.pdf

English

Teaching digital models: secondary technology teachers’ experiences

International journal of technology and design education

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International Journal of Technology and Design Education (2022) 32:1755–1775 https://doi.org/10.1007/s10798-021-09659-5 International Journal of Technology and Design Education (2022) 32:1755–1775 https://doi.org/10.1007/s10798-021-09659-5 Abstract In secondary technology education, models of artifacts, systems and processes, visualized and simulated through digital tools (digital models) are a relatively new element. Technol- ogy teachers teach digital models to meet syllabus criteria of digital competence, applica- ble to for instance problem solving and documentation using digital tools. However, there is a lack of knowledge concerning how teachers use digital models in their teaching, what their intentions are, and what content they choose. It is known, though, that teachers’ expe- riences influence the teaching. Therefore, the aim of this study is to investigate teachers’ experiences of teaching digital models in compulsory school, to contribute to more knowl- edge of teaching in this area. This study takes a phenomenological lifeworld approach, and 12 semi-structured interviews with lower secondary technology teachers form the empiri- cal data. The data were analyzed thematically and the results are four themes of experienc- ing the teaching of digital models, indicating that technology teachers teach with differ- ent aims and purposes; Enhancing and integrating other subjects, Visualizing technology to the pupils, Enabling digital modelling, and Preparing pupils for the future. Further, the results also indicate that the content and methods of teaching differ and that teachers did not experience digital models as one single idea but as an amalgam of multiple ideas. These findings can be used as a basis for further research and development of teaching con- cerning digital models. Keywords  Digital models · Technology education · Phenomenology · Thematic analysis · Experience Teaching digital models: secondary technology teachers’ experiences Helen Brink1   · Nina Kilbrink1   · Niklas Gericke1 Accepted: 11 February 2021 / Published online: 2 March 2021 © The Author(s) 2021 * Helen Brink [email protected] 1 Karlstad University, Karlstad, Sweden Introduction Technology education in lower secondary school takes many different forms in different countries and the syllabuses also differ. However, a common aspect of technology educa- tion in several counties is the use of models, both as a pedagogical tool and as a teach- ing content, for instance in New Zealand, Ireland, and Sweden (Ministry of New Zealand 2017; Department of education & skills, 2017; Skolverket 2011; Nia and De Vries 2016). 56789) 3 23451 H. Brink et al. 1756 Models in technology education are used to visualize technological systems, objects, and artifacts. Models are also used in design processes, when pupils are solving different prob- lems. Today, more and more teachers include digital models (models visualized on a digi- tal screen) in their teaching to meet the syllabus criteria concerning digital competence (Skolverket 2011). In this teaching the use of digital models can be divided into teaching in technology where technology is developed and sometimes teaching about technology where a digital model can be used for assessing or examine technology (Kilbrink 2013; Svensson 2011). The concept teaching digital models should be understood as teaching in and/or about digital models. Teaching digital models also includes pupils designing and modelling using digital tools, i.e. digital modelling. When introducing digital technologies to a content, it will influence both teaching and content. As digital technologies are intro- duced and evolve in technology education, the teachers’ knowledge of these technologies and how to teach them also needs to be developed (Mishra and Koehler 2006). This is a major change in the teaching profession. The former analogue technologies were more sta- ble (Mishra and Koehler 2006). Research shows that it is difficult for teachers to learn not only hardware and software, but also to learn how to use digital technologies in different situations and in an educational manner (Koehler and Mishra 2005). However, very little is known about how technology teachers teach digital models in different situations. f Research in Swedish schools shows that technology teachers are free to design their technology education based on personal and local references, but at the same time, many teachers express that they are uncertain in their professional practice and also uncertain concerning how they should interpret the syllabus (Bjurulf 2008; Hartell 2015). Further, technology education has no national test to support equivalent teaching in Sweden. Introduction As a result of that, technology education takes many different forms in different schools, and it can also differ a great deal in the same school (Skolinspektionen 2014). There is also a recent report (Skolinspektionen 2019), showing that the use of digital tools in lower sec- ondary technology education, which is compulsory, differs between schools. Some of the inspected schools use digital tools in the designated areas mentioned in the technology syl- labus and some schools inspected have a more developed pedagogy considering the use of digital tools, which might mean for instance that pupils can work with different tools depending on individual needs. Further findings noted by the school inspectorate are that different schools have different conditions for collaborating on planning lessons where digital tools are used. Some schools have well planned conferences while the technology teachers in other schools rarely meet at all. In addition, a great many areas in technology education are unreflected, in the sense that the pupils are doing things without reflecting upon either the content or their learning (Skolinspektionen 2014). It is important to find out if these conditions, mentioned above, affect the teaching of digital models. f Further, learning is always about something (Marton 2015) and teaching is one way (but not the only way) to organize work in the classroom in order to make learning possible. Something is to be learned in some particular way. Teaching consists of content, something meant to be learned, and methods, ways for pupils to achieve the content. It is known that teachers’ experiences of the subject matter and teaching content influence teaching (Mar- ton and Tsui 2004; Mishra and Koehler 2006). That can be explained by the fact that differ- ent persons experience a phenomenon in the lifeworld (the world where a person acts and interacts with different subjects and objects) in different ways depending on e.g. previous experiences and knowledge (Bengtsson 2013). Finding out more about teachers’ experi- ences of teaching digital models can help establish a common knowledge base and a com- mon language among the teachers, and knowledge in this area needs to be enhanced. With more knowledge of teachers’ experiences, content and teaching—that is, the what-aspect Teaching digital models: secondary technology teachers’… 1757 and how-aspect of teaching digital models—can be discussed. Introduction Since digital models, visual- ized through digital tools, is a relatively new teaching area, is it important to understand more about teachers’ experiences of their teaching and how they interpret the syllabus. Results from this study can help develop the technology teacher profession and contribute to discussions of what should constitute the core content of technology education, as well as how teaching this specific area can be done. Aim and research question The aim of this study is to describe lower secondary technology teachers’ experiences of teaching digital models using digital tools. Very little is known about this teaching con- ducted in compulsory schools today. With this aim as the point of departure, the following research question was formulated: q How do lower secondary technology teachers experience the teaching of digital mode How do lower secondary technology teachers experience the teaching of digital models? The studied phenomenon is teaching digital models in lower secondary technology education, and the results presented in this article are descriptive themes of teachers’ experiences. The studied phenomenon is teaching digital models in lower secondary technology education, and the results presented in this article are descriptive themes of teachers’ experiences. Background In this article, teaching digital models should be understood as sometimes teaching in and sometimes teaching about digital models, and moreover, sometimes simultaneous teaching in and about digital models. A model is a description or a representation of something, something real and con- crete or something not yet realized, a mental idea (Gilbert et al. 2000). A model is not an exact replica of the original, but hold compromises and simplifications depending on the purpose and the use of the model. Technicians and engineers can use models to develop artefacts or processes, and the model can represent some special feature or function. Models in technology are for instance used for visual support when solving problems, to predict actions or events, to make decisions, and to communicate (Nor- ström 2013; Elmer and Davies 2000; Seery 2017). This communication can be useful both when developing products and artifact but also in relation to already made objects. Only describing a product or a technical solution in words is often not enough; there is a need for sketches, drawings, or models to understand and mediate technology (Sjöberg 2013). Models are in that sense a language, and a way to conceive technological solu- tions (Davies and Elmer 2001). This language is specific for technology and constitutes part of the core of technological knowledge. In the context of technology education, models can be used in a similar manner and have a more descriptive approach.f A model can be a representation used in many different modalities; concrete, ver- bal, mathematical, visual, and gestural (Gilbert et al. 2000). Several of these modalities can also be represented digitally, through a digital tool, visualized on a digital screen, as a digital model. That is how digital models should be understood in this study, as represented through a digital tool, visualized on a digital screen. How then, is teaching digital models conducted, according to technology teachers? What are the aims and pur- poses, what content is chosen, and in what activities are the pupils supposed to engage? These questions, why teach something?, what should be taught? and how can teaching be planned and enacted?, are central for teaching irrespective of subject (Sjøberg 2010; Uljens 1997), and thereby relevant also for this study, which aims to describe teachers’ experiences of teaching digital models. Further, technology education is also characterized by an interest in technology. Background There are studies pointing to teachers’ beliefs about and understandings of technological knowledge as important for technology education (de Vries 2005). It is important to help pupils to acquire a conceptual basis, and teachers should ask themselves What do I mean with technological knowledge in this area? (de Vries 2005). Similarly, it is also likely that teachers’ beliefs and understandings are important for teaching digital models. To better understand the results of this study, technological knowledge and the character of the tech- nology subject will be clarified.i i Mitcham (1994) defines technological knowledge as having both practical and cognitive dimensions and he continues to describe technology as an activity, producing and using technology, activities with hand and mind. De Vries (2005) argues that teaching techno- logical knowledge should also include a normative component, to teach pupils to assess the functions of artifacts (in a school context that can be functions of a model). A model, representing something in technology education, can be seen as a bridge between theory and the lived world, and Nia and DeVries (2016) suggest that models have an epistemic function; technological knowledge can be developed by using and manipulating models. So, technology education has a dual nature (Gibson 2009); it is both practical and theoreti- cal and technological knowledge consists of a mix of both. Yet another way to describe technological knowledge, characteristic for technology education, is to distinguish between pupils learning in technology and pupils learning about technology (Kilbrink 2013; Svensson 2011). Knowledge in technology concerns handling and developing technology and knowledge about technology concerns assess- ing and examining technology. A teaching content in technology education can then be both in and about technology. Pupils engage in a technical activity while developing knowledge of a specific content. The practical activities also deepen other aspects of technology for the pupils, such as methods of technology and developments of prod- ucts and artifacts (Bjurulf 2011). Theoretical material should be used in technology 3 3 H. Brink et al. 1758 education together with more practical activities in order to achieve holistic learning (Gibson 2009) and research elsewhere also points to the importance of interweaving theoretical and practical activities in technology education (Kilbrink 2013). Techni- cal problem solving using digital models can be such an activity, both theoretical and practical, both learning in and about technology. The Swedish context The study presented in this article has been conducted in Swedish lower secondary schools. In Sweden, lower secondary school, years 7–9 for pupils aged 13–15 years, is a part of the compulsory school. The compulsory school is governed by a national and common curriculum, with syllabuses for each subject. Technology education in Sweden has changed regularly during the last decades. In 1980, technology become a subject of its own for the first time. Before 1980, it was included as part of the science subjects (Lgr 80; Lpo 94; Skolverket 2011). Technology education is, as a result of the separa- tion, a relatively young subject and lacks a distinct identity (Hagberg and Hultén 2005; Sjöberg 2013). In the current curriculum, Lgr11 (Skolverket 2011), changes in the tech- nology syllabus were introduced in relation to pupils’ documentation of their technical development. The core content for years 7–9, under the heading Working methods for developing technological solutions, includes the following phrase: “Documentation in the form of manual and digital sketches […] with physical and digital models” (Skolver- ket 2011). Different schools have developed technology education differently, and as a result of that, teaching differs a great deal. Modes of teaching can even differ in the same school, depending of which teacher is teaching (Skolinspektionen 2014, 2019). Moreover, about half of the in-service technology teachers in lower secondary school have formal education for teaching the technology subject (Skolverket 2019). From the above, it is clearly important to investigate teachers’ experiences of teaching digital models, to gain more knowledge and understanding in this area. Background Ahl- bom (2011) points to the importance in education of raising or enhancing pupils’ inter- est in technological learning. So, to achieve successful learning, teachers need to start in and with the pupils’ interest and at the same time develop that same interest. Ginestíe (2018) also points to the importance of developing pupils’ interest in technology, in relation to an understanding of how digital tools can be used in society and how digital tools can be used to solve different problems. One often used digital tool, in society and in many technological professions, is computer aided design (CAD) where three- dimensional objects (digital models) can be created. Technology teachers now use CAD in technology education, to meet the criteria of digital competence in syllabuses. Some schools in Sweden have access to a 3D-printer (Skolinspektionen 2019), which means that digital models can be printed to a physical model in plastics. A printed physical model can be helpful when it comes to supporting pupils’ understanding of the iterative cycle of product development (Novak and Wisdom 2018), where different steps in the process are repeated; idea generation, sketches, building, testing et cetera. 1 1 3 Teaching digital models: secondary technology teachers’… 1759 In addition to CAD, digital models can also be created through algorithms, or sequences of actions (Åkerfeldt et al. 2018). Algorithms can be described with text or with words, or coded with texts or blocks into a program. When describing or coding, it is called programming. Programming can thereby contain both problem solving and coding (Åkerfeldt et al. 2018). Programming has recently entered the Swedish syllabus (Skolverket 2011) with a focus on problem solving and electronical components and how they can be programmed, but also digital competence. Barak (2018) writes that technology education that uses micro control cards, such as Micro:bit or Arduino, is successful when it comes to developing pupils’ ability to design, solve problems, and be creative. But Barak (2018) also adds that education can only be successful if the pupils have a basic understanding of the content; otherwise, there is a risk that the teaching becomes unreflected by the pupils, and the learning absent. l A brief presentation of the compulsory Swedish secondary school technology educa- tion will be presented next to provide a deeper understanding of the context in which this study was conducted. Theory and method In this section, the theoretical and methodological principles used in this study are out- lined, starting with a description of the phenomenology of the lifeworld, continuing with sample and data collection and finally, concluding with descriptions of the thematic analy- sis conducted. 3 H. Brink et al. 1760 Phenomenology of the lifeworld With the phenomenological lifeworld (Bengtsson 2013) as ontological and epistemological approach, a world experienced by a person is pluralistic. It is neither material nor mental, but consists of a complex intertwining of different subjects, objects, and experiences in a regional world, where the person acts. In this regional world which includes things, people, and activity, interactions occur, creating new experiences. All this constitutes the person’s reality, and consequently different persons experience different realities (Bengtsson 2013), depending on their perspectives, positions, and previous experiences. Hence, in this study “experience” is a pertinent concept, understood as the analytical construct of the respond- ents’ knowledge, beliefs, purposes, and intentions related to teaching digital models. The concept “phenomenon” should be understood as “that which appears” (Bengtsson 2013, p. 6), meaning that something must be understood by someone. Teaching digital models is the phenomenon in this study. It has a content and can be experienced, and the experiences can be described and interpreted. Empirical studies are, according to this approach, important in order to increase knowledge of the phenomenon studied (Bengtsson 2013; Kvale and Brinkmann 2014). However, investigating the teaching of digital models is limited to one particular reality, namely teachers’ experiences, formulated in the research question. Semi-structured interviews were chosen to correspond to the ontological assump- tion and the aim of this study, and to answer the research question. The method fostered a deeper understanding of teachers’ lived experiences of teaching digital models and was considered to offer the best opportunity to make teachers’ experiences visible. The empiri- cal data were therefore collected through interviews, where the participants described the phenomenon from their point of view. Since teachers plan and design their lessons, they did during the interviews, in a reflected manner, describe teaching digital models based on their knowledge, beliefs, purposes, and intentions. 1 3 Sample and data collection Technology teachers in Sweden are not a homogenous group, and it is known that teachers design their teaching based on local and personal preferences (Bjurulf 2008; Hartell 2015). Twelve technology teachers, five males and seven females, were strategically chosen for this study (Alexandersson 1994; Kvale and Brinkmann 2014). The strategy was to inter- view teachers who say that they teach digital models to ensure that they have experiences of the phenomenon investigated. Teachers with different backgrounds were chosen to cover the wide spectrum of teachers in service, concerning gender, teaching experience, and the size of the school. They all had formal teacher education, but not all in the technology subject. The interviewed teachers have been in service between 1 and 19 years and they teach different subjects in combination with technology. Table 1 is showing information about the participants. The aim of this study is not to distinguish between different teach- ers’ teaching, but to collect different experiences and analyze them thematically. f The interviews were open in character, with a guide (see Appendix 1) for the questions (Kvale and Brinkmann 2014). The guide helped sustain focus on the phenomenon, that is teaching digital models, and it also supported the interviewees in their reflections and helped them grasp different aspects of the phenomenon. The guide included questions focused on the what-aspect and the how-aspect of the phenomenon, meaning that the inter- viewed teachers described not only what they teach and offered examples and explanations 1 3 Teaching digital models: secondary technology teachers’… 1761 Table 1   Information about the interviewed technology teachers Teacher Gender Formal education in technology subject Teaching expe- riences in year Teacher 1 Female No 1 Teacher 2 Male Yes 3 Teacher 3 Female No 7 Teacher 4 Male Yes 19 Teacher 5 Female Yes 18 Teacher 6 Female Yes 10 Teacher 7 Male Yes 8 Teacher 8 Male Yes 11 Teacher 9 Male No 5 Teacher 10 Female Yes 5 Teacher 11 Female Yes 12 Teacher 12 Female Yes 15 regarding what content they choose in this specific area, but also how they perform and enact that teaching and what activities they prepare for the pupils. These aspects gave the researchers deeper insights into the underlying structures of the interviewed teachers’ expe- riences during the analysis phase (Alexandersson 1994). Sample and data collection The interviews were designed to provide the teachers with opportunities to share their point of view, to answer the ques- tions in their own way, and to clarify and exemplify in depth. The interviews were held at the teachers’ schools, in a place decided by the teacher, in order to make the partici- pant feel comfortable and relaxed. The interviews ran between 35 and 60 min and were recorded digitally. At some of the interview occasions, the researcher was given a view of the technology education classrooms. These conversations were not recorded, but gave the researcher a deeper understanding of the teachers’ explanations. All twelve interviews were conducted by the first author, during October 2018 and September 2019, and yielded rich data which allowed for themes to be established. After nine interviews, no new themes were established, and the three latter interviews gave no further information. Therefore, the data collection ended at twelve interviews. Data analysis The interviews were transcribed in full by the first author. Transcription constitutes a trans- formation from spoken language to written, and require interpretation by the person tran- scribing (Kvale 1996). Therefore, the aim of this study guided the transcriptions to focus on what the interviewees said, on the content in their explanations. Another focus in the transcriptions has been readability. When investigating an under-researched area (as in this study) a rich description of the data set is useful (Braun and Clarke 2006), and an inductive method without pre-existing coding frames for reporting patterns or themes is also helpful (Braun and Clarke 2006). Therefore, a thematic analysis was used in this study, to report the life world experiences, meanings, and realities of the participants. The analysis was performed in two steps. In the first step, themes were extracted from the data set. In the second step, the synthesis, an in- depth analysis extracted teaching aspects within the themes. 1 3 3 H. Brink et al. 1762 In the first step of the analysis, the researchers listened to the audio files and read the transcriptions several times in parallel with the interviews. The material was studied iteratively in order for the researchers to familiarize themselves with the data. Initially, a short descriptive note was added to segments or elements from the data set associated with the phenomenon studied (teaching digital models). The notes then were organized into meaningful groups and coded. Examples of these initial codes are design, abstract, basic, interest, software, programming, and print. This coding was open, meaning that the researchers analyzing the material had no pre-determined codes. This coding pro- cess continued systematically with all twelve transcriptions, however the three latter transcriptions did not generate any new codes. The codes were organized continuously with respect to similarities and common features, and then reduced and organized into themes. The themes were finally reviewed, defined, and named in an iterative process including all three authors and constitute the results from the first analyzing step. Since the themes represent teachers’ experiences of their own aims and purposes related to the teaching of digital models, further analysis was needed to answer the research question and reach in-depth understanding.i In the second step of the analysis, the synthesis, the first author once again read the twelve transcriptions. This second reading focused on teaching content and methods of teaching within each theme. Data analysis Excerpts, words, and sentences from the interviewed teach- ers, associated with a specific theme, were labeled. Some examples of teaching content and methods of teaching found and labeled during the reading are product development, digital simulations, films, and 3D-printer. These labels were coded as teaching aspects and each theme was assigned a number of important teaching aspects. However, the transcripts show that the teaching aspects from different themes sometimes were simul- taneous described, which resulted in an overlapping of themes. Sometimes two of the themes overlapped, sometimes three.l The empirical data form four themes of experiences that reflect the interviewed teachers’ aims and purposes in teaching digital models. The themes also contain dif- ferent teaching aspects, that is teaching content and methods of teaching, related to the teachers’ aims and purposes. The resulting synthesis describes how the four themes relate to each other and outlines where aspects from different themes are used for vari- ous purposes. The results will be presented next. Enhancing and integrating other subjects The empirical data of this study show that teaching digital models in lower secondary technology education is used to enhance and integrate other subjects in school. Digi- tal models seem to be useful for integrating other subjects and are appreciated when it comes to creating common projects. Above all, some of the interviewed teachers discuss mathematics and crafts as important subjects to enhance and integrate in rela- tion to technology education. Mathematical concepts are given a major focus in teach- ing and pupils can work with concepts such as angles, measurements, and coordinate systems. Pupils also work with their spatial ability, with geometrical objects, and with different dimensions. Teacher 7 gives voice to this focus: I will work with X, Y and Z-axis. I will work with the concept volume, so I can connect it to mathematics, to lessons in mathematics. So they [pupils] see where they can use it in other subjects. […] Yes, but it is interesting that, math, I think I do it very much in technology. Trying to make them [pupils] understand why they need to learn mathematics. (Teacher 7) Craft is integrated with technology partly to create physical models of the digital models, designed by the pupils in digital environments. In the technology subject, pupils design for instance a piece of jewelry digitally in a CAD-program and can there- after print it in a 3D-printer. In crafts, the printed jewelry model is used for molding. You know, if you take it and put it into sand, and then mold so… A form, so they [pupils] can do the same jewelry in tin. (Teacher 1) Another common project, described during the interviews, is programming using a micro control card. Teacher 5 described that the aim of teaching digital models in tech- nology is to generate data for mathematical calculations. … and they can program it [Micro:bit] to measure temperature. To measure both out- and inside temperature. After that, pupils will do calculations in math. So, we thereby connect technology and math. (Teacher 5) … and they can program it [Micro:bit] to measure temperature. To measure both out- and inside temperature. After that, pupils will do calculations in math. So, we thereby connect technology and math. (Teacher 5) More similar projects and activities have been mentioned during the interviews, where digital models in technology generate data for mathematical calculations and/ or diagrams. Results The aim of this article is to describe different experiences of teaching digital models in lower secondary technology education. The results are organized to present the four themes, including the interviewed teachers’ aims and purposes in teaching digital mod- els, first. Thereafter the synthesis is presented, describing teaching aspects and relations between the themes. Technology teachers experience the teaching of digital models as the following themes: (a) Enhancing and integrating other subjects, (b) Visualizing technology to the pupils, (c) Enabling digital modelling, (d) Preparing pupils for the future. 1 3 Teaching digital models: secondary technology teachers’… 1763 Each theme is presented with excerpts selected to show examples of how teach- ers describe their teaching and to show a variety from the sample. The excerpts are referred to a specific teacher, (Teacher 1) to (Teacher 12), but should be understood as a representation of the presented experience, or the teaching aspects within the theme. However, the same teacher can have experiences referring to different themes, as an individual teacher can teach with different aims and purposes. In every excerpt, focus is on the teaching of digital models, unless otherwise stated. Visualize technology to the pupils Some teachers in this study experience that it is abstract to teach technical systems. To make technical systems more tangible and concrete, teachers expressed that they use digital models. According to some of the interviewed teachers, digital models provide an opportunity to visualize what otherwise would be difficult to show, and digital mod- els also make difficult technical systems or technical solutions more graspable. In the theme Visualize technology to the pupils, some interviewed teachers use ready-made digital models to show pupils technology that already exists. By ready-made digital models, interviewed teachers mean movies, Youtube clips, or simulations, used to repre- sent some part of the real world. The purpose of using these ready-made digital models is to bring about discussions in class of how technology is being used or what the pur- pose of a certain technology is. The aim of the lessons is also to identify advantages and disadvantages in technology and discuss them as an activity in class. One interviewed teacher says: I have some movies […]. So, from there we can discuss. Copying. […] to start a dis- cussion, to start a conversation with them [the pupils] about the benefits, dangers, we can copy almost everything soon. (Teacher 8) Films provide a good introduction to dialogues and conversations in class, according to some interviewed teachers with experiences in this theme, since questions can be asked based on what has been seen. Simulations are also mentioned as useful in teaching for dia- logues and conversations. It becomes illustrative, of how it works. I mean, if you teach technical systems and talk about the internet and things like that, to make it more distinct. It is so abstract. […] Well, then it is a clear model, of how it is in reality. How it is, seen from behind. (Teacher 3) Simulations give pupils a fast response and quick feedback on learning the specific teaching content, some interviewed teachers say. For instance, pupils can observe if the bridge under construction is going to break or hold, or if the lamp in the electric circuit is going to light up or not. In conclusion, the aim of teaching digital models according to this theme is to make complicated technical systems more graspable and concrete, with support of digital pic- tures, movies, and simulations. Enhancing and integrating other subjects One result from this study is therefore that teachers are teaching digital models as a means to enhance and integrate other subjects. Yet another result is that teaching digital models can visualize technology to the pupils, and this theme will be described in the following section. 1 3 H. Brink et al. 1764 Visualize technology to the pupils The support enhances discussions in class, and ready- made digital models or programs are often used for that. In the next part, the third theme will be described, that is teaching where pupils themselves create digital models: digital modelling. Enabling digital modelling From the empirical data collected in this study, it is clear that technology teachers can teach digital models to give pupils opportunities to learn the process of modelling. In other words, this theme is about enabling digital modelling. Teaching activities include design- ing in CAD or programming. Modelling and programming are iterative, even when the process is digital, and teachers say they want pupils to realize that the process is not linear. Pupils test, try, retry, and redo during this teaching, some interviewed teachers say. Teacher 1 3 Teaching digital models: secondary technology teachers’… 1765 7 gives an example of programming as a digital model in the following excerpt, when talk- ing about a task where pupils are programming a game: 7 gives an example of programming as a digital model in the following excerpt, when talk- ing about a task where pupils are programming a game: … when we program the games, you can say that you create a digital model of the reality, so to speak. If you are a figure catching apples falling down, you make a digi- tal image of your idea. That is a model, a digital image of it. (Teacher 7) When describing the teaching of digital modelling, some interviewed teachers say they initially start teaching the specific software used. To be able to model digitally, using digi- tal tools, pupils need to handle the software. When teaching programming, teachers give examples of how they use a simple interface, like block-programming instead of text-based programming. The reason for using block-programming is that pupils otherwise risk get- ting caught in syntaxes, even though teachers say they want to move on to text-program- ming. Electronic components or robots are also sometimes used when teaching digital modelling, for exemplifying and concretizing programming, and for pupils to work with physical objects. Some teachers said in interviews that when programming, simulations on a screen alone are not enough for learning. Concrete and physical material as different components are experienced by teachers as necessary to complement teaching, and many teachers have bought electronic components for that occasion. Teachers describe that digital models, made by pupils in CAD, are simple with basic geometrical forms, like jewelry or name-tags. From these basic objects, pupils are free to choose and design new objects to model, often based on their interest. Enabling digital modelling What is designed is not important, since the learning content is to handle the software, some interviewed teach- ers say. Pupils often need to learn more advanced functions in the software on their own. The following excerpt shows that the focus among some interviewed teachers is that the software, not the designed object, is the learning content: In one of the basic tasks I teach, pupils will design a key tab, with free design, to test the program. […] The task is to understand the program. (Teacher 2) Sometimes, according to the interviewed teachers, pupils can create a physical model from their digital original. They print the objects in a 3D-printer. The 3D-printer is also used to exemplify contemporary and quick ways to create physical objects. Well, they see a modern technique. That you can, like do a model. So. It is for show- ing how it works. (Techer 12) Well, they see a modern technique. That you can, like do a model. So. It is for show- ing how it works. (Techer 12) The theme of enabling digital modelling is experienced as associated with different teaching aspects. Interviewed teachers say that they need to start teaching the software for pupils to be able to design digitally, program digitally, or print a physical object. The itera- tive process of modelling is experienced by the pupils while modelling. The fourth and last theme identified in this study concerns teachers’ experiences of preparing pupils for the future. Preparing pupils for the future Some participating teachers in this study described during the interviews that they find it important to prepare pupils for the future, for higher education, for professions, and also for day-to-day life. Teachers say they want to present different alternatives for higher education and different possible engineering professions using digital mod- els. They also want to inspire pupils to pursue a technological interest, according to 3 H. Brink et al. 1766 the interviews. Another important aspect of teaching, according to some interviewed teachers, is that pupils need to be informed of the fact that more and more products and artefacts today are manufactured through digital drawings and digital models. Pupils also need to be informed of how to relate to programmed systems, since a great many things today consist of programmed items. These are important knowledge for being able to act as an adult, for any citizen, teachers say. the interviews. Another important aspect of teaching, according to some interviewed teachers, is that pupils need to be informed of the fact that more and more products and artefacts today are manufactured through digital drawings and digital models. Pupils also need to be informed of how to relate to programmed systems, since a great many things today consist of programmed items. These are important knowledge for being able to act as an adult, for any citizen, teachers say. Teaching digital models is described mainly in two ways; either with practical tasks using digital tools, where pupils themselves create models or programs; or with a more theoretical approach using films, pictures, or simulations, where discussions become the dominant part. In the interviews, teachers share experiences showing that concern- ing both the practical and the theoretical teaching, discussions are general. The aim of the teaching is to stimulate interest and inform pupils about different digital tools, to let pupils use digital tools and software, and to inspire them to consider pursuing tech- nical careers. The following excerpt has been chosen to illustrate how one interviewed teacher describes the teaching of digital models for preparing pupils for the future. I would like as many [pupils] as possible to attend technological education in upper secondary school. […] But much of the teaching is to show them what exists, getting them interested. (Teacher 3) Using digital models is a good choice for inspiring and stimulating interest, teachers say, because many pupils think it is fun and novel. Preparing pupils for the future Showing new technologies can give pupils better options for their choice of upper secondary school programme. In order to encourage pupils to consider pursuing technological careers, teachers tell pupils about staffing needs, and what knowledge are required to be employed. Corporations need people who can draw digitally. And, they print everything in 3D, first, before they mold these hydraulic presses. Modelling and printing. (Teacher 8) Furthermore, teachers point out during the interviews that it is important to learn digital models from a more general perspective, not only to pursue higher education. Teachers teach digital models so that pupils can interpret different kinds of models, commonly used in society. General knowledge of how digital models are being used in different professions is described when teachers in the interviews talk about teaching everyday use of technology. The examples given are common, but noteworthy, teachers express that general knowledge is important even for those pupils not choosing a future technological career, since all people need to feel comfortable around technology and understand a little about structures underlying programmed systems. They [pupils] need to know this. First of all, know little, knowledge about tech- nology, and about what is going on around the world. To understand what is hap- pening. (Teacher 2) So, technology teachers are teaching digital models and use ready-made models in teaching, as a method for inspiring pupils, to create an interest in further technology education, to encourage pupils to consider technological professions, and for more general knowledge about technology. In this theme, teaching is both practical and theo- retical, but the aim is to provide information about, show, and present modern technol- ogy, as well as to generate an interest in technology. Preparing pupils for the future 1 3 1 3 Teaching digital models: secondary technology teachers’… 1767 Table 2   Four themes of technology teachers’ experiences of teaching digital models, and corresponding teaching aspects Themes – aim of teaching digital model Teaching aspects– teaching content and/or methods Enhancing and integrating other subjects Mathematical concepts Generating data for mathematical analysis Preparations for crafts Visualizing technology to the pupils Concretize technology Basis for discussions Enabling digital modelling Iterative process Handling software Physical objects Preparing pupils for the future Technological education programmes Technological professions General technological knowledge a) Enhancing and integrating other subjects c) Enabling digital modelling b) Visualizing technology to the pupils d) Preparing pupils for the future a + c b + c c + d b + d b + c + d Fig. 1   Combinations and relations of technology teachers’ experiences of teaching digital models b) Visualizing technology to the pupils b + c + d Fig. 1   Combinations and relations of technology teachers’ experiences of teaching digital models Synthesis When teaching for integrating other sub- jects, the 3D-printer can also be used, according to interviewed teachers, so that pupils can print physical models and use them for molding. Enabling digital modelling then provides enhancing and interacting other subjects, shown in Fig. 1 as (a + c). Examples of common aspects are handling software, physical objects, mathematical concepts, generating data for mathematical analysis, and preparations for crafts, see Table 2. Teacher 11 explains a situ- ation where mathematical concepts and preparations for craft are the teaching content and handling the software is the method. But you take very simple objects, that they [pupils] have designed on their own. […] They started with a name tag. A name tag they later could do in crafts. That is why it [the name tag] has to have right dimensions. (Teacher 11) Teaching that aims for enabling digital modelling and visualizing technology to the pupils is multifaceted Fig. 1 (b + c). Interviewed teachers say that it is important for pupils to understand that modelling is an iterative process which moves back and forth between generating ideas, producing sketches, modelling, and testing. Pupils are encouraged to test and redo on their own when results are not as expected. Teachers use different software and programs in the activities, like CAD or block-programming. Teaching then concerns han- dling the software and the iterative process at the same time. Some teachers also express that teaching that enables digital modelling includes making a physical object from the dig- ital object. The focus of the described activities is to show modern and novel methods for technological manufacturing, and to concretize technology. Examples of common aspects are concretizing technology, iterative process, handling software, and physical objects, see Table 2. One means of using 3D-printed physical objects when teaching to enabling digital modelling, can also be to prepare pupils for the future, Fig. 1 (c + d). Teachers describe in the interviews that one intention with technology education is to raise an interest among pupils for technology and for technological educations and professions. They attempt to stimulate interest by telling the pupils about different professions that use printed models before production, and pupils are allowed to try these methods, hopefully to develop an interest. Synthesis Learning is always about something, and teaching is about making learning possible through using different methods. In this text, teaching content and methods are called teaching aspects, and the aim and purpose of teaching a specific area are associated with some specific teaching aspects. Therefore, teaching digital models also involves different teaching aspects which reflect teachers’ aims and purposes in their teaching. fl In this study, the aims and purposes of teaching digital models are described in four themes, partly overlapping each other. The overlaps is the result of different teaching aspects associated with more than one theme. Technology teachers can teach digital models with one or more of the themes in focus simultaneously. Before describing the combinations and relations between the themes, the teaching aspects of teaching digi- tal models need to be presented and related to the four themes. The teaching aspects have already been introduced and mentioned in the statements of the themes above, but are also summarized in Table 2 below. The four themes are presented to the left, 3 3 H. Brink et al. 1768 and teaching aspects are presented to the right. In the following text, the connections between the themes of Fig. 1 will be described based on the connected teaching aspects of Table 2. and teaching aspects are presented to the right. In the following text, the connections between the themes of Fig. 1 will be described based on the connected teaching aspects of Table 2. The four themes are related and they have common teaching aspects. This is illustrated in Fig. 1 where the themes are represented by the circles and the overlapping areas are representations of teaching aspects, that is teaching content and methods, that are used for fulfilling multiple aims and purposes. i Teachers experience that teaching digital models is a good way of enhancing and inte- grating other subjects, and they use software like CAD and block-programming for that purpose. They also sometimes use hardware like micro-control cards when teaching. Pupils are thereby working with digital models when they provide mathematics with data, when they work with mathematical concepts, or when they make substrates for manufacturing in crafts. Using digital models for integrating technology with other subjects requires pupils to be engaged in activities with the digital models. They are modelling digitally, even though modelling is not the aim of the teaching. Synthesis Enabling digital modelling is also used in activities when teachers teach program- ming, where the aim is to prepare pupils for the future, for the programmed contemporary society. Teachers teach to make pupils understand that programmed machines or systems are man-made with different purposes, and that the machines or systems are just as good as they are made. Examples of common aspects are handling software, physical objects, 1 3 Teaching digital models: secondary technology teachers’… 1769 and technological educations and professions, see Table 2. Teacher 10 describes a situation where pupils are programming. The teaching content is handling the software, and at the same time, handling the software is a method for discussions about programming, another teaching content: and technological educations and professions, see Table 2. Teacher 10 describes a situation where pupils are programming. The teaching content is handling the software, and at the same time, handling the software is a method for discussions about programming, another teaching content: It is about them being confident as adults. […] You cannot just trust these programs. They get as good as they are made. (Teacher 10) Teachers, teaching for visualizing technology, often use ready-made digital models as a basis for discussions, such as films, animations, or different applications. In those activi- ties, when teachers are discussing together with the pupils, abstract technology becomes graspable. Pupils can see and be aware of technology used in everyday surroundings. These discussions prepare pupils for the future, according to the interviewed teachers, by making pupils conscious of the technology that surrounds them, shown in Fig. 1 (b + d). Above all, teachers point out that teaching abstract concepts and abstract systems can be scaf- folded with digital models. Knowledge of abstract concepts and systems is important for all pupils, even for those pupils not interested in higher education in technology. Examples of common aspects are basis for discussions, concretizing technology, and general technologi- cal knowledge, see Table 2. Teachers participating in this study can also teach digital models with teaching aspects from three of the themes simultaneously, to fulfill different aims and purposes, illustrated in Fig. 1 (b + c + d). Enabling digital modelling can be taught together with preparing pupils for the future and visualizing technology to the pupils. Synthesis One example of this is when teachers teach programming with for instance Micro:bit; pupils are engage in activities with digital modelling, and at the same time, electronical components make programming more concrete, as discussions about programmed applications and systems. This example contains the same teaching aspects from three different themes of aims and purposes. f The synthesis shows that teaching digital models is not centered on only one objective, and teaching content and methods may differ, but also overlap between the various aims and purposes. Hence, Fig. 1 indicates that the teachers did not experience the teaching of digital models as one single idea but as an amalgam of multiple ideas that can mean dif- ferent things in different situations or contexts. Digital models, as a concept and a practice, have many meanings for the teachers of this study. Discussion Previous research has highlighted the risk that the technology subject when integrated with mathematics and science may serve to enhance the goals for those other subjects and that integration may also detract from the integrity of technology as a subject (Williams 2011). During the interviews, the examples described by the teachers often point to the learning objectives of other subjects. Using digital models as a way of showing technological solutions to others and to oneself, as a language of technology (Davies and Elmer 2001), is not an explicit goal/ aim in teaching digital models, according to the results of this study. Thereby, one char- acteristic of technology may be missing, if pupils are not made aware of the role and function of models; to communicate, to support ideas, to transform mental ideas to con- crete models, and to make the ideas available to others. As stated above, Swedish tech- nology education differs a great deal between different schools (Skolinspektionen 2014, 2019). Based on the findings of the present study, it can now also be stated that educa- tion concerning the teaching of digital models also differs. There are different experi- ences of teaching digital models, which can be interpreted as an indication that there is no consensus regarding teaching content or methods of teaching digital models, the progression of the teaching content, or the levels of knowledge that pupils are supposed to acquire. Teachers did not share a common knowledge base during the interviews. The aim and purpose of teaching digital models, the four themes, seem more important than the specific content. i Previous research has pointed to the importance of interweaving theoretical and practical activities in technology education (Kilbrink 2013). And that is what happens when teachers for instance enabling digital modelling and prepare pupils for the future. Pupils engage in a practical activity when modelling using digital tools, and get insights into the programmed contemporary world or different professions. The results show that pupils are given opportuni- ties to learn in and about technology. Furthermore, teachers include a normative component (de Vries 2005), when digital models representing different kinds of technology are discussed in terms of consequences and values in technology education. In this discussion, it is impor- tant to highlight that teachers express that they need pedagogical support to visualize tech- nological content that pupils find abstract. Discussion The point of departure for this article is to present technology teachers’ experiences of teaching digital models. By comparing previous findings and knowledge of models in tech- nology education, digital design using CAD, programming and the character of technology subject, with the empirical data from this study, discussions about the resulting themes can ensue and be understood. One strength of research with a lifeworld approach is the pos- sibility to discuss the themes in relation to other studies and theories (Bengtsson 2013). Mishra and Koheler (2006) explain that introducing new technologies to a content will influence the teaching of that content. Teaching models is for some teachers now teaching digital models, and technology teachers’ experiences of this new teaching are important to clarify. The results of this study, the four themes and the teaching aspects, answer the peda- gogical questions of why?, what? and how? in relation to the teaching of digital models. 1 3 3 H. Brink et al. 1770 The interviewed technology teachers have answered questions about what and how, but the main focus in their responses has been why they teach as they do, and why they choose content as they do. It seems more important for the interviewed teachers to jus- tify their teaching than just to explain it. This insight can be related to the role of the technology subject in Swedish schools. Technology education has a vague identity and, as stated above, there is no central core of content (Hagberg and Hultén 2005; Sjöberg 2013). Teachers seem to try to strengthen the role of the subject by justifying their peda- gogical choices and arguing why their teaching is important.i Another finding of this study is that digital models are a means for enhancing and integrating other subjects, such as mathematics and crafts. Integrating other subjects gives pupils a holistic grasp of the teaching content, but what are the consequences of letting the technology subject being used as backup for other subjects? What will hap- pen with the core content and with the identity of the technology subject, consider- ing the fact that these aspects are already unclear (Hagberg and Hultén 2005; Sjöberg 2013)? Teachers are struggling to promote the technology subject on its own merits on the one hand, and on the other hand, they are using it as a means for other purposes in other subjects. 1 3 Discussion Teachers want to concretize this difficult teaching content, and digital models are considered appropriate for that aim. Digital models often open up for discussions about advantages and disadvantages of the specific technology of interest to 1 3 Teaching digital models: secondary technology teachers’… 1771 the pupils and the teaching content can be varied a great deal, for instance to ask how search terms are bouncing on the internet, and what strengths are involved in lattice constructions.i the pupils and the teaching content can be varied a great deal, for instance to ask how search terms are bouncing on the internet, and what strengths are involved in lattice constructions.i To continue, technology teachers find it important sometimes to concretize the digital mod- els, made by pupils when modelling digitally. They print the designed objects in a 3D-printer to show modern manufacturing methods, or they use electronic components to show con- nections between programming and controlling. In this way, pupils are given opportunities to work with the iterative cycle of product development (Novak and Wisdom 2018), as they can try out, redo, and change their objects or programmed codes. This is also an example of teachers interweaving theory and practical activities (Kilbrink 2013), and an example of com- bining the teaching of technological knowledge with technological activities (Mitcham 1994), producing and using technology. Since the technology subject has a dual nature with both practical and theoretical activities (Gibson 2009), is it surprising that the dual nature is not mentioned when the interviewed teachers describe their teaching. Could it be that technology teachers usually do not discuss the subject with colleagues? Norström (2014) suggests that without a professional common base of what technological knowledge can be, and without a professional common base of terminology, “discussions and transfer of information become unnecessarily difficult” (Norström 2014, p. 33). The results of this study corroborate the lack of a common conceptual basis concerning digital models. The overall result from this study is that there is not one way to experience digital models, and therefore not one way to teach digital models. Digital models are sometimes used as a learning objective (for instance learning the software), and sometimes used as a means for other learning objectives (for instance providing inspiration for further technological studies). Moreover, some teachers do not have organized conferences or shared arenas to discuss the technology subject with others (Skolinspektionen 2019). Conclusions The results presented in this article show that technology teachers teach digital models with different aims and purposes, that the teaching content differs, and that pupils are given different prerequisites for learning digital models. The thematic analysis of the interviews resulted in four descriptive themes of the interviewed teachers’ experiences of teaching digital models; Enhancing and integrating other subjects, Visualizing tech- nology to the pupils, Enabling digital modelling and finally, Preparing pupils for the future. Each theme also has teaching aspects, such as the teaching content and methods used. Hence, there is not one way to experience the teaching of digital models, and a conclusion from this study is that discussions about this area need to be more nuanced and specific to provide a common knowledge base in this area. Since technology is rap- idly changing and developing, the meaning of concepts such as digital models can also change over time. It is therefore necessary to understand how digital models are expe- rienced, understood and interpreted in education today, to be able to understand any future developments. More specific discussions can also help highlight the technologi- cal outcomes in integration with other subjects. Further, more nuanced discussions can better include what requirements are reasonable for pupils in lower secondary school, regarding some specific part of digital models, for instance CAD, or programming, and could also include digitalization at a more general level. If actors in technology educa- tion start to discuss these issues in a more nuanced and specific manner, more knowl- edge of pupils’ learning, individualization, the level of teaching, planning and designing lessons, and different applications or programs can help enhance and develop the tech- nology subject. Further, if teachers are talking about the same thing when discussing these questions, it can help transfer the teachers’ own knowledge about digital models into teaching, and it can help pupils to acquire a conceptual basis. Technology education is researched both nationally and internationally, and many areas are well defined, but not the process when pupils in lower secondary school cre- ate and design in digital environments. When interviewing technology teachers for this study, many of them talked about CAD as an important area of education. It gave us insights into the need for more knowledge about teachers’ experiences of teaching involving CAD. Discussion Some technology teachers have to make individual decisions about their teaching. It seems possible that these results explain why it becomes difficult to discuss teaching digital models, when the teachers mean different things in different situations, but use the same word, and further, when the concept digital models is used in the technology subject syllabus without a clear definition (Skolverket 2011). This can explain and be one reason why technology teachers have trouble interpreting the syllabus (Bjurulf 2008; Hartell 2015), that the concept digital models is vague and not well defined. To improve the technology subject, and improve the teaching of technology, discussions of teaching digital models should be more nuanced and specific. Teaching design using digital design tools is one example of an area that is more nuanced, digital visualization of electroni- cal circuits is another. Using digital tools to examine and test sustainable constructions is also another area to discuss separately. These are just examples of teaching digital models, which cannot be discussed as one type of teaching. If discussions become more nuanced and more specific, our knowledge of pupils’ optimal learning or teachers’ best practice can increase. i However, Skolinspektionen (2014) pointed out in their report that a great many areas in technology education are unreflected, but from the results of this study, it can be stated that technology teachers are reflecting. They are reflecting upon why they teach digital models, as the four themes indicate, but there is a lack of reflection concerning the choice of content and method. 1 3 1 H. Brink et al. 1772 Conclusions Therefore, to get a more holistic view of technology education, aspects of creating and designing in digital environments need to be elaborated on in future studies. Another topic for further research can be to investigate how the use of digital models affect pupils’ learning or interest in technology. Appendix 1 See Table 3. 1 3 Teaching digital models: secondary technology teachers’… 1773 Teaching digital models: secondary technology teachers’… Table 3   Interview guide Question Supplementary questions Can you describe an example or assignment where pupils work with models or modelling using digital tools? Do you have more examples? Have you taught something else previously? How do you explain digital models and digital modelling to the pupils? How did you choose your assignment? What convinced you to choose that? Do you need to do any preparations for that assign- ment? How do you work with progression in that area? How do you find out what the pupils already know? What does it mean to you, to teach this? What do you want the pupils to learn (from the chosen assignment)? How does this learning become visible? What else can pupils learn from this assignment? How do you explain the assignment to the pupils? What words and concepts do you use? Are there specific words or concepts you think belong to the assignment? What usually causes difficulties for the pupils? How do you help them? Are there other ways to explain? What else do pupils find difficult? What are the technological areas in your assignment, when you are teaching digital models or digital modelling? How can the digital tool contribute to an under- standing of that technological area? What programs or applications do you use? Why did you choose that? Were there more reasons? What competences do you find important for you to have when teaching digital models or digital modelling? Anything else? How do you organize assignments with digital mod- els and digital modelling in the classroom? What motives are there for that way to work? What is your role in the classroom, when pupils are working with the assignment? Can you give more examples? Can you clarify? Can you tell me more? What do you mean with …? Can you describe that? Can you develop that? When you say… what … how…? How do you concretize that? Author contributions  All authors contributed to the study conception and design. The first draft of the man- uscript was written by Author 1 and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding  Open access funding provided by Karlstad University.. This work was supported by Region Värm- land and Karlskoga Kommun. Appendix 1 Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not 1 3 H. Brink et al. 1774 permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. References bom, H. (2011). Teknikintresserad? Jag?. In Hansson, Nordlander, Skogh (Red.), Teknikutbildning för framtiden – perspektiv på teknikundervisningen i grundskola och gymnasium. Stockholm: Liber. Åkerfeldt, A., Kjällander, S., & Selander, S. (2018). Programmering– introduktion till digital kompetens i grundskolan. Liber AB.i Alexandersson, M. (1994). Den fenomenografiska forskningsansatsens fokus. In Starrin, B., & Svensson, P. G. (1994). Kvalitativ metod och vetenskapsteori. Lund: Studentlitteratur. Bengtsson, J. (2013). With the lifeworld as ground. A research approach for empirical research in education: The Gothenburg tradition. 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https://discovery.ucl.ac.uk/1447209/1/Cavoli_VoR_linking_%20transport_health_and_sustainability.pdf

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Linking transport, health and sustainability: Better data sets for better policy-making

Journal of transport & health

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Clemence Cavoli a,n, Nicola Christie a, Jennifer Mindell b,1, Helena Titheridge a Clemence Cavoli a,n, Nicola Christie a, Jennifer Mindell b,1, Helena Titheridge a a Civil, Environmental & Geomatic Engineering, University College London, Gower Street, WC1E 6BT London, UK b Epidemiology & Public Health, University College London, HSSRG, Department Epidemiology & Public Health, 1–19 Torrington Place, London WC1E 6BT, UK a Civil, Environmental & Geomatic Engineering, University College London, Gower Street, WC1E 6BT London, UK b Epidemiology & Public Health, University College London, HSSRG, Department Epidemiology & Public Health, 1–19 Torrington a r t i c l e i n f o Article history: Received 3 December 2013 Received in revised form 25 July 2014 Accepted 4 August 2014 Available online 16 September 2014 Keywords: Transport Health Sustainability Data sets Article history: Received 3 December 2013 Received in revised form 25 July 2014 Accepted 4 August 2014 Available online 16 September 2014 Keywords: Transport Health Sustainability Data sets Article history: Received 3 December 2013 Received in revised form 25 July 2014 Accepted 4 August 2014 Available online 16 September 2014 Keywords: Transport Health Sustainability Data sets The impact transport has on our physical and mental health and on the environment is increasingly recognised by academics, practitioners and decision-makers. To inform policy-making and research, it is crucial to have access to sufficient comprehensive datasets linking these topics. Large scale surveys rarely combine questions on transport, health and sustainability, limiting their usefulness in research and policy-making. This project set out to identify the gaps in administrative and survey datasets to link health outcomes to travel behaviour and the quality of the environment; investigate the impact this has on research and policy-making; assess how these gaps might be addressed; and identify what the needs are for joined datasets. To achieve this, the project interviewed key decision-makers and stakeholders across England, including civil servants within national or local government and third sector organisa- tions. The results highlight gaps within national datasets; the insufficient links between health, transport, and sustainability datasets; and the need for further joined data in various fields, in particular active travel, health and wellbeing. Participants suggested several solutions, including better harmonisa- tion of surveys and data fusion. & 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY licens (http://creativecommons.org/licenses/by/3.0/ Contents lists available at ScienceDirect Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.jth.2014.08.001 2214-1405/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). n Corresponding author. Tel.: þ44 2076791590/7526169742. E-mail address: [email protected] (C. Cavoli). 1 Dr Mindell's salary is paid in part to work on the Health Survey for England. The funders played no part in this work. 2 In this paper we refer to the following definition of sustainability as stated by the World Wide Fund for Nature: “Improvement in the quality of human life within the carrying capacity of supporting ecosystems”. World Wide Fund for Nature. 1993. Sustainable Use of Natural Resources: Concepts, Issues and Criteria. Gland, Switzerland. ( ) dell's salary is paid in part to work on the Health Survey for England. The funders played no part in this work. n Corresponding author. Tel.: þ44 2076791590/7526169742. http://dx.doi.org/10.1016/j.jth.2014.08.001 2214-1405/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativec Journal of Transport & Health 2 (2015) 111–119 Journal of Transport & Health 2 (2015) 111–119 ( ) 1 Dr Mindell's salary is paid in part to work on the Health Survey for England. The funders played no part in this work. 2 In this paper we refer to the following definition of sustainability as stated by the World Wide Fund for Nature: “Improvement in the quality of human life within the carrying capacity of supporting ecosystems”. World Wide Fund for Nature. 1993. Sustainable Use of Natural Resources: Concepts, Issues and Criteria. Gland, Switzerland. p // g/ /j j 4-1405/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecomm rs. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). http://dx.doi.org/10.1016/j.jth.2014.08.001 2214-1405/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). paper we refer to the following definition of sustainability as stated by the World Wide Fund for Nature: “Improvement in the qua acity of supporting ecosystems”. World Wide Fund for Nature. 1993. Sustainable Use of Natural Resources: Concepts, Issues and C http://dx.doi.org/10.1016/j.jth.2014.08.001 http://dx.doi.org/10.1016/j.jth.2014.08.001 2214 1405/& 2014 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons org/licenses/by/3 0/) Dr Mindell s salary is paid in part to work on the Health Survey for England. The funders played no part in this work. 2 In this paper we refer to the following definition of sustainability as stated by the World Wide Fund for Nature: “Improvement in the quality of human life within the carrying capacity of supporting ecosystems”. World Wide Fund for Nature. 1993. Sustainable Use of Natural Resources: Concepts, Issues and Criteria. Gland, Switzerland. E-mail address: [email protected] (C. Cavoli). 2 In this paper we refer to the following definition of sustainability as stated by the World Wide Fund for Nature: “Improvem carrying capacity of supporting ecosystems”. World Wide Fund for Nature. 1993. Sustainable Use of Natural Resources: Concepts 1.1. Is there a need for joined up research? 1.1. Is there a need for joined up research? Measuring multidirectional relationships between health outcomes and the way we travel is becoming increasingly important. For instance, cycling as a way of commuting is increasing in many cities (Transport for London, 2011), but how is this affecting injury rates? Does it improve physical and/or mental health? Are the benefits of cycling outweighed by issues such as air pollution or injury? Recent research tackles some of these questions and highlights the importance of cross-disciplinary analysis. For example, mounting evidence focuses on the link between active travel, in particular cycling and walking, and reduced health issues such as obesity (Donaldson, 2004; Mindell et al., 2011a; Woodcock et al., 2011). Studies have shown that the health benefits of cycling outweigh the risks (De Hartog et al., 2010; Hillman, 1993). The economic impact of active travel has also been studied. Some researchers have estimated that the National Health Service (NHS) in England and Wales could save up to d17 billion within 20 years if active travel were to increase (Jarrett et al., 2012). Links have also been established across the fields of environmental sustainability,2 health and transport. Correlations between low carbon and active travel policies have been identified (Mindell et al., 2011b). Research has also highlighted the impact that environmental factors or the ‘built environment’ (e.g. neighbourhood, school, street) can have on people's physical and mental health (Booth et al., 2005; Law et al., 2007; Srinivasan et al., 2003). For instance, obesity or diabetes are associated with the level of deprivation or the walkability of an area (Booth et al., 2005; Law et al., 2007) and disadvantaged areas also tend to have high rates of collisions (Christie et al., 2007; Edwards, 2006; Graham et al., 2005; Watkins, 2012). C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 112 The quality of data collected in national surveys in England has also been questioned (Mindell et al., 2012; Ward et al., 2002; Woodcock et al., 2013). Problems with cycling and walking statistics include over-counting of serious travel-related cycling injuries in hospital data, and not counting pedestrian falls as transport injuries (Mindell et al., 2012; Ward et al., 2002; Woodcock et al., 2013). Many researchers highlight that data about cycling and walking need to be better collected (Mindell et al., 2012) or better analysed (Bhatia and Wier, 2011; Mindell et al., 2012). 1. comparing assets and drawbacks of main surveys; 1.2. Aims of the study This project set out to identify the gaps in datasets to link transport, health, and environmental sustainability; investigate the impact these have on research and policy-making; determine how these gaps might be addressed; and identify what the needs are for joined datasets. 2. identifying national survey gaps; 1.1. Is there a need for joined up research? ) In the field of health and active travel, several gaps have also been highlighted. The former National Obesity Observatory identified key issues related to physical activity data collection (National Obesity Observatory, NHS, 2009). That report considered many surveys, including the Health Survey for England, Active People Survey, and the National Travel Survey and identified that there is insufficient data about the impact cycling and walking has on health (National Obesity Observatory, NHS, 2009; Woodcock et al., 2011). In the field of obesity, studies highlighted the need for more research on obesity and the built environment (Booth et al., 2005; Dugdill et al., 2009). Research suggests that there are many gaps and issues in datasets which need to be addressed, plus a need to join and analyse datasets Research suggests that there are many gaps and issues in datasets which need to be addressed, plus a need to join and analyse datasets in a more systematic and strategic way. However, gaps and issues related to health, environmental sustainability and transport across UK datasets have not been comprehensively identified and recommendations to fill these gaps not sufficiently addressed. 2. Methods: key stakeholders consultation Between October 2012 and April 2013, key stakeholders in the fields of transport, health and sustainability in England were interviewed. In total 27 people were interviewed: 22 formally, using semi-structured interviews (See Annex 1) and five informally. Prospective participants were selected purposively with a view to representing a variety of stakeholders who use national datasets in these fields. Among those interviewed, 11 individuals specialised in transport (including safety), eight in health, seven in environmental sustainability, and two were from data providers. All the participants' fields of expertise and interests crossed into at least one other of the three areas of transport, health, and sustainability (including social and economic sustainability). In terms of transport, the focus of enquiry was personal travel and mobility (defined as the ability to move between places); the choice of participants from the transport field reflected this. Participants from the following roles/types of institutions were represented:  10 researchers in academia, institutes/schools and centres  5 local government policy makers  4 national policy makers  4 third sector organisations  1 parliamentary group  2 data providers The semi-structured topic guide included three parts (see Annex 1). The first focused on the data sets' advantages and disadvantages, and general questions about aims and objectives, outcomes and impacts of UK national policies related to data sets. The second part was dedicated to establishing the questions, themes or topics that participants would like to see addressed in national surveys. The last part of the questionnaire collected participants' views about different solutions and ideas on whether there should be a platform for centralising surveys or information about surveys and the potential for data fusion. Finally, participants were asked to describe what their ideal survey would be and whether this survey should focus on the local or national level. Fig. 1. y The following questions were addressed in the topic guide:  What datasets do participants use and what do they think are the weaknesses of each of those dat  What are the strengths and limitations of the data sets stakeholders use?  What are the strengths and limitations of the data sets stakeholders use? What impact do these limitations have on policy making evaluation a What are the strengths and limitations of the data sets stakeholders use? What impact do these limitations have on policy-making, evaluation and research? 3.1. Gaps in national datasets The most frequently used datasets were STATS19 (police road traffic collision dataset), Road Casualties (Department for Transport routine statistics based mainly, but not exclusively, on STATS19 data), and Hospital Episode Statistics (HES, National Health Service hospital healthcare data) - all administrative data – and the National Travel Survey (NTS), Active People Survey, and the Health Survey for England – all random sample population surveys. A brief description of each survey is shown in Table 1. Several gaps were highlighted by the participants, as illustrated in Table 2: The majority of the participants mentioned that one of the main limitations of current national data sets is the limited sample size, which does not provide data on a small geographical scale. As one of the stakeholders stated: too often ‘assumptions that are made from the national datasets cannot be applied to the local population’ (participant 8, Health). London policy makers commented that in the case of London, it is such a ‘unique case that national survey results are not necessarily representative’ (participant 1, Health and Transport). More generally, data sets are not ‘fine grained enough’ to represent certain minorities, or look at certain ‘specific issues’ such as unusual injuries (participant 11, Transport and Environment; participant 13, Health). Three data sources in particular were pointed out: the National Travel Survey (NTS), Active People Survey and reported road casualties. y ( ) p y p In addition, participants mentioned that there is a lack of longitudinal data sets amongst national surveys. As one participant said there is a lack of ‘Consistency and continuity” (participant 17, Transport). Participants insisted that it is important and useful to have longitudinal data, collecting information from the same individuals over time and made suggestions such as establishing a ‘baseline and measure every five years’ (participant 13, Health). Data harmonisation describes the process of reconciling different measurements of the same parameter (e.g. age, ethnicity, travel time etc.) between different types of survey to make them mutually compatible. Participants commented on the lack of harmonisation between surveys and the different definitions and categories which are a barrier to harmonisation such as time inconsistencies and definition of basic characteristics such as age group, the definition of ‘urban areas’, as well as level of education. 4. providing solutions and ideas Each matrix was divided into sub-themes and then systematically cross-analysed to identify key themes (Miles et al., 2014). These themes were also reviewed by another researcher to check for consistency. 3 The recommendation has recently changed and is now 150 min of moderately vigorous activity per week. 3. Existing data sets: limitations and impact on policy making 3.1. Gaps in national datasets 2. Methods: key stakeholders consultation What impact do these limitations have on policy-making, evaluation and research? What impact do these limitations have on policy-making, evaluation and research?  To what extent is there a need for a survey that explores the interrelationship between mobility  To what extent is there a need for a survey that explores the interrela What gaps in knowledge would such a data set help address? y p What gaps in knowledge would such a data set help address? g p g p  What do stakeholders think about data fusion or the establishment of a platform which would centralise all surveys? g p g p keholders think about data fusion or the establishment of a platform which would centralise all surveys  What do stakeholders think about data fusion or the establishment of a p  What do stakeholders think about data fusion or the establishment of a platform which would central  What would the ideal survey be like? Participants were interviewed with informed consent. Interviews were audio taped and transcribed. Informal interviews were first conducted to gain a general understanding of the situation. This helped shape the semi-structured questionnaire. Data was coded and analysed for thematic content. Key themes are exemplified in the paper using verbatim anonymised quotations from participants. Key gaps in national data sets were identified through the thematic analysis of the interviews. To conduct the analysis of the interviews a framework matrix was developed based on the key research questions addressed by the topic guide and then populated with responses from participants. The four themes of the matrix were: 113 C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 3. investigating what questions participants would like to add to surveys; and 4. providing solutions and ideas HEALTH Local government (2) Data provider (1) Parliamentary group (1) Academia (4) SUSTAINABILITY Local government (1) Primary care trust (1) Professional Institute (1) Academia (2) Non-Governmental Organisation (2) TRANSPORT National government (1) Local government (1) Transport consultancy (1) Academia (4) Non-Governmental Organisation (1) Data provider (1) • • • • • • • • • • • • • • • • Fig. 1. List of participants. Fig. 1. List of participants. Fig. 1. List of participants. 3. investigating what questions participants would like to add to surveys; and 3. investigating what questions participants would like to add to surveys; and 4. 2. Methods: key stakeholders consultation providing solutions and ideas 3.1. Gaps in national datasets Between surveys there were inconsistencies over the definition of physical activities, with the Department of Health measuring 30 min of activity3 whereas the Active People Surveys talk about 20 min. One of the key gaps identified by participants is that active travel is not sufficiently addressed through national surveys. Even though current data sets provide, to some extent, a big picture of the situation, more data sets are needed to fully understand and measure active travel and its impact on health. This is especially true for walking and cycling as well as active commuting under 30 min. Participants noted that current national data sets do not differentiate between cycling and walking as leisure, a way to commute, or other travel. As stated by a participant: ‘Getting a holistic picture of how much walking and cycling contributes to people's overall activity and for what purpose is quite difficult.’(participant 10, Health). A key missing element related to walking is insufficient data on short walking journeys between different modes of transport. Walking as part of ‘multi part journeys’ is not adequately recorded and as a result the ‘value of walking is under-recognised’ (participant 13, Health). According to many participants, cycling is equally under-represented in national surveys. They felt that it was not sufficiently researched C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 114 Table 1 National surveys and routine datasets relating to health and transport in the UK. Road casualty data ‘STATS 19’ Routinely collected data on road collisions reported to the police where someone has been injured (STATS19 system). Includes information on mode of travel, age and gender of all drivers an all injured persons, circumstances of collisions, types of vehicle involved and severity of injury. Published annually. Hospital Episode Statistics Routinely collected data on all admissions, outpatient appointments and A&Ea attendances at NHS hospitals in England. It is a record-based system that covers all NHS trusts in England. The database processes over 125 million records annually. Data includes diagnoses and procedures (operations), and patient demographics. Road traffic casualties can be identified through the International Classification of Disease external cause codes. National Travel Survey Data on personal travel patterns in Great Britain. Based on an annual household survey of approximately 20,000 people from 8000 households. Active travel needs to be better addressed The four areas are not sufficiently linked Health and Transport lack connection and not well recorded (see below): ‘Statistics about cycling are often confusing,’ (participant 10, Health). The National Travel Survey, in particular, includes journeys only on routes along which motor vehicles can travel, and therefore undercounts walking and cycling trips and distances, given the marked rise in such journeys on traffic-free routes in the past decade (Sustrans, 2013). and not well recorded (see below): ‘Statistics about cycling are often confusing,’ (participant 10, Health). The National Travel Survey, in particular, includes journeys only on routes along which motor vehicles can travel, and therefore undercounts walking and cycling trips and distances, given the marked rise in such journeys on traffic-free routes in the past decade (Sustrans, 2013). Many participants highlighted the lack of data sets which link health and transport, the need for ‘public health policies to get information from transport and vice versa’ (participant 15, Transport). Some participants stated that highlighting the relationship between travel and health is a key way to change people's behaviour and that the link between transport and obesity needs to be better highlighted. It was also mentioned that changes on the ground, such as infrastructure, are not sufficiently linked ‘with actual health outcomes’ (participant 11, Transport and Environment). Other participants talked about the need to link the fields of transport, health and sustainability. As stated by one: ‘We need to understand the bigger picture and determine what has the biggest impact on health. Is it air quality? Or inactivity? So if for example it was the case that it was a city where air quality was causing much greater health impacts than road traffic collisions or physical inactivity then the policy makers should be focusing their efforts on air quality. But if it's a city where actually it's physical inactivity that's the biggest impact on health, then that's where the efforts should go.’(participant 1, Health and Transport). Several participants indicated that there are flaws in injury data sets. The most frequent issues are linked to reported cycling casualty rates. Datasets such as STATS19 indicate that ’the number of casualties increases but it is linked to the increase in the number of cyclists’ (participant 10, Health). Reacting to a newspaper article about the increase in cycling collisions during the Olympics one participant said: ‘You've got to put that in the context of exposure stats. Active travel needs to be better addressed The four areas are not sufficiently linked Health and Transport lack connection […] It might actually have become safer to cycle over the same period because so many people were out on their bikes.’ (participant 3, Transport and Environment). 3.2. Impact on policy making and research Table 1 Table 1 National surveys and routine datasets relating to health and transport in the UK. Table 1 National surveys and routine datasets relating to health and transport in the UK. Road casualty data ‘STATS 19’ Routinely collected data on road collisions reported to the police where someone has been injured (STATS19 system). Includes information on mode of travel, age and gender of all drivers an all injured persons, circumstances of collisions, types of vehicle involved and severity of injury. Published annually. National Travel Survey Data on personal travel patterns in Great Britain. Based on an annual household survey of approximately 20,000 people from 8000 households. Data is based on interviews and seven day travel diaries and covers how, why, when and where people travel as well as factors which affect personal travel such as car availability, driving licence holding and access to key services. Published annually. Active People Survey Data on the number of people taking part in sport across the nation and in local communities. Telephone based interviews among people age 14 and over throughout the year. The key measure is the ‘1 x 30’ indicator, the percentage of the adult population participating in sport, at moderate intensity, for at least 30 minutes on at least four days out of the last four weeks. Cycling is included if done at least once a week at moderate intensity for 30 minutes. Published annually. y . y. Health Survey for England Data on health (e.g. Body Mass Index, longstanding illness) and health-related behaviours in adults and children living in private households in England. Published annually. y y health (e.g. Body Mass Index, longstanding illness) and health-related behaviours in adults and children living in private households i Published annually. Table 2 Gaps in National Data Sets. Conceptual gaps Active travel needs to be better addressed The four areas are not sufficiently linked Health and Transport lack connection 3.1. Gaps in national datasets Data is based on interviews and seven day travel diaries and covers how, why, when and where people travel as well as factors which affect personal travel such as car availability, driving licence holding and access to key services. Published annually. Active People Survey Data on the number of people taking part in sport across the nation and in local communities. Telephone based interviews among people age 14 and over throughout the year. The key measure is the ‘1 x 30’ indicator, the percentage of the adult population participating in sport, at moderate intensity, for at least 30 minutes on at least four days out of the last four weeks. Cycling is included if done at least once a week at moderate intensity for 30 minutes. Published annually. Health Survey for England Data on health (e.g. Body Mass Index, longstanding illness) and health-related behaviours in adults and children living in private households in England. Published annually. a A&E: Attendance at hospital Accident and Emergency department (Emergency Room). 4.1. Physical activity, active travel and health Participants expressed an interest in including more general questions about health. For instance, a question such as ‘How is your health in general?’ could be included in many non-health surveys (participant 3, Transport and Environment). Most participants also insisted that Participants expressed an interest in including more general questions about health. For instance, a question such as ‘How is your health in general?’ could be included in many non-health surveys (participant 3, Transport and Environment). Most participants also insisted that there is a necessity to obtain more details about physical activity and active travel, especially in relation to obesity. There is a need to better link certain areas such as ‘Physical activity and dietary combination’ or the impact active travel has on health (participant 8, Health). Issues such as the ‘Impact of walking and cycling on overall activity level and links between active travel and obesity’ were raised (participant 11, Transport). p ) Participants showed much interest in obtaining data about overall physical activity during the entire day and in understanding people's daily routine. There is a perceived need to have a ‘Physical activity diary’ for people to record their physical activities, from leisure to commuting, including short commuting trips (participant 21, Transport). Participants felt that this would help identify: ‘What proportion of it (physical activity) is coming from work, what proportion of it is coming from leisure activities, what type of leisure activity…’ (participant 1, Health and Transport). Fears related to cycling were also mentioned. Issues such as whether ‘unpleasant experiences cyclists suffer whilst they cycle prevents them from cycling? (Such as getting abuse from drivers or threats)’ were raised (participant 2, Transport and Health). Stakeholders would like to be able to ‘pick up changes in views about fear of traffic or other disincentives to cycling’ (participant 13, Health). Other questions such as ‘Why do people cycle?’ or ‘Who cycles?’ were suggested (participant 10, Health). One participant noticed: ‘We have seen the cyclists increased by 20% [in London] in terms of counts on the road; but who are those people cycling?’ (participant 4, Transport). Behavioural changes related to cycling were also mentioned or questions such as ‘Do people's walking and cycling patterns change across the life course? What contribution is it making to their health?’ Suggestions were made such as having ‘cohort studies of cyclists’ (participant 10, Health; participant 2, Transport and Health). 4.1. Physical activity, active travel and health Travel to work and especially to school was mentioned many times as being a key theme which needs to be better addressed in surveys. Many participants voiced their disappointment at the fact that the Department of Education stopped collecting data about children travelling to school at a local level. It was a ‘really invaluable resource’ (participant 10, Health). One participant highlighted the fact that independent mobility for children ‘is the sign of a healthy society’ and also that ‘habits need to be taken at a young age.’ (participant 12, Health and Transport). A key theme raised by many participants was measuring well-being, especially ‘psychological well-being’. Well-being as described by participants referred to psychological or mental well-being, such as levels of depression or effect on mood, as well as quality of life and happiness. Participants felt that there is a need to address and measure well-being in surveys. In relation to well-being, one participant stated: ‘One of the social aims is not just to have a population which is sort of physically functioning in health terms but also have a sense of purpose or a sense of happiness[…] then logically you need metrics to assess how well you are doing on that’ (participant 3, Transport and Environment). In addition, participants highlighted that behavioural change is a key issue and should be better measured. There is a focus on collecting hard data but ‘that failed to take account of the nature of changes that are needed along the way when you have got long term shifts in population level behaviours.’ (participant 13, Health). Questions such as ‘In the long term, who has changed behaviour, why and how?’ need to be raised (participants 4, Transport). In the field of transport it would be useful to further tackle reasons to travel as well as behavioural issues related to cycling. Participants showed much interest in perceived safety, especially in relation to cycling and walking. Areas such as ‘risk perception’ and ‘attitudes to road safety’ need to be addressed (participant 20, Health). One participant redefined the phrase road safety: ‘Casualty reduction is not road safety. Road safety is more than that, it is freedom from fear or harm of injury on or around the road environment.’ (participant 12, Health and Transport). Table 3 Key themes which need to be better addressed. 3.2. Impact on policy making and research The majority of participants felt that the limitations of existing datasets influence and limit research to a large extent. One participant stated that: ‘We should be able to tell a compelling story about the impact of transport on health but at the moment it is a struggle for us to find basic data’ (participant 1, Health and Transport). Most participants felt that policy objectives are not sufficiently addressed by surveys, although some interviewees were unsure. Some areas were quoted as missing the data to enable the evaluation of national policy targets, particularly health, transport and carbon emissions. In the field of health and transport, there is a gap between national policies which ‘are giving priorities to obesity and physical activity’ and the data sets available (participant 6, Transport). There is also a need for more data sets about cycling, since many policies have launched schemes for increased investments. Most participants mentioned that policy outcomes are not sufficiently measured by national data sets. In general ‘there is a lack of evaluation’ and this is especially true in certain fields such as walking, cycling, carbon emissions, air quality, behavioural changes, which are ‘hardly measured’, and more generally long term impacts (participant 2, Transport and Health; participant 13, Health). Participants pointed out that evaluation is crucial, that ‘Policies should be prospectively evaluated’ and that ‘pre- and post-evaluation of interventions’ are very useful (participant 2, Transport and Health; participant 6, Transport). As one participant said: ‘we need to know what is the value for C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 115 money of schemes. We need to understand the value on the return of our investment’. Identifying whether investments in policies or projects benefit society is key (participant 6, Transport). 4. New data sets: users' needs Many participants expressed their interest in linking transport, health, and sustainability; ‘It is useful that those relationships are explored’ (participant 7, Health). It is important to establish ‘how changes in one area, for instance health, can have a spill over other areas, such as environment.’ (participant 22, Sustainability). Participants were asked which areas or subjects need to be better addressed by existing surveys. Four key themes emerged from the content analysis, as illustrated by Table 3 below. 4.1. Physical activity, active travel and health 4.1. Physical activity, active travel and health Physical activity, active travel and health Well-being, behaviours and attitudes Linking mobility, health and sustainability Background information, identifiers and underrepresented groups General health levels Behavioural questions, attitude and perception Carbon emissions, air quality and energy Basic background characteristics (age, gender) More details about physical activity and active travel Perceived safety Pleasantness of the environment where people commute More data about deprived groups and ethnicity Cycling Life changing event (Transition, moving home…) Transport and the economy People's location Travel to school Social and economic sustainability Using data from new technology 4.3. Under-represented groups and background information Many participants would like surveys to focus more on age, especially older people and young people. One participant suggested that surveys should look at ‘How we can keep people active and happy in their old age (especially since people are living much longer)’ (participant 20, Health). Linking ageing and mobility was also raised. Many participants talked about the importance of including deprived groups in national survey data sets. These disadvantaged or very disadvantaged groups included the disabled, those with limited mobility or accessibility issues, those on lower incomes or education levels and the socially excluded. Participants reported that it would be useful to identify how disability affects people's daily activity (participant 20 H l h) I i l i d d h ‘H l h i li i f i l i d i li i ’ ( i i 8 H l h) 20, Health). It is also very important to understand the Health implication of isolation and marginalisation (participant 8, Health). Ethnicity was also an important factor mentioned by participants. Deprivation and ethnicity are often tied together. According to some participants, ethnic, social and religious segmentation should be given more importance in national surveys; although being able to analyse data by specific ethnic groups would require a very large survey. ) y p p f g Ethnicity was also an important factor mentioned by participants. Deprivation and ethnicity are ofte participants, ethnic, social and religious segmentation should be given more importance in nationa analyse data by specific ethnic groups would require a very large survey. It was often mentioned that obtaining ‘geographic positioning’ would be extremely useful (participant 22, Sustainability). Participants argued that having access to people's postcode and the location of, for example, trip destinations would provide a wealth of information such as ‘where people want to go’ or ‘tracing people's movements’ (participants 4, Transport). It is especially important in the case of STATS19 road casualty data, where the location of the collision is specified. However, participants were aware that obtaining such data ‘might not be realistic’ due to anonymity issues (participants 4, Transport). 4.2.2. Energy Participants highlighted the need to obtaining data sets about energy savings and understanding the value of home retrofitting. In addition, one participant commented on the need to join data about electricity and gas usage with vehicle consumption. This would help to answer questions such as ‘are people who drive the most also the people who have the highest electricity and gas bills?’ (participant 3, Transport and Environment). Some participants also addressed the link between health, physical activity (especially obesity) and carbon consumption (active travel versus driving a car). One concluded by saying: ‘Often what is going to help people's health is also going to help the planet’ (participant 20, Health). p (p p ) The ‘pleasantness of the environment where people commute’ was mentioned as being a key issue (participant 3, Transport and Environment). It is also related to perception of safety and well-being, as stated by one participant, commuters ‘don't necessarily want to be walking on a busy road but you might consider walking (to go to work) down a more pedestrianised street’ (participant 1, Health and Transport). Participants suggested addressing questions such as ‘Does a change in people's environment improve their life?’ (participant 22, Sustainability). 4.2.1. Air quality 4.2.1. Air quality q y According to some, the impact pollutants emissions have on health is not sufficiently tackled in national surveys. Although participants did not mention how to address this issue through national surveys, they detected a specific need for further data sets related to ‘asthma and cancer in relation to particulates’ (participant 6, Transport) or the need for more solid evidence ‘about the efficiency of 20 miles an hour on fuel efficiency and pollution’ (participant 12, Health and Transport). Table 3 C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 116 4.2. Environmental sustainability 4.2. Environmental sustainability When asked which questions would you liked to see addressed in national surveys, the majority of the participants mentioned environmental sustainability. Three key issues were specifically raised: air quality, energy, and the pleasantness of the environment. 5.1. Data fusion: ideas and obstacles 5.1. Data fusion: ideas and obstacles Almost all participants mentioned that data fusion offers solutions to many problems and gaps in national data sets. Data fusion describes the process of merging existing databases into a single participant level database to provide a deeper understanding of a specific issue. It involves matching data, i.e. pairing participants on the basis of common characteristics, usually key demographics (age, gender, area, deprivation/social class, ethnicity, education). This integration of data is based on the principle that these common characteristics can reliably predict behaviour. The outcome of data fusion, like any modelling process, needs to be validated by looking at whether its predictions are accurate. In addition, careful checks need to be made to take into account differences in the measurement of matching criteria. Expressions such as ‘harmonising’, ‘synchronising’, ‘combining’ or ‘integrating’ surveys were very often used by participant. One participant stated: ‘We need to create data sets that relate to each other. The more holistic benefits are, the better case for investment it is’ (participant 22, Sustainability). Others mentioned that: ‘Aggregation of data is very important to reach conclusions on a global scale’, and: ‘It is about making the data talk to each other and link up’ (participant 18, Transport and Environment; participant 1, Health and Transport). Linking data sets can also reduce survey costs, although there were some misconceptions of the advantages of data fusion, for example one participant thought that it would ‘increase or boost the sample size’ (participant 8, Health). As illustrated in Table 4 below, participants described the advantages data fusion could provide, giving practical examples and identifying potential obstacles. Several suggestions were made regarding which data sets and surveys to combine. According to man some transport questions would be very useful. Several participants also noticed that the gaps within STATS19 could be solved by cross-referencing STATS19 with HES on the basis of time, data and transport injury (participant 9, Transport and Environment). However, HES does not provide information about people's ed that the gaps within STATS19 could be solved by cross-referencing STATS19 with HES on the basis of participant 9, Transport and Environment). However, HES does not provide information about people's C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 117 Table 4 Data fusion. 5.1. Data fusion: ideas and obstacles Data fusion Advantages  Increase sample size  Reduce costs  Likely to reduce burden on participants Linking surveys  Link NTS and HES  Link NTS and Active People Survey  Link Stats 19 and HES  Link Health and Transport Surveys Practical ideas  Use the same sample  Add identical questions or aggregate data  Harmonise definitions  Establish common ‘identifiers’  Link data from complementary surveys Obstacles  Might be burdensome for participants  Might increase costs general health status, only about the diseases or injuries they have when admitted to hospital. In addition, HES has limitations: official analyses of ‘transport injuries’ exclude pedestrian falls but HES overestimates travel related cycling injuries (Mindell et al., 2012). 5.2. How can data sets from different surveys be combined? 5.2.1. Participants offered different solutions to enable data fusion, such as using the same sample Adding identical questions to different surveys was offered as a solution to ease ‘cross data analysis’. This is easier with harmonised definitions and categories between surveys. The ONS (Office for National Statistics) harmonisation team was created because ‘people are becoming more and more aware of data linkage’ (participant 23, Transport, Health and Environment). Participants noted that at the government level, data fusion ‘already exists to some extent’ and a ‘lot of questions are already harmonised, for example about ethnicity’ (participant 23, Transport, Health and Environment). When not asked of the same participants, this enables information from one source to be linked to different information from a second source for people with identical answers to those identical questions and other characteristics in common. Aggregating data about the same individuals from different sources was offered as another solution. For example, to link the NTS and HES, you could ask participants in NTS for permission to link their responses to HES. There might be limitations since the sample will be different for each survey but ‘as long as you are aware of that and as long as you are aware of the limitations the variation in sample sizes can have and you can take that into account and interpret the results’ (participant 8, Health). 5.1. Data fusion: ideas and obstacles The participant added: ‘We may not be able to make a decision with absolute precision but we are going to be more informed than we would be without it.’ Several participants said that the best solution is to find common identifiers or ‘intelligent comparators’ (participant 8, Health). Participants felt that surveys should have ‘common indices where the datasets can be properly interrelated and agreed upon unified identifiers’ so that surveys can be interrelated through ‘certain core attributes’ (e.g. geographic locations, demographics, personal identification) (participant 22, Sustainability). As suggested by one participant, ‘the collection of certain indices could be mandatory such as the National Insurance Number or very specific demographics such as date of birth’ (participant 22, Sustainability). f (p p y) Linking data sets from complementary sources was recently used by the NTS team as a way to reduce the survey size, which they had to do for economic reasons. As said by a person in charge of the NTS ‘One of the big savings we could make within the questionnaire is we can link data from other data sources so we do not actually have to ask the respondents.’ (participant 23, Transport, Health and Environment). For example, the NTS used to ask participants ‘how far it is to their local services’; now they will be using the participant's postcode and link it to Accessibility Indicator data which is done separately (participant 23, Transport, Health and Environment). As a result, ‘it reduces the questionnaire length, gives us better quality data and also reduces the burden on the respondents’. Data fusion might often require getting people's permission to obtain certain data (participant 23, Transport, Health and Environment). 6. Discussion Discussions with stakeholders revealed varying levels of knowledge about the data collected by different surveys and some confusion about operational definitions, such as the recommended physical activity levels according to the Department of Health (see Section 3.1). However, such confusion could be overcome by using a sophisticated online platform as proposed in this paper. This would make surveys more accessible and understandable for users. Some of the participants’ suggestions may be difficult to implement. For example, using the same sample for different surveys would generally be too burdensome for survey participants and would impact on response rates. Some survey questions which were proposed may be too broad – or too specific – to be included in national surveys. Suggested questions such as “Does cycling improve your well-being”, may need to be more specific to study individuals’ perceptions of how cycling affects wellbeing, while separate questions on cycling, wellbeing and confounding factors would be required in a single survey for epidemiological assessment of associations between cycling and wellbeing. Some participants suggested linking surveys with routine data (e.g. NTS with HES). This would not increase sample size but would increase the range of data available for those survey participants, for far lower costs than additional survey items. There are many examples where data linkage has been achieved and has shed new light on the relationship between transport exposure and health outcomes (e.g. Ward et al. 2002). It also has the advantage of providing subsequent health outcome data, of greater aetiological importance than cross-sectional associations seen in surveys, where reverse causality cannot be excluded (such as greater wellbeing resulting in more cycling, rather than vice versa). A specific suggestion from several interviewees was for the establishment of a simple, comprehensive online platform describing existing surveys. In theory, this already exists for national surveys in the form of the UK Data Service, which provides comprehensive information about the methods and content of each of these surveys, detailed definitions of the variables, and access to anonymised datasets. Implications of our research include the need for greater publicity about the UK Data Service. However, this does not include the routine, administrative data, so would not provide the desired platform for all relevant datasets. 5.3. Platform centralising all surveys: suggestions and barriers 5.3. Platform centralising all surveys: suggestions and barriers When asked whether establishing an online platform summarising all existing surveys and offering links to surveys would be helpful, many participants agreed that it would, especially in order to gather all the information about surveys which is ‘too often spread out’ (participant 14, Transport safety). ‘There needs to be a map as to where all these resources are’ added one participant (participant 8, Health). Another person added that there is also a ‘Need to invent mechanisms so that people from different fields are aware of the surveys being elaborated’ (participant 22, Sustainability). Participants suggested that an online platform could ‘provide routes to access to surveys’ (participant 2, Transport and Health). In addition to providing a list, this platform could include relevant information or ‘insights’ about each survey, ‘A paragraph about each survey and an explanation about how each survey relates to each other would be helpful’ (participants 22, Sustainability; participants 1, Health and Transport). The platform could also clearly identify links between surveys and possibilities for data fusion. Examples of existing platforms were given such as: the Public Health Observatories and the National Obesity Observatory (all now part of Public Health C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 118 England), UK Data Service, data.gov.uk, ONS, Economics and Social Research Council (ESRC) Question Bank, the Road Safety Observatory, the Road Safety Knowledge Centre, and the European Road Safety Observatory. 7. Conclusion This study identified a number of gaps in national sources of data (and the clear recognition of the limitations that these gaps caused), the need for new data sets, and proposals for a number of practical solutions. Limitations of current surveys were: insufficient sample size, a lack of harmonisation between surveys, and insufficient links between health, transport, and sustainability. Participants agreed that these deficiencies adversely influence and limit the research, and compromise the ability to evaluate the objectives of national policies. In addition, participants expressed their doubts about the capacity existing surveys have to meet national policy objectives. These limitations could lead to insufficient understanding of how best to meet national policy objectives and the implications of policies under consideration for implementation. This could result, for example, in poor policy decisions with unintended consequences for health and the environment. The majority of the participants recognised the strong need for joined up research in the fields of transport, health and sustainability. Topics that were highlighted included active travel and its link to health and well-being. More specific themes such as the pleasantness of the environment where people commute were also raised. Many also acknowledged the importance of better representation for certain groups of the population, especially older people and those from deprived areas. When asked for solutions and ideas, participants highlighted the need to harmonise all surveys by establishing common indices across surveys to facilitate data fusion, which was mentioned as being key for the future of national surveys. The ONS data harmonisation programme may go some way addressing this. Other ways forward include increased funding of research aimed at improving data fusion techniques. 6. Discussion One of the strengths of this study is the breadth of stakeholders who participated and the many commonalities in points raised by a range of individuals, such as the need for increased data on walking or the need for harmonised definitions across surveys. As a result, the study successfully identified a series of gaps in national data sources which need to be tackled, and several key topics which should be addressed in surveys. y One limitation of the study is that we did not interview sufficient people responsible for organising surveys. Although many participants are very familiar with national surveys and the making of these surveys, they lack the necessary technical understanding to explain the detail of processes such as data fusion. As a result, topics such as the barriers to data fusion could not be discussed at length in the study, yet it is key to improving future surveys (Bleiholder and Naumann, 2009). Although limited to England or UK data sources, most of the underlying principles will also apply in other countries interested in similar cross-cutting policy issues (such as the implications of encouraging low carbon and active travel modes), particularly those relating to gaps in national surveys (Merom et al., 2010) or methods to address these gaps such as data fusion (Bleiholder and Naumann, 2009; Dong and Naumann, 2009). Annex 1. Topic Guide I. Existing data sets (1) Do you use datasets to explore the relationship between mobility, safety, health, and sustainability? a. If yes what data sets do you use? b. What are the advantages and disadvantages of current datasets? I. Existing data sets I. Existing data sets (1) Do you use datasets to explore the relationship between mobility, safety, health, and sustainability? a. If yes what data sets do you use? b. What are the advantages and disadvantages of current datasets? 119 C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 C. Cavoli et al. / Journal of Transport & Health 2 (2015) 111–119 c. How, if at all, are these datasets linked? c. How, if at all, are these datasets linked? c. How, if at all, are these datasets linked? (2) To what extent are existing surveys at the National level able to meet the aims & objectives of our National Policies? (3) To what extent are existing surveys able to analyse the relationship between policies and outcomes? (4) To what extent do the limitations of existing datasets influence and limit research? ( ) g y y p p (4) To what extent do the limitations of existing datasets influence and limit research? (4) To what extent do the limitations of existing datasets influence and limit research? What kind of new data sets are needed? (1) Possibility to Gather/Combine existing data sources a. What should it look like? b. What are the possible difficulties/barriers? c. How would we overcome them? d. What is the best forum to gather all the actors responsible for surveys at the National level (2) Add new information to existing data sets a. Would it be feasible to add data to existing survey? b. If yes, how should we do it? (3) National Survey a. What should the ideal survey be like according to you? b. What are the possible difficulties/barriers? c. How would we overcome them? d. To what extent do we need to give priority to obtaining National Data versus Local Data? d. What is the best forum to gather all the actors responsible for surveys at the National level a. Would it be feasible to add data to existing survey? b. If yes, how should we do it? at o a Su ey a. What should the ideal survey be like according to you? a. What should the ideal survey be like accordi b. What are the possible difficulties/barriers? c. How would we overcome them? d. To what extent do we need to give priority to obtaining National Data versus Local Data? d. 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At Least Five a Week: Evidence on the Impact of Physical Activity and its Relationship to Health. Departmen Dong, X.L., Naumann, F., 2009. Data fusion: resolving data conflicts for integration. Proc. VLDB Endow 2, 1654–1655. At Least Five a Week: Evidence on the Impact of Physical Activity and its Relationship to Health. Department of Health, Lond n, F., 2009. Data fusion: resolving data conflicts for integration. Proc. VLDB Endow 2, 1654–1655. ugdill, L., Crone, D., Murphy, R., 2009. References Mindell J Watkins S Cohen J 2011a Health on the move Policies for Health Promoting Transport Transport and Health Study Group Stockport (2) Miles, M.B., Huberman, A.M., Saldaña, J., 2014. Qual. Data Anal. Methods Sourcebook. Mindell, J., Cohen, J.M., Watkins, S., Tyler, N., 2011b. Synergies between low-carbon and healthy transport policies. Proc. Inst. Civil Eng. Transp. 164, 127–139. Mindell J Watkins S Cohen J 2011a Health on the move Policies for Health Promoting Transport Transport and Health Study Group Stockport (2) Mindell, J., Watkins, S., Cohen, J., 2011a. Health on the move, Policies for Health Promoting Transport. Transport and Health Study Group, Stockport (2). Mindell, J.S., Leslie, D., Wardlaw, M., 2012. Exposure-based, “like-for-like” assessment of road safety by travel mode Using routine health data. PLoS One 12 (7), e50606. http: //dx.doi.org/10.1371/journal.pone.0050606. Mindell, J., Watkins, S., Cohen, J., 2011a. Health on the move, Policies for Health Promoting Transport. Transport and Health Study Group, Stockport (2). Mindell, J.S., Leslie, D., Wardlaw, M., 2012. Exposure-based, “like-for-like” assessment of road safety by travel mode Using routine health data. PLoS One 12 (7), e50606. http: //dx.doi.org/10.1371/journal.pone.0050606. Mindell, J.S., Leslie, D., Wardlaw, M., 2012. 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Reviewing the Influence of Welding Setup on FE-Simulated Welding Residual Stresses

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Introduction Finite element (FE) simulation of welding has evolved rapidly in the last decades, although the major goal has ever since remained the same, i.e. the calculation of welding residual stresses (WRS) and plastic strains. The possibility to take into consideration even more effects of real welding in the simulation increased therewith as well. Nevertheless, welding is a complex multiphysics process and many different phenomena (electromagnetic, thermal, mechanical etc.) take place, as it was thoroughly described by Francis et al. [1]. A new challenge has therefore arisen for modern engineers: take into consideration only those factors who exhibit a non- negligible influence on welding residual stresses, depending on the desired preciseness level of each investigated case, in order to balance computational cost and enable better overview of the modelling approach by the practitioner. Straightforward modelling approaches can provide precise results, as long a proper choice of the factors to be taken into consideration is made [2]. Lindgren proposed a concept of filtering simulated factors based on the desired precision [3], from down to basic up to very accurate levels of simulation, by making, nevertheless, a qualitative and not quantitative evaluation. The influence of each simulated factor on the welding residual stresses can differ in each investigated case. For example, the influence of clamped edges, which is proven to have a significant effect on the transverse WRS [4] of butt-welds, is expected to be less significant in the case of fillet welds. Therefore, an investigation of the influence of individual factors on WRS is necessary for a further improvement of welding simulation techniques. The present study reviews previous and new FE simulations in order to evaluate the influence of selected factors of welding simulation. A review of selected welding parameters and modelling approaches, regarding their influence on WRS, is carried out, with the objective to offer guidance to the practitioner. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 license. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under license by Materials Research Forum LLC. Reviewing the Influence of Welding Setup on FE-Simulated Welding Residual Stresses Stefanos Gkatzogiannis1,a,*, Peter Knoedel1,b and Thomas Ummenhofer1,c 1KIT Steel & Lightweight Structures, Research Center for Steel, Timber & Masonry, Otto- Ammann-Platz 1, D-76131 Karlsruhe, Germany [email protected], [email protected], [email protected] Keywords: Welding Residual Stresses, FE Simulation, Boundary Conditions, Heat Input, Material Model, Aluminum, Steel Keywords: Welding Residual Stresses, FE Simulation, Boundary Conditions, Heat Input, Material Model, Aluminum, Steel Abstract. Previous and new simulations of welding residual stresses with the finite element method are reviewed in the present study. The influence of modelling mechanical boundary conditions, erroneous prediction of the weld heat source coefficient and the influence of microstructural changes in aluminum welds are investigated. The results are analyzed so that concrete suggestions regarding the investigated factors, acting as guidance to the practitioner, can be presented. Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Residual Stresses 2018 – ECRS-10 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC ://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 Reviewing the Influence of Welding Setup on FE-Simulated Welding Residual Stresses Stefanos Gkatzogiannis1,a,*, Peter Knoedel1,b and Thomas Ummenhofer1,c 1KIT Steel & Lightweight Structures, Research Center for Steel, Timber & Masonry, Otto- Ammann-Platz 1, D-76131 Karlsruhe, Germany astefanos gkatzogiannis@kit edu bpeter knoedel@kit edu cthomas ummenhofer@kit edu Theoretical Background As already previously discussed, welding is a multiphysics problem, but simulation of WRS requires modelling only of some aspects of the welding process. The thermal behavior and possible microstructural changes are investigated along with the mechanical behavior of a 197 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 component when WRS are under the scope. These three fields, which are presented in Fig. 1 are interacting bidirectionally with each other. Practical models, predicting the WRS with satisfying preciseness by only simulating the interaction of thermal on microstructural and mechanical behavior and of microstructural field on the mechanical behavior have been developed [2]. The considered interactions are presented in Fig. 1. Ignoring the reverse influence of microstructural and mechanical behavior enables solution of the problem with a sequential unidirectionally coupled thermal–mechanical analysis [2]. A description of the numerical background is avoided in the present case for sake of space, but it is thoroughly provided in previous work of the authors [2]. Modelling of the thermal field includes the simulation of the weld heat source, the heat transfer inside the investigated component and heat losses to the surrounding environment (boundary conditions). The Goldak’s double ellipsoidal is the state-of-the-art approach for modelling the weld heat source [5]. The effective heat input is predicted by multiplying the power of the heat source with a coefficient of the weld heat source. Proposed values of this coefficient for various weld types are proposed in [6]. Nevertheless, different values usually in a range of ±10 % for same welding types are found elsewhere (see [7] etc.). Heat transfer inside the component is governed by three dimensional Fourier’s law of heat conduction. Heat losses are modelled according to Newton’s law of cooling. A transient thermal analysis is carried out, whereby the temperature history of the nodes of the FE model are saved at each solution step. Fig. 1: Investigated fields and respective interactions in an engineering approach for arc welding simulation, TRIP stands for transformed induced plasticity [2] Fig. 1: Investigated fields and respective interactions in an engineering approach for arc welding simulation, TRIP stands for transformed induced plasticity [2] Regarding the modelling of the microstructural changes that take place, different models have been proposed in the past. Investigated Factors Results from previous and new FE analyses, which were all carried out according to the above- described theoretical background, are presented in the current study in order to review specific aspects of weld simulation. FE commercial software ANSYS [13] was applied in all cases. Solid 8 node elements “solid 90” and “solid 180” were applied for the transient thermal and the static structural analyses respectively in all cases. The following aspects are investigated: • Modelling of mechanical boundary conditions (BC): A single-pass weld component of Swedish steel HT36 (equivalent to S355) with dimensions of 2000 mm x 1000 mm x 15 mm was simulated in [10] (component A in the present study). Geometry of the weld section is presented in Fig. 2. The component was welded with submerged-arc welding (three electrodes welding consecutively), an electric power of 98 kW and a welding speed of 25 mm/s (150 cm/min; 3.92 kJ/mm heat input). Applied material parameters are given in [2]. Clampers were modelled either by fixing the respective nodes in all directions or by applying linear spring elements with stiffness of 106 N/mm to all nodes in the clamped area in the longitudinal and transverse direction and fixing their vertical displacement. The results are compared with measurements for an identical component found elsewhere [7]. • Thermal input and welding sequence of multi-pass welds: Influence on the WRS, which can be caused by possible erroneous modelling of thermal heat input is investigated in [9]. The 5- pass X-grooved butt-weld with dimensions 300 mm x 300 mm x 10 mm by AISI 316L, which is presented in Fig. 2, was simulated (component B in the present study) and different cases of differentiating heat input were modelled. The component was initially considered to be welded with an electric power of 4.3 kW, a welding speed of 5 mm/s (30 cm/min; 0.86 kJ/mm heat input) and the welding sequence A-B-C-D-E. Applied material parameters are given in [9]. Effective heat input was changed by either increasing the welding speed, or reducing the heat input (see Table 1). The results are reviewed from a different scope in the present study in order to estimate the possible error of calculated WRS under the assumption that an under- or overestimation of ± 10 % of the weld heat source’s efficiency has taken place. Theoretical Background A straightforward engineering approach, which was proposed by the authors of the present study [2], provided results with sufficient preciseness and can be applied for the simulation of various materials [8], [9], [10]. Main feature of the approach lies on the assignment of material models to the finite elements inside the fusion zone (FZ) and the heat- affected zone (HAZ) during cooling down based on predominant parameters of the thermal cycle, in order to simulate the modified mechanical behavior of the transformed microstructure. The selection of material models is based on predictions of the transformed microstructure according to continuous cooling transformation (CCT) diagram or assessment of microstructure through measurements. The importance or negligibility of microstructural changes during modelling of ferritic or austenitic steels respectively has been confirmed in the past [2], [9]. Softening in the HAZ of aluminum alloys was as well successfully modelled elsewhere [8]. Nevertheless, a quantification of this influence on WRS is not known to the authors of the present study. The same problem arises as well when high strength steels are regarded. Liu et al. [11] and Lee and Chang [12] simulated weldments by ASTM A514 (yield strength of 717 MPa) with phase changes and a high strength carbon steel with yield strength of 790 MPa by neglecting them respectively. 198 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC Finally, during modelling of the mechanical field, the nodal temperature history from the transient thermal analysis is applied as external thermal strains. Solution takes place in a static structural analysis, whereby temperature dependent material parameters and the restraints applied to the real welded component are modelled [10]. Usually, components are clamped down during welding. Fixing of the respective nodes in the FE models is a common approach of modelling but deviates from physical restraining reality. An alternative approach applied elsewhere [10] is the use of linear spring elements, restraining the respective nodes (see Fig. 4). Further steps of the mechanical solution are based on classical finite element theory for nonlinear materials. Investigated Factors • Microstructural modelling: Numerical studies regarding welding of Aluminum alloy initially presented at [8], are repeated in the present study, by neglecting this time the phase transformations, in order to evaluate qualitatively and quantitatively the influence of recrystallization in the HAZ of aluminum alloys on the simulated WRS. A single-pass V grooved component of EN AW-6060 welded with an electric power of 3.1 kW and a welding speed of 10 mm/s (60 cm/min; 0.31 kJ/mm heat input) is modelled. • Microstructural modelling: Numerical studies regarding welding of Aluminum alloy initially presented at [8], are repeated in the present study, by neglecting this time the phase transformations, in order to evaluate qualitatively and quantitatively the influence of recrystallization in the HAZ of aluminum alloys on the simulated WRS. A single-pass V grooved component of EN AW-6060 welded with an electric power of 3.1 kW and a welding speed of 10 mm/s (60 cm/min; 0.31 kJ/mm heat input) is modelled. Results and Discussion The calculated longitudinal WRS for the HT36 component A are presented in Fig. 4. “Meas.” stands for measured WRS found in [7], while “Fixed” and “Springs” are referring to the respective applied modelling approach of BC. In both cases, tensile longitudinal stresses, higher than the nominal yield limit of the material at room temperature are met in the FZ and HAZ and 199 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Proceedings 6 (2018) 197-202 compressive stresses in the areas away from the weld. In the case of modelling with spring elements, tensile stresses of even up to 700 MPa are met (yield strength in the weld area is higher than the nominal 355 MPa due to the phase changes taken place – increase of bainitic and martensitic phases), while for the fixed case the maximum tensile stress is not higher than 450 MPa. Although both approaches produce similar results qualitatively, the agreement of the spring model with the measurements inside the weld section is clearly better. Similar improvement due to use of spring elements was observed as well in the case of calculated transverse WRS [10]. Fig. 2: Investigated component A – left: Geometry and clampers, dimensions are given in mm – right: Applied spring elements for the simulation of clampers Fig. 2: Investigated component A – left: Geometry and clampers, dimensions are given in mm – right: Applied spring elements for the simulation of clampers The calculated longitudinal WRS for the AISI 316L component B are presented in Fig. 4. Similar profiles of longitudinal WRS are calculated in all investigated cases (see Table 1). Tensile and compressive WRS are met near and away from the weld respectively. The increase of the heat input rate, either by increase of heat input or reduction of welding speed, shifts down the calculated profile of WRS. Nevertheless, the largest deviation from the initial profile (H1) is met in the case of reduced welding speed down to 50 % (case V2). A difference of up to 150 MPa or 28 % between the peak tensile stresses of those two cases is observed. Similar but not so significant differentiation of the transverse WRS profiles was observed as well [9]. Results and Discussion g p [ ] Table 1: Reviewed investigated cases found in [9] Investigated case Model Investigated variable welding parameters H1 initial welding parameters Fig. 3: Investigated component B, dimensions are given in mm V1 welding speed – 25 % reduced V2 welding speed – 50 % reduced Q1 heat input – 25 % increased Q2 heat input – 50 % increased Fig. 3: Investigated component B, dimensions are given in mm The calculated WRS for the EN AW 6060 component C are presented in Fig. 5 for the cases of taking into consideration and neglecting the recrystallization in the HAZ. In the case of the longitudinal WRS the calculated profile differs significantly, both qualitatively and quantitatively, near the weld. Neglecting the microstructural changes, leads to a lower calculated peak stress and a shift of this peak stress from the boundaries between HAZ and parent material in the middle of the component. In the case of the transverse WRS, neglecting recrystallization causes a shift up of the calculated profile of up to 100 MPa. Conclusions The following conclusions were drawn, based on the above-presented results: • Applied approach for modelling of clampers during a weld simulation can have a significant effect on the calculated WRS, at least in the case of butt-welds. The use of spring elements for modelling the longitudinal and transverse restraints in the clamped area produces results • Applied approach for modelling of clampers during a weld simulation can have a significant effect on the calculated WRS, at least in the case of butt-welds. The use of spring elements for modelling the longitudinal and transverse restraints in the clamped area produces results 200 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 that show better agreement with experimentally measured WRS. Moreover, as higher tensile WRS are calculated, this approach lies on the safe side. This modelling approach is therefore suggested for adoption in practical applications as well. suggested for adoption in practical applications as well. Fig. 4: Longitudinal WRS – left: Component A – right: Component B, resulting WRS on the top of the component, for the investigated cases presentes in Table 1 -200 -100 0 100 200 300 400 500 600 700 800 -80 -60 -40 -20 0 Longitudinal WRS [MPa] Distance from weld centerline [mm] Meas. [7] Meas. in the weld [7] Fixed Springs -400 -200 0 200 400 600 0 10 20 30 40 50 Longitudinal WRS [MPa] Distance from weld centerline [mm] H1 V1 V2 Q1 Q2 -200 -100 0 100 200 300 400 500 600 700 800 -80 -60 -40 -20 0 Longitudinal WRS [MPa] Distance from weld centerline [mm] Meas. [7] Meas. in the weld [7] Fixed Springs -400 -200 0 200 400 600 0 10 20 30 40 50 Longitudinal WRS [MPa] Distance from weld centerline [mm] H1 V1 V2 Q1 Q2 Distance from weld centerline [mm] Fig. 4: Longitudinal WRS – left: Component A – right: Component B, resulting WRS on the top of the component, for the investigated cases presentes in Table 1 -200 -150 -100 -50 0 50 100 150 200 -150 -130 -110 -90 -70 -50 -30 -10 Longitudinal WRS [MPa] Rec No rec Fig. 5: WRS of the alluminum component C with and without modelling of recrystilisation in the HAZ, collored areas of the component show modelling of the microstructural changes, A: parent material, B: HAZ 50 % recrystalized, C: HAZ 100 % recrystlized, D: FZ. Acknowledgement The above-presented work was carried out as a part of the framework of a PhD thesis at Karlsruhe Institute of Technology [16]. Conclusions -200 -150 -100 -50 0 50 100 150 200 -150 -130 -110 -90 -70 -50 -30 -10 Longitudinal WRS [MPa] Distance from weld centerline [mm] Rec No rec -200 -150 -100 -50 0 50 100 150 200 0 20 40 60 80 100 120 140 Transverse WRS [MPa] Distance from weld centerline [mm] Rec No rec Fig. 5: WRS of the alluminum component C with and without modelling of recrystilisation in the HAZ, collored areas of the component show modelling of the microstructural changes, A: parent material, B: HAZ 50 % recrystalized, C: HAZ 100 % recrystlized, D: FZ. • A possible erroneous modelling of the weld heat source could lead to an underestimation of the WRS and therefore non-conservative results. An increase of 50 % of the heat input rate led to a reduction of 28 % of the peak tensile stress (worst case scenario). Assuming a linear behavior, an overestimation of 10 % of the heat source coefficient would lead to an underestimation of 5.6 % of the peak tensile stresses. This deviation lies in the boundaries of the acceptable numerical error, of practical weld simulations (± 10 % [2]). Therefore, applying values for the coefficient of weld heat source from literature or previous measurements during the simulation of WRS, is considered valid. • Aluminum welding simulations neglecting the recrystallization in the HAZ, produce erroneous results. A lower longitudinal peak stress is calculated and is present at the middle of the component. Simulations considering the microstructural changes show that the peak stress is met on the boundaries of the HAZ, exhibiting a much more crucial case, regarding fatigue strength of the investigated component. Therefore, neglecting recrystallization in the HAZ of aluminum weld during weld simulation is not conservative and should be avoided. 201 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Residual Stresses 2018 – ECRS-10 Materials Research Forum LLC Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Forum LLC doi: http://dx.doi.org/10.21741/9781945291890-31 Materials Research Proceedings 6 (2018) 197-202 doi: http://dx.doi.org/10.21741/9781945291890-31 References [1] J.A. Francis, H.K.D.H. Bhadeshia, P.J. Withers, Welding residual stresses in ferritic power plant steels, Material Science and Technology 23 (2007) 1009-1020. https://doi.org/10.1179/174328407X213116 [2] P. Knoedel, S. Gkatzogiannis, T. Ummenhofer, Practical aspects of welding residual stress simulation, Journal of Constructional Steel Research 132 (2017) 83–96. https://doi.org/10.1016/j.jcsr.2017.01.010 [3] L.-E. Lindgren, Computational Welding Mechanics - Thermomechanical and Microstructural Simulations, Woodhead Publishing in Materials, first ed., Cambridge England, 2007. https://doi.org/10.1201/9781439824092 [4] S. Kou, Welding Metallurgy, John Wiley & Sons, Inc., second ed., Hoboken, New Jersey, 2003. [5] J.A. Goldak, A. Chakravarti, M. Bibby, A new finite element model for welding heat sources, Metall. Trans. B 15 (1984) 299–305. https://doi.org/10.1007/BF02667333 [6] J.N. Dupont, A.R. Marder, Thermal efficiency of arc welding processes, Weld. J. 74 (1995) 406–416. [7] B. Andersson, Thermal Stresses in a submerged-arc welded joint considering phase transformations, Trans. ASME 100 (1978) 356–362. https://doi.org/10.1115/1.3443504 [8] P. Knoedel, S. Gkatzogiannis, T. Ummenhofer, FE simulation of residual welding stresses: Aluminum and steel structural components, Key Engineering Materials 710 (2016) 268-274. https://doi.org/10.4028/www.scientific.net/KEM.710.268 [9] S. Gkatzogiannis, P. Knoedel, T. Ummenhofer, Influence of welding parameters on the welding residual stresses, Proceedings of the VII International Conference on Coupled Problems in Science and Engineering, Rhodes Island, Greece, June 12–14 (2017) 767–778. [10] S. Gkatzogiannis, P. Knoedel, T. Ummenhofer, FE welding residual stress simulation - Influence of boundary conditions and material models. EUROSTEEL 2017, September 13–15, 2017, Copenhagen, Denmark, (2017), Ernst & Sohn Verlag für Architektur und technische Wissenschaften GmbH & Co. KG, Berlin. Influence of boundary conditions and material models. EUROSTEEL 2017, September 13–15, 2017, Copenhagen, Denmark, (2017), Ernst & Sohn Verlag für Architektur und technische Wissenschaften GmbH & Co. KG, Berlin. [11] W. Liu, J. Ma, F. Kong, S. Liu, R. Kovacevic, Numerical modeling and experimental verification of residual stress in autogenous laser welding of high-strength steel, Lasers Manuf. Mater. Process. 2 (2015) 24–42. https://doi.org/10.1007/s40516-015-0005-4 [12] C.H. Lee, K.H. Chang, Prediction of residual stresses in high strength carbon steel pipe weld considering solid-state phase transformation effects, Computers and Structures 89 (2011) 256–265. https://doi.org/10.1016/j.compstruc.2010.10.005 ] ANSYS® Academic Research, Release 18.2, Help System, ANSYS, Inc., (2018) [14] S. Gkatzogiannis, Finite Element Simulation of High Frequency Hammer Peening, Ph.D. thesis (in progress), KIT, Karlsruhe Institute of Technology, Department of Civil, Geo and Environmental Sciences, KIT Steel & Lightweight Structures, 2018. 202

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Considerations on the consequences of stresses related to the challenges of professional and private life

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Pruszyński Jacek J., Pruszyńska Irena B., Włodarczyk-Pruszyńska Inga Z. Considerations on the consequences of stresses related to the challenges of professional and private life. Journal of Education, Health and Sport. 2022;12(6):67-79. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2022.12.06.006 https://apcz.umk.pl/JEHS/article/view/JEHS.2022.12.06.006 https://zenodo.org/record/6512029 Pruszyński Jacek J., Pruszyńska Irena B., Włodarczyk-Pruszyńska Inga Z. Considerations on the consequences of stresses related to the challenges of professional and private life. Journal of Education, Health and Sport. 2022;12(6):67-79. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2022.12.06.006 https://apcz.umk.pl/JEHS/article/view/JEHS.2022.12.06.006 https://zenodo.org/record/6512029 urnal has had 40 points in Ministry of Education and Science of Poland parametric evaluation. Annex to the announcement of the Minister of Education and Science of December 21, 2021. No. 32343. rnal's Unique Identifier: 201159. Scientific disciplines assigned: Physical Culture Sciences (Field of Medical sciences and health sciences); Health Sciences (Field of Medical Sciences and Health Scienc Punkty Ministerialne z 2019 - aktualny rok 40 punktów. Załącznik do komunikatu Ministra Edukacji i Nauki z dnia 21 grudnia 2021 r. Lp. 32343. Posiada Unikatowy Identyfikator Czasopisma: 201159. Przypisane dyscypliny naukowe: Nauki o kulturze fizycznej (Dziedzina nauk medycznych i nauk o zdrowiu); Nauki o zdrowiu (Dziedzina nauk medycznych i nauk o zdrowiu). Punkty Ministerialne z 2019 - aktualny rok 40 punktów. Załącznik do komunikatu Ministra Edukacji i Nauki z dnia 21 grudnia 2021 r. Lp. 32343. Posiada Unikatowy Iden Przypisane dyscypliny naukowe: Nauki o kulturze fizycznej (Dziedzina nauk medycznych i nauk o zdrowiu); Nauki o zdrowiu (Dziedzina nauk medycznych i © The Authors 2022; ; This article is published with open access at Licensee Open Journal Systems of This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 20.04.2022. Revised: 25.04.2022. Accepted: 02.05.2022. Considerations on the consequences of stresses related to the challenges of professional and private life Jacek J. Pruszyński MD, PhD, Department of Geriatrics and Gerontology, School of Public Health, Center of Postgraduate Medical Education, Warsaw, 01-826 Poland; [email protected], ORCID: 0000-0003-2123-6488 Irena B. Pruszyńska DVM, Department of Cancer Biology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, 02-787 Poland; ORCID: 0000-0003-0784-8066 Inga Z. Włodarczyk-Pruszyńska MD, II Department of Radiology, Medical University of Warsaw, Warsaw, 02-091 Poland; ORCID: 0000-0003-2719-1815 Keywords: work, risk of addiction, supportive professions, workaholism, burnout This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Pruszyński Jacek J., Pruszyńska Irena B., Włodarczyk-Pruszyńska Inga Z. Considerations on the consequences of stresses related to the challenges of professional and private life. Journal of Education, Health and Sport. 2022;12(6):67-79. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2022.12.06.006 https://apcz.umk.pl/JEHS/article/view/JEHS.2022.12.06.006 https://zenodo.org/record/6512029 Abstract There is a group of people for whom work is an important factor in building their self-esteem. Often, such people include workers from the group of “supportive” professions, which promote behaviours that increase the risk of addiction to work. The factors contributing to this are the constant rush along with the individual tendency for perpetual self-control and meticulous checking of each performed activity. People who are idealistic about their duties, set the bar high, take too much responsibility and at the same time have low self-esteem, fall into the trap of addiction more often. The inability to independently regulate the time of their work means that they are driven by a constant internal compulsion to perform work and to think about it, combined with the accompanying feeling of discomfort in situations when the ability to deal with matters related to work is limited. Burnout can be seen as a compensatory 67 symptom that occurs when recognition and approval are not present in a person's life, and low self-esteem leads to an unrealistic pursuit of excellence, recognition and acceptance. A noticeable tell of burnout is the loss of features that usually characterize a person involved in their activities such as creativity, conscientious fulfilment of obligations, willingness to cooperate with others, communication skills, the ability to deal with stressful events and the ability to make independent decisions. The abovementioned skills are disrupted as a result of a number of mental, physical and behavioural changes associated with the burnout syndrome. Recovering from addiction requires complex therapy, improvement of interpersonal relationships and creation of an appropriate environment for functioning in professional and personal life. Einleitung. Es kann davon 68 ausgegangen werden, dass die meisten Religionen und Kulturen ein gemeinsames Konzept einer Arbeitsethik zu haben scheinen, die als Verpflichtung zu Anstrengung und Streben nach Exzellenz definiert ist [4]. Bereits im 19. Jahrhundert beschrieb der Schriftsteller Paul Lafargue ein solches Phänomen: übermäßige Arbeitslust, fiebrige Arbeitssucht als das schlimmste Übel der damaligen Zeit, als Ursache für die Verschlechterung der geistigen und körperlichen Verfassung. In seinem Text „Das Gesetz zur Faulheit“ (1883) kritisierte er die Arbeitsmoral des Durchschnittsbürgertums und die damit verbundenen Folgen der Überproduktion. Die Kritik an Paul Lafargue scheint bis heute gültig zu sein. Viele Menschen arbeiten immer noch bis zum Umfallen. Die Zahl der Workaholics wächst von Jahr zu Jahr [5]. Eine weitere frühe Beschreibung des Burnout-Phänomens war die 1953 veröffentlichte Fallstudie einer Krankenschwester, die in einer psychiatrischen Abteilung arbeitete. Durch angespannte Arbeitsereignisse und die Auswirkungen verschiedener Stressoren begann sie sich müde, geistig und körperlich hilflos zu fühlen, sie wurde skeptisch gegenüber den Bedürfnissen der Patienten und war mit ihren beruflichen Aufgaben unzufrieden [6]. Kurz nach dieser Veröffentlichung wurde die Kurzgeschichte „A Burn-Out Case“ von Graham Greene geschrieben, in der der arbeitsmüde Protagonist, ein weltberühmter Architekt, der unter Symptomen leidet, die für das Burnout-Syndrom charakteristisch sind, beschließt, seinen derzeitigen Lebensstil komplett zu ändern, Er kündigt seinen Job und lebt im afrikanischen Busch [7]. Die ersten Bücher über Suchtarbeit erschienen in den 1970er Jahren in den USA. Ein Artikel im Wall Street Journal aus dem Jahr 1971 hob Mitarbeiter hervor, denen es schwer fällt, abends das Büro zu verlassen und nach Hause zu gehen. Trotzdem waren die damaligen Berichte über Arbeitssucht eher anekdotisch. Einige amerikanische Forscher gehen sogar so weit, Workaholismus als erstrebenswert anzusehen, nicht nur für Unternehmen, sondern auch für den Einzelnen [8]. Es gibt mehrere Verbindungen zwischen obsessivem Arbeitsengagement (Workaholismus) und dem Burnout-Syndrom. Arbeitssucht kann als ein bestimmter Bewusstseinszustand betrachtet werden, der zum Verlust des Wirklichkeitsbezugs führen kann. Der Workaholic vernachlässigt in seiner übermäßigen Begeisterung für die Arbeit Familie, Freunde, Erholung und seine eigenen Interessen. Alle Angelegenheiten, die nicht mit dem Berufsleben zu tun haben, sind seiner Meinung nach von geringer Bedeutung. Häufiger verfallen in die Arbeitssucht Menschen, die idealistisch an ihre beruflichen Aufgaben herangehen, die Messlatte für sich selbst hoch legen und meist zu viel Verantwortung übernehmen [9]. Einleitung. Sigmund Freud wurde gefragt, was seiner Meinung nach ein normaler Mensch können müsse. Er hat geantwortet: "Lieben und arbeiten". Spezialist für Psychoanalyse hat so die Eckpfeiler des menschlichen Daseins, den Lebenssinn, genannt. Noch heute sind sich die meisten westlichen Länder einig, dass Arbeit die zentralste Qualität des Menschen sein kann. Wenn wir eine neue Person kennenlernen, wird eine der ersten Fragen nach dem Beruf sein. Unsere Arbeit ist von großer Bedeutung für unser Selbstverständnis [1]. Der Begriff der Arbeit als landesweites Phänomen findet sich in der Literatur vor allem in Bezug auf die Japaner. Die Gründe für lange Arbeitszeiten in Japan sind nicht nur wirtschaftlicher (z. B. organisatorischer Mehrarbeitsbedarf durch zunehmenden Wettbewerb), sondern auch soziokultureller Natur. Arbeit ist für die Japaner der Prozess der Erfüllung von Verpflichtungen gegenüber der Gesellschaft und sich selbst als soziales Wesen. Mitarbeiter sind wie alte Samurai, die die Absichten ihrer Vorgesetzten ausgeführt haben, ohne Fragen zu stellen, Beschwerden oder Einwände zu melden. In der japanischen Kultur ist die am Arbeitsplatz verbrachte Zeit oft ein symbolischer Ausdruck der Unterwerfung unter die Autorität des Managements und der Loyalität gegenüber der Organisation [2]. g y g g g Arbeit kann allgemein als das Gegenteil von Freizeit definiert werden. Manche Menschen können sich ihr Leben ohne Arbeit nicht vorstellen, denn nur dank ihr können sie sich verwirklichen, für andere ist es nur die Notwendigkeit, ihr Überleben zu sichern. Man könnte sagen, die einen leben um zu arbeiten und die anderen arbeiten um zu leben. Für viele Menschen ist die Arbeit eine zeitraubende Tätigkeit und eine Gelegenheit, Kontakte zu knüpfen und persönliche Zufriedenheit zu Erlangen [3]. Arbeit kann zu einem zentralen Bezugspunkt im Leben eines Menschen werden, wenn er davon ausgeht, dass Arbeit wichtiger ist als andere Lebensbereiche. Der zentrale Arbeitsplatz steht in negativem Zusammenhang mit der Ethik der Freizeitgestaltung. Es ist davon auszugehen, dass eines der Elemente, die die Zustimmung zum Workaholismus ausmachen, in den Glaubensgrundsätzen zu finden ist, wonach Arbeit den Gläubigen rettet und das Schwelgen in Vergnügungen ewige Verdammnis bringt. Der Anspruch, dass Arbeit eine Tugend und Spiel Sünde ist, prägt auch heute noch die Mentalität mancher Gesellschaften. Die Arbeitsethik ermutigt die Menschen, hart zu arbeiten, viele Stunden damit zu verbringen, und spiegelt sich in Redewendungen wider, wie z. B. eine Person, die sich ihrer Arbeit „verschrieben“ hat. Einleitung. Zu diesen Personen unter anderem Gesundheitspersonal, die aufgrund der Art ihrer Arbeit wie in den meisten anderen helfenden Berufen Verhaltensweisen zeigen, die das Risiko einer Arbeitssucht erhöhen. Dazu gehören ununterbrochene Eile, gepaart mit einem Hang zu ständiger Selbstbeherrschung und akribischer Kontrolle jeder ausgeführten Tätigkeit. In den Vereinigten Staaten von Amerika wurde in einem breiten Spektrum medizinischer Fachrichtungen zwischen 25 % und 60 % Burnout gemeldet [10]. Eine große Gruppe von Burnout-Opfern sind neben medizinischem und veterinärmedizinischem Personal, deren Arbeit auch eine emotionale Einbindung in die Probleme anderer erfordert, also Lehrer, Geistliche, Polizisten, Gesundheits- und Sozialarbeiter. Arbeitssucht Der Begriff „Workaholic“ wurde erstmals 1968 von Pastor (und Psychologieprofessor) Wayne Oates verwendet. Er definierte Workaholismus als „Arbeitssucht, Zwang oder unkontrolliertes Bedürfnis, ohne Unterbrechung zu arbeiten“, während er erkannte, dass Arbeitssucht ein Ersatz für Alkohol sein kann und auf ähnlichen psychologischen Mechanismen beruht [11,12]. 1979 beschrieb der Arzt Mentzel das Phänomen der Arbeitssucht und erklärte, dass sich der 69 süchtig machende Aspekt der Arbeit jedoch von der Wirkung des Alkohols unterscheide, da er keine unmittelbaren gesundheitlichen Probleme verursache. Die Nachwirkungen unterscheiden sich jedoch nicht wesentlich. Es ist sichtbar: „… dieses Verhalten ein Workaholic ist einem Alkoholiker verblüffend ähnlich." Mentzel bemerkte auch, dass die absolute Menge an geleisteter Arbeit der Einstellung zur Arbeit selbst untergeordnet sei. Berger beschrieb die Diagnose von Arbeitssucht wie folgt: „Der Schlüssel ist das Konzept des Kontrollverlusts, der das Maß an Wahlfreiheit und Willensfreiheit im Verhalten bei der Arbeit darstellt. Arbeitssucht wird nicht daran gemessen, was und wie viel der Betroffene tut, sondern daran, was er nicht kann“ [13]. Seitdem wurden viele Definitionen von Workaholismus entwickelt, definiert als Arbeitssucht, Arbeitsabhängigkeit, Arbeitsbesessenheit oder Arbeitsverlangen. Workaholismus definierte man unter anderem als ständigen übermäßigen Einsatz von Zeit und Energie in die Arbeit sowie exzessives und zwanghaftes Arbeiten, das durch innere oder äußere Faktoren verursacht wird und das möglicherweise zu psychischen, gesundheitlichen und sozialen Beeinträchtigungen sowie nachteiligen Auswirkungen auf das Arbeitsumfeld führt. Es wurde auch versucht, zwischen Arbeit und Arbeitssucht quantitativ zu unterscheiden, indem man die Dauer der ersteren auf 50 Stunden pro Woche begrenzte oder annahm, dass es sich bei einer Arbeit von mehr als 11,5 Stunden pro Tag um Arbeitssucht handelt [14]. Es ist anzunehmen, dass Arbeitssucht mit dem Phänomen einer gestörten bewussten Zeiterfahrung zusammenhängt, da eine übertriebene Zustimmung zur Verlängerung von Arbeitszeiten auf Kosten des Ausschlusses anderer Lebensformen vorliegt [15]. Man hat den Eindruck, dass Workaholics sich hauptsächlich auf die Gegenwart und die nahe Zukunft konzentrieren. Die ferne und fernste Zukunft ist ebenso wie die Vergangenheit nicht mehr Gegenstand ihres Interesses, als wollten diese Menschen ständig in der Gegenwart leben, die für sie zu einem Ersatz für die wahre Ewigkeit wird. Die Unfähigkeit, die Zeit ihrer Arbeit selbstständig zu regulieren, führt dazu, dass Workaholics einen ständigen, inneren Zwang (starkes Bedürfnis) zur Verrichtung von Arbeit oder anderen damit zusammenhängenden Tätigkeiten und den Zwang zum Nachdenken über die Arbeit verbunden mit dem begleitenden Gefühl von Unbehagen (Unwohlsein) empfinden, sobald sie arbeitsbezogenen Angelegenheiten eingeschränkt nachgehen können. Arbeitssucht Die Beziehung eines Workaholics zu seiner Arbeit stellt eine ernsthafte Konkurrenz für andere wichtige Lebensbeziehungen dar, und die Arbeit wird zu einer Quelle größerer Befriedigung als das Familienleben und andere bisherige Beziehungen und Leidenschaften. Das Verhalten eines Arbeitssüchtigen (der die meiste Zeit beruflichen Angelegenheiten widmet) kann auch ein Versuch sein, vorhergesagte negative Szenarien, die in seiner Arbeit auftreten können, und folglich negatives Denken über sich selbst zu verhindern. Das Selbstwertgefühl eines Workaholic ist daher bedingt – er muss bestimmte Voraussetzungen erfüllen, um sich zu beweisen und ein Scheitern zu vermeiden, das, wenn es auftritt, stark erfahren wird [16]. Nach der rational-emotiven Verhaltenstherapie von Albert Ellis sind die Annahmen einer übermäßig involvierten Person: Wenn ich nicht meine ganze Zeit der Arbeit widme, werde ich meine Aufgaben und meinen Job nie so gut wie andere erledigen können, und ich muss einen guten Eindruck machen, die Erwartungen anderer Menschen erfüllen und sie in der Wirksamkeit der Handlungen sogar übertreffen [17]. Als Folge solcher Annahmen arbeitet der Workaholic immer mehr, obwohl mehr als 11 Stunden am Tag bei der Arbeit zu verbringen, neben vielen ungünstigen Phänomenen, auch das Risiko erheblich erhöht, die Qualität seiner Aktivitäten zu verschlechtern und sogar unbeabsichtigte, aber wichtige Fehler zu begehen für das Ergebnis seiner Arbeit durch einen dauerhaft überlasteten Mitarbeiter. Besonders wichtig ist es in Berufen wie Arzt, Zahnarzt, Krankenpfleger oder Tierarzt. 70 Workaholismus, der die Funktionsfähigkeit und Gesundheit und manchmal das Leben eines Workaholics bedroht, ist, wie andere Süchte, ein Krankheitszustand. Gesundheit als Bedingung für die effiziente und effektive Teilnahme eines Individuums am gesellschaftlichen Leben und seine Fähigkeit, wertvolle Aufgaben zu erfüllen, und Krankheit als "schädliche Dysfunktion" scheint am nützlichsten zu sein, um die Bedrohung durch Arbeitssucht durch Störung der psychischen, Soziales und körperliches Wohlbefinden der Betroffenen [18]. Das aktuelle Gesellschaftssystem, das auf die Idee des Fortschritts ausgerichtet ist und auf dem Prinzip des Unternehmertums und der Funktionalität basiert, in dem der Erfolg eines Individuums als das Streben nach einer Karriere, Anerkennung der Umwelt oder einer guten sozialen Position verstanden wird, übersetzt diese Bestrebungen in die Notwendigkeit ständiger Aktivität. Diese Aktivität drückt sich vor allem in der Arbeit aus, die eine Art Fetisch ist und für viele Menschen an sich der primäre Wert ist. Arbeitssucht Es ist anzunehmen, dass die Situation, in der zwischen dem Begriff „beruflich mehr engagiert als andere“ und dem Begriff des „erfolgreichen Menschen“ ein Gleichheitszeichen gesetzt wird, eine der Ursachen für die Verbreitung der Suchtform wie Arbeitssucht ist, insbesondere wenn Arbeitsengagement mit einem von der Gesellschaft positiv wahrgenommenen Lebensstil gleichgesetzt wird [19]. Begünstigt wird dies durch das in den Medien verbreitete Bild einer „beruflich engagierten“ Person und implizit einer Person, die „Karriere macht“. Dieser Haltung steht der Begriff der Freiheit entgegen, der in der europäischen Kultur als unveräußerliches Recht eines jeden Menschen gilt. Workaholismus zerstört die Freiheit des Einzelnen dadurch, dass die Betroffenen der Suchtgewalt erliegen, die das Erreichen anderer Lebensziele erschwert oder sogar unmöglich macht. Arbeitssucht und Burnout-Syndrom Zu den wichtigen Faktoren, die den mentalen, emotionalen und körperlichen Zustand von Menschen mit Burnout-Syndrom beeinflussen, gehören die ungewöhnliche (unregelmäßige, Schicht-, Nacht-)Arbeitsweise und die Besonderheit der Arbeitsbelastung, die in den meisten helfenden Berufen üblich ist; das Tempo und die Arbeitsbedingungen, der Mangel an institutioneller Unterstützung und die Unvorhersehbarkeit der Auswirkungen von Handlungen [27]. Die Burnout-Beschreibung von Freudenberger berücksichtigte die Bedeutung persönlicher Eigenschaften und Motivationen, die den Mitarbeiter beeinflussen. Menschen, die sich ihrer Arbeit widmen, spüren einen inneren Druck, der sie motiviert, so viel wie möglich zu geben. Eine solche Haltung kann den bei diesen Menschen oft vorkommenden Eindruck erklären, dass die eigenen Bedürfnisse und Wünsche den Bedürfnissen und Wünschen der Umwelt untergeordnet sind [28]. Erfolglose Versuche, die an sich selbst gestellten unrealistisch hohen Erwartungen zu erfüllen, lösen Schuldgefühle aus, die wiederum dazu führen können, dass man versucht, diesen Zustand durch noch stärkere Beteiligung an den eigenen Aktivitäten zu kompensieren. In Anbetracht der zunehmenden Verbreitung von Burnout verwendet Freudenberger in seinem Buch „Burn-Out: the high cost of high achievement” die folgenden Begriffe, um es zu beschreiben: „verarmen, körperliche und geistige Ressourcen erschöpfen, sich selbst zerstören durch exzessives Streben nach Zielen aufgrund unrealistischer Erwartungen, die man sich selbst auferlegt oder die einem von außen durch soziale Werte auferlegt werden“. Diese Begriffe implizieren nicht die Notwendigkeit eines Zusammenhangs zwischen Burnout und Arbeit [29], sodass der Burnout-Begriff weiter gefasst werden kann als ein Konflikt zwischen einem idealisierten Selbstbild und dem realen, unvollkommenen „Ich“, dem dieses idealisierte Bild widerspricht. Burnout kann sich in diesem Fall als Folge dieser Verleugnung herausstellen. Das Burnout-Syndrom kann als spezifische Form oder Anfangsstadium einer Stressdepression definiert werden. Der ursprüngliche Begriff beschreibt die Erschöpfungsdepression, die durch ständigen Arbeitsstress entsteht. Hier sind Frauen oft zusätzlich haushalts- und familiären Belastungen ausgesetzt. Durch den über die Jahre akkumulierten Stress reicht oft schon ein relativ kleiner Triggerfaktor aus, um den Krankheitsausbruch auszulösen [30]. Eine weitere Person, die das Problem des Burnouts erforschte, wenn auch in einem etwas engeren Rahmen, hauptsächlich bezogen auf den arbeitsbedingten Burnout, war die Sozialpsychologin Christina Maslach [31,32]. Gemeinsam mit ihren Kollegen von der University of Berkeley forschte sie zum Burnout von Menschen, die in Berufen mit hohem, chronischem Stress arbeiten. Für beruflichen Burnout ist laut Maslach arbeitsbedingter Stress verantwortlich. Diese Definition ging zunächst davon aus, dass Burnout zwei Komponenten hat: Depersonalisation und emotionale Erschöpfung. Im Zuge der weiteren Forschung kam eine weitere Komponente hinzu, nämlich das Gefühl mangelnder persönlicher Leistung [33]. Arbeitssucht und Burnout-Syndrom Wie bereits erwähnt, kann Arbeitssucht zum Burnout-Syndrom führen. Anspruchsvolle Kunden, ständige Erreichbarkeit oder zu kurze Fristen für die Erledigung von Aufgaben – die Arbeit setzt den Mitarbeiter ständig unter Druck. Immer mehr Menschen kämpfen mit diesen Anforderungen [20]. Das Burnout-Syndrom wurde erstmals 1974 in zwei Artikeln beschrieben, einer von Herbert J. Freudenberger [21] und der andere von Sigmund Ginsburg [22]. Freudenberger, der den Begriff „Burnout“ zu einem populären Begriff machte, welcher in die wissenschaftliche Literatur einging, definierte ihn als einen Zustand, der durch ein Gefühl von geistiger und körperlicher Erschöpfung, Ungeduld, übermäßiger Reizbarkeit, verbunden mit Zynismus und chronischer Langeweile, einer Neigung zur Isolation und Unterdrückung von Emotionen gekennzeichnet ist [23]. Der Begriff „Burnout“ bezieht sich auf die Zeit, als Freudenberger sich selbst als Vorbild für berufliche Fehlfunktionen erkannte. Er selbst beschrieb die Situation, in der er sich damals befand: „Du engagierst dich in der Arbeit, weil du es von dir selbst verlangst, deine Mitarbeiter es von dir verlangen und die Bevölkerung, der du Dienst leistet, es von euch verlangt. Wie in solchen Fällen üblich, werden immer mehr Anforderungen an einen immer kleineren Personenkreis gestellt. Dadurch baust du nach und nach in die Menschen um dich herum und in sich selbst ein, dass du unentbehrlich bist. Du engagierst dich immer mehr in das, was du tust. Bis du dich schließlich, genau wie ich, in einem Zustand der totalen Erschöpfung wiederfindest” [24]. Bei der Diskussion der Verhaltenssymptome des Burnout-Syndroms erwähnte Freudenberger Schwierigkeiten bei der Kontrolle seiner Emotionen, leichtes Verfallen in Irritation und Frustration, Wutausbrüche und eine Haltung des Misstrauens und der Paranoia, in der man einerseits das Gefühl haben kann, dass jeder möchte dich verletzen, und andererseits ein Allmachtsgefühl erfahren, das zu übermäßig riskanten Aktivitäten führen kann. Weitere Merkmale von Menschen mit Burnout, auf die Freudenberger aufmerksam machte, waren Sturheit, Starrheit und Inflexibilität sowie der Glaube, in ihrem Job alles durchgemacht zu 71 haben und mehr zu wissen als jeder andere. Freudenberger stellte den Prozess des Burnouts selbst als Kreislauf dar, der beinhaltet: Bewährungszwang, zunehmendes Arbeitsengagement, Vernachlässigung der eigenen Bedürfnisse, wachsenden Konflikt zwischen den Bedürfnissen des Arbeitnehmers und den Anforderungen der Arbeit, Verzerrung (Veränderung) der Lebenswerte, Rückzug und Isolation, spürbare Verhaltensänderungen, Gefühl der Depersonalisierung, Verlust des Selbstwertgefühls und das Gefühl innerer Leere, Depression und schließlich ausgewachsenen Burnout [25,26]. Arbeitssucht und Burnout-Syndrom Weitere Studien zeigten das Auftreten von Burnout-Symptomen bei Vertretern einer zunehmenden Zahl von Berufen – darunter Lehrern, Militärs, Polizisten, Führungskräften [34], Ärzten [35] und Krankenschwestern [36]. 72 Die Häufigkeit von Burnout hängt von Beruf, Geschlecht und Wohnsitzland ab. Bei Tierärzten in den Vereinigten Staaten von Amerika zeigten 2/3 der weiblichen Korrespondenten frühe Burnout-Symptome, verglichen mit dem nationalen Durchschnitt für diesen Beruf von 67 % [37]. Eine weitere in den Vereinigten Staaten von Amerika durchgeführte Studie berichtete von einer Prävalenz von Burnout bei Tierärzten von 50,2 % [38]. In Belgien leiden 14,4 % der Tierärzte an Burnout, aber anders als in den USA sind Männer stärker gefährdet als Frauen [39]. In Deutschland ergab eine Befragung von Tierärzten, dass die Berufsbelastung um 42 % höher war als die der Allgemeinbevölkerung [40]. In der Humanmedizin berichteten 46 % der Ärzte in den USA über mindestens ein Burnout-Symptom [41]. Im Vereinigten Königreich wies etwa ein Drittel der Ärzte Burnout- Symptome auf [42]. Die Ergebnisse aus Großbritannien waren mit Studien aus Katar vergleichbar [43]. In Belgien wurde nur bei 6 % der Ärzte das Burnout-Syndrom diagnostiziert, und weitere 13 % gehörten zur Risikogruppe, dieses Leiden zu entwickeln [44]. Burnout wurde bei 10 % bis 78 % des europäischen Pflegepersonals festgestellt 45]. Langfristige Stresssituationen zu Hause oder am Arbeitsplatz können Menschen ans Ende ihrer Kräfte bringen. Die Gründe dafür können unterschiedlicher Art sein und hängen von der beruflichen Situation und den Charaktereigenschaften des Anwalts ab [46]. Anzeichen und Abläufe beim Burnout-Syndrom In der Öffentlichkeit bedeutet Burnout, dass man schon einmal etwas erreicht hat, während Depressionen fälschlicherweise mit Schwäche in Verbindung gebracht werden. Burnout wird oft nicht als eigenständige Krankheit gesehen, sondern als Risikofaktor für verschiedene psychische Probleme [49]. In der internationalen Klassifikation der Krankheiten (ICD-10, International Classification of Diseases, Version 10) wird Burnout nicht als eigenständige Krankheitseinheit definiert, sondern unter Ziffer 73 geführt. Dort steht der Begriff „Burnout“ unter dem Oberbegriff „Probleme mit der Bewältigung von Schwierigkeiten mit dem Leben“. Aufgrund der Schwere des Burnout-Problems haben sich bereits viele spezifische Methoden zur Vorbeugung und Behandlung entwickelt. Es gibt Publikationen, die die Stadien der Überwindung von Burnout beschreiben, wobei es zunächst darum geht, sich der Problematik bewusst zu werden, die die pathologische Berufstätigkeit eines Workaholics schafft. Der nächste Schritt sollte darin bestehen, Prioritäten festzulegen, sich realistische Ziele zu setzen, mit seiner Energie und Gefühlen richtig umzugehen. Die nächste Stufe bezieht sich auf die Wiedererlangung der Kontrolle über das Leben durch die Verwirklichung persönlicher Bedürfnisse und Wünsche und die Reflexion über das eigene Verhalten und den Weg der beruflichen Erfüllung. Weitere Maßnahmen sind das Erlernen von Durchsetzungsvermögen und Nein-Sagen zur Abwehr von Überforderungen und Ansprüchen aus der Umwelt. Auf der letzten Etappe geht es um Erholung und Regeneration. Ein wichtiges Element dieser Zeit ist die Nutzung von Pausen, deren Aufgabe es ist, Beginn und Ende der durchgeführten Aktivitäten klar zu markieren und so einer Situation vorzubeugen, in der es keine Klarheit mehr darüber herrscht, an wie vielen und was für Themen arbeiten wir derzeit. Ebenso wichtig sind in dieser Phase verschiedene Entspannungsformen und Hobbies, die Erholung und Ablösung von beruflichen Pflichten ermöglichen. Weitere wichtige Elemente der Burnout-Bekämpfung sind der Verzicht auf Arbeit an freien Tagen, Sonn- und Feiertagen sowie die Erweiterung des eigenen Betätigungsfeldes. Es kann von Vorteil sein, den Arbeitgeber oder die Art der Arbeit zu wechseln und den Kontakt mit Menschen zu knüpfen, die eine andere Lebenseinstellung vertreten [50]. Symptome emotionaler Erschöpfung, Depersonalisation und Funktionsminderung - sollten den Verdacht auf ein Burnout-Syndrom wecken, insbesondere wenn andere somatische Ursachen ausgeschlossen werden. Eine umfassende Burnout-Therapie sollte Psychoedukation, Entspannungsformen, Psychotherapie mit Analyse von Belastungsfaktoren in Beruf und Privatleben sowie somatische Therapie umfassen [51]. Ziel der Therapie ist es, den Patienten zu befähigen, seine Situation zu verstehen und seine Verhaltensmuster zu ändern. Anzeichen und Abläufe beim Burnout-Syndrom Wenn wir von Burnout sprechen, stellen wir uns meist einen überengagierten Mitarbeiter oder Unternehmer vor, der sich maßlos seinen belastenden Arbeitsaufgaben gewidmet hat und dabei völlig ausgebrannt ist. Allerdings muss verstanden werden, dass Burnout nur ein Synonym für die Auswirkungen dauerhafter Überlastung ist, nicht nur im Berufsleben, sondern auch im Privatleben. Das kann dem Präsidenten eines großen Unternehmens oder einer Hausfrau passieren, und immer häufiger betrifft dieses Problem sogar Menschen, die noch nicht mit der Arbeit begonnen haben, also Studenten. Die durchgeführten Untersuchungen deuten darauf hin, dass mehr als 10 % der Medizinstudierenden während ihres Studiums an studienbedingtem Burnout leiden [47,48]. Auffällige Anzeichen für Burnout sind der Verlust der Eigenschaften, die eine Person in ihrer Tätigkeit auszeichnen sollten, also Kreativität, gewissenhafte Erfüllung ihrer Pflichten, Kooperationsbereitschaft, Kommunikationsfähigkeit, Belastbarkeit, Offenheit für Veränderungen und die Fähigkeit, eigenständige Entscheidungen zu treffen. Die oben genannten Fähigkeiten sind durch eine Reihe von körperlichen, geistigen und Verhaltensänderungen im Zusammenhang mit dem Burnout-Syndrom gestört, wodurch sich Betroffene einer stereotypen, eiligen Arbeit zuwenden und im Laufe der Zeit eine deutlich ausgeprägte Tendenz zeigen Pflichten so weit wie möglich zu vermeiden. Jeder Job, den wir machen, kann uns aufgrund von unkontrollierbarem Stress ausbrennen lassen. Aus diesem Grund schreiben gesetzliche Arbeitsschutzvorschriften vor, dass alle Arbeitsplätze auf mögliche psychische Belastungen überprüft werden müssen. Betriebliche Abläufe werden ebenso analysiert wie externe Faktoren wie Lärm und Störfaktoren. Das bedeutet auch, dass der Arbeitgeber, wenn in einer solchen Analyse Belastungsfaktoren identifiziert werden, Maßnahmen zum Schutz der Gesundheit der Arbeitnehmer ergreifen muss. Laut Robert-Koch-Institut sind rund 4 % der erwachsenen Deutschen von Burnout betroffen. Viele Burnout-Symptome sind mit Depressionen vergleichbar, resultieren jedoch aus Müdigkeit, Überarbeitung und erschöpfenden Konflikten. Im Gegensatz zur Depression zeigt Burnout ein Gefühl der Depersonalisation und Unzufriedenheit mit den erzielten 73 Ergebnissen. Die Diagnose „Burnout“ wird oft eher akzeptiert. In der Öffentlichkeit bedeutet Burnout, dass man schon einmal etwas erreicht hat, während Depressionen fälschlicherweise mit Schwäche in Verbindung gebracht werden. Burnout wird oft nicht als eigenständige Krankheit gesehen, sondern als Risikofaktor für verschiedene psychische Probleme [49]. In der internationalen Klassifikation der Krankheiten (ICD-10, International Classification of Diseases, Version 10) wird Burnout nicht als eigenständige Krankheitseinheit definiert, sondern unter Ziffer 73 geführt. Dort steht der Begriff „Burnout“ unter dem Oberbegriff „Probleme mit der Bewältigung von Schwierigkeiten mit dem Leben“. Ergebnissen. Die Diagnose „Burnout“ wird oft eher akzeptiert. Anzeichen und Abläufe beim Burnout-Syndrom Eines der wichtigsten Elemente der Therapie ist es, dem Patienten bewusst zu machen, dass sein Verhalten mit zur Entstehung des Burnout-Syndroms beigetragen hat [52]. Sie sollten sich fragen, ob wir der Prävention von Arbeitssucht und Burnout-Syndrom genügend Aufmerksamkeit schenken. Der Aufwand für den Erwerb einer Ausbildung, gefolgt von der ständigen Notwendigkeit, seine Fähigkeiten zu verbessern, ist nur ein Element des chronischen Stresses, dem die meisten Berufstätigen heute ausgesetzt sind. Eine zusätzliche Belastung ist das aktuell geförderte Bild des idealen Arbeitnehmers als voll und ganz seiner Arbeit gewidmeten Menschen. Infolgedessen sind Berufstätige im Laufe ihres Berufslebens daran gewöhnt, dass sie systematisch mit immer mehr Aufgaben belastet werden, die in einem dauerhaft zu kurzen Zeitraum zu erledigen sind. Dennoch ist es meist nicht die Tätigkeit an sich, die zum Burnout führt. Die subjektiven Gefühle des Patienten spielen eine wichtige Rolle bei der Entstehung eines Erschöpfungsgefühls. Die persönliche Belastbarkeit ist eine individuelle Angelegenheit. Das hat viel mit inneren Einstellungen zu tun, aber auch mit Persönlichkeitsmerkmalen. Wir haben bestimmte Persönlichkeitstypen identifiziert, die besonders anfällig für Burnout zu sein scheinen. Oft sind es Menschen, die ausgehend von 74 ihrem Arbeitsideal mit Begeisterung an ihre Aufgaben herangehen. Sie machen ihr Selbstverständnis davon abhängig, ihre Ziele möglichst schnell zu erreichen. Wenn sie ihre Ziele nicht erreichen, werden sie ihre Anstrengungen weiter steigern, motiviert durch den zunehmenden inneren Druck. Ein weiterer Burnout-gefährdeter Persönlichkeitstyp sind Menschen mit geringem Selbstvertrauen. Solche Menschen sind schüchtern, überempfindlich und passiv. Um von anderen Menschen anerkannt zu werden, sind sie bereit, sich den Erwartungen der Umwelt anzupassen. Die Folge dieses Verhaltens ist die Unfähigkeit, selbstbewusst auf die Forderungen anderer zu reagieren, was dazu führt, dass sie „nein“ und in einen Strudel von Forderungen geraten, die sie nicht erfüllen können. Obwohl die beiden Persönlichkeitstypen unterschiedlich sind, teilen sie ein starkes Verlangen nach Anerkennung durch die Außenwelt und Schwierigkeiten, ihre Gefühle auszudrücken. Auch die Unfähigkeit, sich realistische Ziele zu setzen, ist ein Risikofaktor. Sich unrealistisch hohe Ziele zu setzen oder Ziele zu setzen, die nicht mit den eigenen Wünschen übereinstimmen, führt zu Enttäuschung und dem Gefühl, im Leben versagt zu haben. Die Bereitschaft, jede Aufgabe perfekt zu erledigen und sich unentbehrlich zu fühlen, wird ebenfalls als Risikofaktor für Burnout angesehen [53]. Sie sollten auch einen häufigen Fehler berücksichtigen, der als Kategoriefehler bekannt ist. Anzeichen und Abläufe beim Burnout-Syndrom Die Definition des Kategoriefehlers im Oxford Dictionary of Philosophy 1994 lautet wie folgt: „wenn Dinge oder Tatsachen einer Art so dargestellt werden, als ob sie zu einer anderen gehörten„. In den Köpfen vieler Menschen ist ein guter Arbeiter dasselbe wie ein guter Mensch zu sein. Dies führt zu einem verständlichen Wunsch, seine Arbeit immer gut zu machen. Der oben erwähnte Kategorienfehler besteht darin, dass wir, wenn wir uns als gute oder schlechte Mitarbeiter kategorisieren, unberechtigt in eine andere Kategorie springen, weil wir nicht nur das sind, was wir tun, und uns daher nicht nur durch das Prisma unserer Arbeit identifizieren können [54]. Die Entwicklung präziser Denkfähigkeiten, die eine Überverallgemeinerung von Urteilen verhindern, scheint ein Weg zu sein, um Kategorienfehler zu vermeiden. Es gibt noch eine weitere kognitive Verzerrung, die die Wahrscheinlichkeit eines Burnout-Syndroms erhöhen kann. Sie basiert auf der Selektivität unseres Gedächtnisses bezüglich positiver oder negativer Erinnerungen. Eine der Studien, die sich auf diese kognitive Verzerrung konzentrierte, versuchte, Burnout im Hinblick auf Gedächtnisverzerrungen gegenüber emotionalen Informationen zu untersuchen. Diese Studie ergab, dass das Burnout-Syndrom negativ mit der Tendenz korrelierte, sich an positive Wörter zu erinnern, und positiv mit der Tendenz, sich an negative Wörter zu erinnern [55]. Diese Ergebnisse ähneln den Ergebnissen anderer 2015 veröffentlichter Studien, in denen die Autoren die Burnout-Tendenz mit einer zu starken Fokussierung auf negative Informationen und weniger Beachtung positiver Informationen in Verbindung brachten [56]. Zusammenfassung Die Phänomene Arbeitssucht und Burnout bedürfen noch der Erforschung, da noch nicht alle Funktionsstörungen dieser beiden Herausforderungen der modernen Welt vollständig aufgeklärt sind. Das mit beiden Problemen verbundene Stressphänomen nehmen viele Menschen als eine Situation wahr, die eintritt, wenn das unglückliche Nebeneinander von Persönlichkeitstypus und Ermahnungen der Umstände eine Bewältigung unmöglich macht. Es wird davon ausgegangen, dass diese Situationen nicht besonders erschwerend sein müssen, da wiederholte Stressoren, die sich über die Zeit verteilen, auch zu einem Faktor werden können, der schädliche Prozesse auslöst, in deren Folge die Wirksamkeit der durchgeführten Aktivitäten abnimmt. Es ist davon auszugehen, dass die Bedeutung des stressigen Umfelds, obwohl es vor einiger Zeit erkannt wurde, immer noch nicht so gewürdigt wird, wie es verdient, und die Analyse der Ursachen von Überengagement und dem daraus resultierenden 75 Burnout konzentriert sich normalerweise auf die Eigenschaften von Menschen unter diesen Syndromen leiden. Der langfristige Kampf mit Stress, sowohl beruflich als auch nicht beruflich, ist ein wichtiger Faktor, der die Aktivierung ungünstiger Verhaltensmechanismen hervorruft, insbesondere wenn das Umfeld, in dem die jeweiligen Menschen leben, ihre Bestrebungen und Einschränkungen nicht berücksichtigt. 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Compassion satisfaction, burnout, and secondary traumatic stress among full-time veterinarians in the United States (2016-2018). J Am Vet Med Assoc. 2021 Jun 1;258(11):1259-1270. doi: 10.2460/javma.258.11.1259. PMID: 33978434. 39. Hansez I, Schins F, Rollin F. Occupational stress, work-home interference and burnout among Belgian veterinary practitioners. BT. Ir Vet J. 2008;61(4):233-241. Published 2008 Apr 1. doi:10.1186/2046-0481-61-4-233 40. Stress im Tierarztberuf als Gesundheitsrisiko Ergebnis der Online-Umfrage Autoren: Katja Geuenich Ausgabe: 1/2011 S. 4-4 https://www.bundestieraerztekammer.de/btk/dtbl/archiv/artikel/1/2011/stress-im- tierarztberuf-als-gesundheitsrisiko [letzer Zugriff: 03.02.2022]. 41. Shanafelt TD, Hasan O, Dyrbye LN, Sinsky C, Satele D, Sloan J, West CP. Changes in Burnout and Satisfaction With Work-Life Balance in Physicians and the General US Working Population Between 2011 and 2014. Mayo Clin Proc. 2015 Dec;90(12):1600- 13. doi: 10.1016/j.mayocp.2015.08.023. Erratum in: Mayo Clin Proc. 2016 Feb;91(2):276. PMID: 26653297. 42. Soler JK, Yaman H, Esteva M, Dobbs F, Asenova RS, Katic M, Ozvacic Z, Desgranges JP, Moreau A, Lionis C, Kotányi P, Carelli F, Nowak PR, de Aguiar Sá Azeredo Z, Marklund E, Churchill D, Ungan M; European General Practice Research Network Burnout Study Group. Burnout in European family doctors: the EGPRN study. Fam Pract. 2008 Aug;25(4):245-65. doi: 10.1093/fampra/cmn038. Epub 2008 Jul 11. PMID: 18622012. 43. Abdulla, L.; Al-Qahtani, D.M.; Al-Kuwari, M.G. Prevalence and determinants of burnout syndrome among primary healthcare physicians in Qatar. S. Afr. Fam. Pract. 2011, 53, 380–383 78 44. Vandenbroeck S, Van Gerven E, De Witte H, Vanhaecht K, Godderis L. Burnout in Belgian physicians and nurses. Occup Med (Lond). 2017 Oct 1;67(7):546-554. doi: 10.1093/occmed/kqx126. PMID: 29016982. 45. Aiken LH, Sermeus W, Van den Heede K, Sloane DM, Busse R, McKee M, Bruyneel L, Rafferty AM, Griffiths P, Moreno-Casbas MT, Tishelman C, Scott A, Brzostek T, Kinnunen J, Schwendimann R, Heinen M, Zikos D, Sjetne IS, Smith HL, Kutney-Lee A. Patient safety, satisfaction, and quality of hospital care: cross sectional surveys of nurses and patients in 12 countries in Europe and the United States. BMJ. 2012 Mar 20;344:e1717. doi: 10.1136/bmj.e1717. PMID: 22434089; PMCID: PMC3308724. 46. Burnout: Wie merke ich, dass ich betroffen bin? https://www.aok.de/pk/magazin/koerper-psyche/psychologie/burnout-so-merken-sie- ob-sie-betroffen-sind/ [letzer Zugriff: 03.02.2022]. 47. Costa EF, Santos SA, Santos AT, Melo EV, Andrade TM. Burnout Syndrome and associated factors among medical students: a cross-sectional study. Clinics (Sao Paulo). 2012, 67(6):573-580. doi:10.6061/clinics/2012(06). 48. Burnout, die Krankheit unserer Zeit. https://www.temedos.de/journal/burnout-die- krankheit-unserer-zeit [letzer Zugriff: 03.02.2022]. 49. Burnout. https://www.praktischarzt.de/krankheiten/burnout/ [letzer Zugriff: 03.02.2022]. 50. Solecki L, Klepacka P. Przyczyny oraz czynniki sprzyjające występowaniu zespołu wypalenia zawodowego wśród lekarzy zatrudnionych w publicznych sektorach opieki zdrowotnej. Medycyna Środowiskowa. 2017, t. 20, z.1:7-16. 51. von Känel R. Was der Klinikarzt vom Burnout-Syndrom wissen sollte Von der gezielten Fragestellung bis zur Übermittlung der Verdachtsdiagnose. https://www.clinica-holistica.ch/downloads/symposium/klinikarzt_roland-von- kaenel.pdf [letzer Zugriff: 03.02.2022]. 52. BT. Burnout – auf die innere Einstellung kommt es an. https://www.stepstone.de/Karriere- Bewerbungstipps/burnout/ [letzer Zugriff: 03.02.2022]. 53. Ibidem. 53. Ibidem. 54. Rogowski S. Człowiek znaczy słowo. Postępy Psychiatrii i Neurologii. 2006, t. 15, z. 2:1-5. 55. 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English

Severe Gastroparesis following Radiofrequency Catheter Ablation for Atrial Fibrillation: Suggestion for Diagnosis, Treatment, and Device for Gastroparesis after RFCA

Case reports in gastrointestinal medicine

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Hindawi Publishing Corporation Case Reports in Gastrointestinal Medicine Volume 2014, Article ID 923637, 6 pages http://dx.doi.org/10.1155/2014/923637 Hindawi Publishing Corporation Case Reports in Gastrointestinal Medicine Volume 2014, Article ID 923637, 6 pages http://dx.doi.org/10.1155/2014/923637 Hindawi Publishing Corporation Case Reports in Gastrointestinal Medicine Volume 2014, Article ID 923637, 6 pages http://dx.doi.org/10.1155/2014/923637 been reported and retrospective studies have been conducted [4–6]. been reported and retrospective studies have been conducted [4–6]. 1. Introduction The definition of gastroparesis is a delay in emptying of food from the stomach. The causes of gastroparesis are postvago- tomy syndrome of a peptic ulcer, pancreatic adenocarcinoma, mesenteric vascular insufficiency, and systemic disease such as amyloidosis. Most patients with gastroparesis complain of abdominal distension and epigastric discomfort after eating. No ulcers or gastric outlet obstructions are found on an upper endoscopy exam. A gastrointestinal motility study is widely conducted to diagnose gastroparesis.h Correspondence should be addressed to Sang Jin Lee; [email protected] Correspondence should be addressed to Sang Jin Lee; [email protected] Received 18 October 2014; Accepted 11 December 2014; Published 30 December 2014 Academic Editor: Wen-xie Xu Copyright © 2014 D. S. Lee and S. J. Lee. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gastroparesis following radiofrequency catheter ablation (RFCA) is a very rare complication, as only two cases have been reported in the English literature. A 42-year-old man underwent RFCA due to recurrent drug-resistant symptomatic atrial fibrillation. The patient complained of indigestion and early satiety 2 days after the procedure. Contrast-enhanced computed tomography and an upper gastrointestinal series of the abdomen showed a large amount of material remaining in the stomach area. All food material was removed by endoscopy, and the patient received medical treatment. We suggest a flow chart for diagnosis and treatment of AFGS based on the present case and previous cases. Endoscopic medical patent was designed on the basis of this case. Dong Seok Lee1 and Sang Jin Lee1,2 1Department of Internal Medicine, Gangneung Asan Medical Center, University of Ulsan College of Medicine, Gangneung, Republic of Korea 2Department of Internal Medicine, Gangneung Asan Medical Center, Sacheon-myeon, Gangneung, Gangwon-do 210-71 Republic of Korea 1Department of Internal Medicine, Gangneung Asan Medical Center, University of Ulsan College of Medicine, Gangneung, Republic of Korea 2. Case Report A 59-year-old man presented with a history of paroxysmal palpitation for more than a few years. Symptom duration and frequency had been prolonged recently with a documented ECG. He was treated with digoxin, quinidine, propafenone, metoprolol, and amiodarone sequentially. However, his symptoms persisted. He was diagnosed with drug-refractory, symptomatic, chronic AF and underwent RFCA. The patient was transferred to the EP laboratory. After an esophagram, we positioned the catheters and performed a dual septal punc- ture. The baseline ECG indicated normal sinus rhythm. Abla- tion at the left superior pulmonary vein (LSPV) was started and electrical isolation of the LSPV, left inferior pulmonary The radiofrequency catheter ablation (RFCA) procedure is widely performed to treat atrial fibrillation (AF). However, various complications have been reported. Thromboembolic events, tamponade, severe pulmonary vein stenosis, femoral pseudoaneurysm, and atrioesophageal fistula have been reported as complications of the RFCA procedure [1–3]. A few cases of vagus nerve injury due to RF ablation energy have 2 2 Case Reports in Gastrointestinal Medicine (a) (b) (c) (d) (e) Figure 1: (a) Simple abdominal X-ray reveals a large amount of material in the stomach, suggesting severe gastric hypomotility. (b) Abdominopelvic computed tomography reveals a large amount of food material in the stomach, suggesting partial stricture in the postbulbar portion of the duodenum. (c) Endoscopic findings reveal large amounts of food stored in the gastric body and antrum. (d) The food was removed using a cap-fitted endoscope and a net over a period of days. (e) Peristaltic motion of the gastric antrum was normal and the pylorus opened and closed normally. (a) (b) (b) (a) (d) (c) (c) (d) ( ) ( ) (e) (e) Figure 1: (a) Simple abdominal X-ray reveals a large amount of material in the stomach, suggesting severe gastric hypomotility. (b) Abdominopelvic computed tomography reveals a large amount of food material in the stomach, suggesting partial stricture in the postbulbar portion of the duodenum. (c) Endoscopic findings reveal large amounts of food stored in the gastric body and antrum. (d) The food was removed using a cap-fitted endoscope and a net over a period of days. (e) Peristaltic motion of the gastric antrum was normal and the pylorus opened and closed normally. and 1(e)). The food was removed using a cap-fitted endoscope and a net over a period of days. 2. Case Report At the time of paralysis, spe- cific lesions have not been found inside the gastrointestinal tract and biopsy was not performed. With suspected gastro- paresis, he underwent a gastric emptying scan. The gastric emptying scan revealed delayed gastric emptying time with an almost flat time-activity curve. There still remained a large amount of contrast material in the 2-hour delayed image, suggesting severe gastric hypomotility (Figure 2(a)). He was treated with prokinetics (50 mg itopride three times daily) and antiulcer medication (100 mg rebamipide three times daily). After the start of treatment, digestive function and excretion function were improved by 20 percent in one week. Digestive function has been completely recovered in two months later. After normalization of gastrointestinal motility, there were no abnormal findings in the duodenum in follow- up gastroscopy examination (Figure 2(b)). vein, right superior pulmonary vein, and right inferior pul- monary vein as well as a cavotricuspid isthmus block was completed. There was atrial flutter (AFL) on the left atrial side. An activation map was prepared. The AFL went through the roof and the perimitral area. We induced AF with ISO 5. After RFCA, he was admitted to the intensive care unit due to the long procedure time, 7 hours. No immediate complica- tions were detected during the procedure. Sinus rhythm was maintained after the procedure. Patient has no underlying disease except for arrhythmia and did not complain of anxiety every day, before and after RFCA. But he complained of abdominal distension and epigastric discomfort after eating on hospital day 2. Bowel sounds were normal, and gas out was normal, but gastric distension was noted. He complained of epigastric pain, indigestion, and early satiety for 1 week. To prove gastroparesis related with RFCA, we used simple abdomen X-ray, abdominal pelvic CT, and gastroscopy as a diagnostic method. A simple abdominal X-ray revealed a large amount of material in the stomach, suggesting severe gastric hypomotility. Abdominal pelvic CT revealed large amount of food in the stomach (Figures 1(a) and 1(b)).i 3. Discussion Persistent gastroparesis is often a symptom of nerve damage in this area. This is most commonly due to abnormalities or damage in the way the stomach and intestines contract. Decreased production of stomach acid is also a common symptom of this type of nerve damage [7]. other than AF, and digestive function was good. Because there was no underlying disease, we eliminated other causes for the gastroparesis. After treatment, severe abdominal disten- sion findings revealed the gastroparesis findings, and AFGS was diagnosed. Although AFGS can occur based on treat- ment time, the vagus nerve tract, intensity of the electric force, treatment time, and RFCA site, it is reversible. RFCA treatment time is different according to the patient and center. In our center it took about 6 hours and it took up about 7 hours in this case. According to the existing literature, AFGS has been estimated by the treatment site and the amount of energy. Procedure time is also important, because a large amount of energy is transmitted in proportion to the time. In order to reduce the thermal injury to a minimum, we wrapped cooling pack to the portion of the neck in the proce- dure. We will prepare a prospective study on the effectiveness of the cooling pack to minimize the gastrointestinal paralysis on the above variables. Because the position of the vagus nerve is adjacent to the heart, gastroparesis due to the vagus nerve can occur due to various mechanical procedures such as postvagotomy syn- drome for peptic ulcer or pancreatectomy due to pancreatic cancer. However, a vagus nerve injury due to electrical energy is very rare. Shah et al. reported four cases of acute delayed gastric emptying caused by vagus nerve injury after RF ablation for AF [2]. A case report of severe gastroparesis has been published recently [5]. Gastroparesis is observed as food retention via endoscopy and is supposedly caused by damage to the vagal nerve fibers surrounding the distal esophagus, as suggested in previous studies [5, 8]. Thus, delayed gastric emptying is considered a direct noncardiac complication of the ablation procedure.ii It took 6 months for the previous gastroparesis cases to recover to normal [5]. Our case was more serious, as the patient complained of terrible distension. However, gastric function completely recovered in 2 months with a rapid diagnosis, treatment, and exercise. 3. Discussion The vagus nerve begins in the brain and extends into the abdomen. The vagus nerve is responsible for such varied tasks as heart rate, gastrointestinal peristalsis, sweating, and quite a few muscle movements in the mouth, including speech and Endoscopic findings revealed large amounts of food stored in the gastric body. Normal peristaltic motion of the gastric antrum and pylorus was observed (Figures 1(c), 1(d), 3 3 Case Reports in Gastrointestinal Medicine ANT 3min ANT 120 min ANT 30min ANT 60min ANT 90min Aer 3min Aer 30min Aer 60min Aer 90min Aer 120 min (a) ANT 1min ANT 120 min ANT 30min ANT 60min ANT 90min Aer 1min Aer 30min Aer 60min Aer 90min Aer 120 min (b) ANT 3min ANT 120 min ANT 30min ANT 60min ANT 90min Aer 3min Aer 30min Aer 60min Aer 90min Aer 120 min (a) ANT 1min ANT 120 min ANT 30min ANT 60min ANT 90min Aer 1min Aer 30min Aer 60min Aer 90min Aer 120 min (b) Figure 2: (a) Gastric emptying scan reveals delayed gastric emptying time with an almost flat time-activity curve. A large am material remains in the 2-hour delayed image, suggesting severe gastric hypomotility. (b) Gastric emptying scan reveal emptying time, suggesting normal gastric motion. ANT 3min ANT 120 min ANT 30min ANT 60min ANT 90min Aer 3min Aer 30min Aer 60min Aer 90min Aer 120 min (a) ANT 1min ANT 120 min ANT 30min ANT 60min ANT 90min ANT 3min ANT 30min ANT 1min ANT 120 min ANT 30min ANT 60min ANT 90min ANT 1min ANT 120 min ANT 30min ANT 60min ANT 90min ANT 120 min Aer 120 min Aer 120 min in Aer 60min Aer 90min (b) Figure 2: (a) Gastric emptying scan reveals delayed gastric emptying time with an almost flat time-activity curve. A large amount of contrast material remains in the 2-hour delayed image, suggesting severe gastric hypomotility. (b) Gastric emptying scan reveals delayed gastric emptying time, suggesting normal gastric motion. Figure 2: (a) Gastric emptying scan reveals delayed gastric emptying time with an almost flat time-activity curve. A large amount of contrast material remains in the 2-hour delayed image, suggesting severe gastric hypomotility. (b) Gastric emptying scan reveals delayed gastric emptying time, suggesting normal gastric motion. keeping the larynx open for breathing. Digestive problems can sometimes occur as a result of vagus nerve damage. 3. Discussion p We defined atrial fibrillation gut syndrome (AFGS) as symptoms of decreased gastrointestinal motility that occur after RFCA treatment. Our patient had no underlying disease Case Reports in Gastrointestinal Medicine The symptom of the acute gastroparesis History taking and physical examination Likely systemic disease Simple abdomen X-ray and L tube insertion Anatomic study: endoscopic exam with new device and emptying food material GI motility drug (prescribed at low doses) Exercise and motility drug Simple abdomen X-ray and gastric emptying scan follow up Complete recovery Preventive soft diet trial for 7 days in high risk patient (long time of RFCA, recurrent RFCA, multifocal site, the habit of rapid consumption, high RFCA energy) Yes Yes Yes Yes Yes Yes Yes Yes Yes No GI motility study: gastric emptying scan Figure 3: Flow chart of the diagnosis and treatment in our case and previous other cases. Preventive soft diet trial for 7 days in high risk patient (long time of RFCA, recurrent RFCA, multifocal site, the habit of rapid consumption, high RFCA energy) Yes Figure 3: Flow chart of the diagnosis and treatment in our case and previous other cases. is paralyzed but movement of the pylorus is normal. Thus, the bowel is normal on a physical exam but retention of food material interferes with the pylorus. Removal of food is essential to resolve the abdominal distension and problem with the pylorus. According to our experience, endoscopic removal by colonoscopy is recommended for hardened food material. It is believed that the recovery period can be reduced by rapid diagnosis and proper treatment and rehabilitation. We suggest a flow chart for diagnosis and treatment of AFGS based on the present case and previous cases. This flow chart can minimize the complications that occur after the operation in the AFGS high risk group. We recommend a soft diet in the high risk patient, as indicated in Figure 3, because it is easy to remove residual food material in cases of severe gastroparesis. Medical patent has been invented on the basis of the present case (patent number 10-2014-0102131, Republic of Korea) (Figure 4). Patent device is devised to eliminate gastric contents through the esophagus without surgery. By using the patent endoscope tube, sufficient internal space of the esoph- agus is ensured. 4. Conclusion Although many complications can occur during RFCA, it is an absolutely necessary treatment for AF. AFGS is very unfamiliar complications to the cardiologists and gastroen- terologists. It is difficult to predict AFGS on the basis of the patient symptoms. Our patient was the most serious case of AFGS that has been reported to date but shows that AFGS is fully reversible. In addition, this case presents the summary for diagnosing and treating AFGS to the RFCA practitioner. Considering these reversible and frequent AFGS compli- cations, we think AFGS should be added to the common causative category of mechanical gastroparesis. 3. Discussion Via a secure space, grinding endoscopic instrument is inserted and all of the gastric contents can be removed easily such as hematoma, foreign body, and food material. In future, fast diagnosis and treatment of AFGS will be possible. Various gastrointestinal foreign matter removals can be possible without surgery. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. A feature of digestive tract paralysis due to vagus nerve damage is that movement of the wall of the digestive tract 5 Case Reports in Gastrointestinal Medicine 5 210 220 260 230 200 110 140 100 120 130 330 340 300 600 320 310 Clean water Crushed hematoma, gastric content (a) 400 410 420 430 (b) 250 200 240 (c) 110 100 (d) 100 530 540 500 510 520 (100) Insertion tube (110) Crushing unit (120) Recording unit (130) Illumination unit (200) Irrigating and suction unit (210) Operation buttons of crushing (220) First irrigation button (230) Suction button (240) Suction tube (250) Irrigation tube (260) Second irrigation button (300) Storage chamber (310) Collection containers (320) Storage container (330) Pump (340) Valve (400) Guide tube (410) Outer tube (420) Inner tube (500) Crushing device (510) Mixer case (520) Fastening parts (530) Blade moving parts (540) Blade (600) Diagnosis parts (e) 0 400 410 420 430 (b) 210 220 260 230 200 110 140 100 120 130 330 340 300 600 320 310 Clean water Crushed hematoma, gastric content 400 410 420 430 210 220 260 230 200 110 140 100 120 130 330 340 300 600 320 310 Clean water Crushed hematoma, gastric content 200 110 140 100 120 130 330 340 300 600 320 310 Clean water Crushed hematoma, gastric content (a) 400 410 420 430 (b) 250 200 240 (c) 110 100 (d) (b) 110 100 (d) (b) (a) (b) 250 200 240 (c) 110 100 (d) 250 200 240 (c) (d) (c) (c) (d) 100 530 540 500 510 520 (c) (d) 100 530 540 500 510 520 (100) Insertion tube (110) Crushing unit (120) Recording unit (130) Illumination unit (200) Irrigating and suction unit (210) Operation buttons of crushing (220) First irrigation button (230) Suction button (240) Suction tube (250) Irrigation tube (260) Second irrigation button (300) Storage chamber (310) Collection containers (320) Storage container (330) Pump (340) Valve (400) Guide tube (410) Outer tube (420) Inner tube (500) Crushing device (510) Mixer case (520) Fastening parts (530) Blade moving parts (540) Blade (600) Diagnosis parts (e) Figure 4: Continued. Conflict of Interests 6 Case Reports in Gastrointestinal Medicine 6 110 130 120 300 100 220 230 200 210 260 (100) Insertion tube (110) Crushing unit (120) Recording unit (130) Illumination unit (200) Irrigating and suction unit (210) Operation buttons of crushing (220) First irrigation button (230) Suction button (240) Suction tube (250) Irrigation tube (260) Second irrigation button (300) Storage chamber (310) Collection containers (320) Storage container (330) Pump (340) Valve (400) Guide tube (410) Outer tube (420) Inner tube (500) Crushing device (510) Mixer case (520) Fastening parts (530) Blade moving parts (540) Blade (600) Diagnosis parts (f) 110 130 120 300 100 220 230 200 210 260 210 Figure 4: (a) Schematic control flow of the gastric content removing device; (b) endoscopic guide tube for evacuating gastric content; (c) irrigation and suction unit of the gastric contents; (d) mixer parts of crushing unit; (e) fastening parts of crushing unit; (f) appearance of the gastric content removing device. Acknowledgment [5] S. W. Choi, S. H. Kang, O. S. Kwon et al., “A case of severe gastroparesis: indigestion and weight loss after catheter ablation of atrial fibrillation,” Pacing and Clinical Electrophysiology, vol. 35, no. 3, pp. e59–e61, 2012. The authors acknowledge the financial support for patent by The authors acknowledge the financial support for patent by Foundation for Industry Cooperation, University of Ulsan. The authors acknowledge the financial support for patent by Foundation for Industry Cooperation, University of Ulsan. [6] H. Knopp, U. Halm, R. Lamberts et al., “Incidental and ablation- induced findings during upper gastrointestinal endoscopy in patients after ablation of atrial fibrillation: a retrospective study of 425 patients,” Heart Rhythm, vol. 11, no. 4, pp. 574–578, 2014. References [1] C. Pappone, H. Oral, V. Santinelli et al., “Atrio-esophageal fistula as a complication of percutaneous transcatheter ablation of atrial fibrillation,” Circulation, vol. 109, no. 22, pp. 2724–2726, 2004. [7] K. L. D. Moore and F. Arthur, Clinically Oriented Anatomy, 7th edition, 2014. [8] C. F. Pisani, D. Hachul, E. Sosa, and M. Scanavacca, “Gastric hypomotility following epicardial vagal denervation ablation to treat atrial fibrillation,” Journal of Cardiovascular Electrophysi- ology, vol. 19, no. 2, pp. 211–213, 2008. [2] D. Shah, J.-M. Dumonceau, H. Burri et al., “Acute pyloric spasm and gastric hypomotility: an extracardiac adverse effect of per- cutaneous radiofrequency ablation for atrial fibrillation,” Jour- nal of the American College of Cardiology, vol. 46, no. 2, pp. 327– 330, 2005. [3] N. Dagres, G. Hindricks, H. Kottkamp et al., “Complications of atrial fibrillation ablation in a high-volume center in 1,000 procedures: still cause for concern?” Journal of Cardiovascular Electrophysiology, vol. 20, no. 9, pp. 1014–1019, 2009. [4] T. J. Bunch, K. A. Ellenbogen, D. L. Packer, and S. J. Asirvatham, “Vagus nerve injury after posterior atrial radiofrequency abla- tion,” Heart Rhythm, vol. 5, no. 9, pp. 1327–1330, 2008.

https://openalex.org/W2054026360

https://ojrd.biomedcentral.com/counter/pdf/10.1186/1750-1172-3-12

English

McCune-Albright syndrome

Orphanet journal of rare diseases

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Published: 19 May 2008 Orphanet Journal of Rare Diseases 2008, 3:12 doi:10.1186/1750-1172-3-12 This article is available from: http://www.ojrd.com/content/3/1/12 © 2008 Dumitrescu and Collins; licensee BioMed Central Ltd. © 2008 Dumitrescu and Collins; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract McCune-Albright syndrome (MAS) is classically defined by the clinical triad of fibrous dysplasia of bone (FD), café-au-lait skin spots, and precocious puberty (PP). It is a rare disease with estimated prevalence between 1/100,000 and 1/1,000,000. FD can involve a single or multiple skeletal sites and presents with a limp and/or pain, and, occasionally, a pathologic fracture. Scoliosis is common and may be progressive. In addition to PP (vaginal bleeding or spotting and development of breast tissue in girls, testicular and penile enlargement and precocious sexual behavior in boys), other hyperfunctioning endocrinopathies may be involved including hyperthyroidism, growth hormone excess, Cushing syndrome, and renal phosphate wasting. Café-au-lait spots usually appear in the neonatal period, but it is most often PP or FD that brings the child to medical attention. Renal involvement is seen in approximately 50% of the patients with MAS. The disease results from somatic mutations of the GNAS gene, specifically mutations in the cAMP regulating protein, Gs alpha. The extent of the disease is determined by the proliferation, migration and survival of the cell in which the mutation spontaneously occurs during embryonic development. Diagnosis of MAS is usually established on clinical grounds. Plain radiographs are often sufficient to make the diagnosis of FD and biopsy of FD lesions can confirm the diagnosis. The evaluation of patients with MAS should be guided by knowledge of the spectrum of tissues that may be involved, with specific testing for each. Genetic testing is possible, but is not routinely available. Genetic counseling, however, should be offered. Differential diagnoses include neurofibromatosis, osteofibrous dysplasia, non- ossifying fibromas, idiopathic central precocious puberty, and ovarian neoplasm. Treatment is dictated by the tissues affected, and the extent to which they are affected. Generally, some form of surgical intervention is recommended. Bisphosphonates are frequently used in the treatment of FD. Strengthening exercises are recommended to help maintaining the musculature around the FD bone and minimize the risk for fracture. Treatment of all endocrinopathies is required. Malignancies associated with MAS are distinctly rare occurrences. Malignant transformation of FD lesions occurs in probably less than 1% of the cases of MAS. puberty (PP) [1,2]. It was later recognized that other endo- crinopathies, including hyperthyroidism (reviewed in [3]), growth hormone (GH) excess [4,5], renal phosphate wasting with or without rickets/osteomalacia [6] and Review McCune-Albright syndrome Claudia E Dumitrescu and Michael T Collins* Open Access McCune-Albright syndrome Claudia E Dumitrescu and Michael T Collins* Address: Skeletal Clinical Studies Unit, Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, USA Email: Claudia E Dumitrescu - [email protected]; Michael T Collins* - [email protected] * Corresponding author Received: 29 November 2007 Accepted: 19 May 2008 Received: 29 November 2007 Accepted: 19 May 2008 Published: 19 May 2008 Epidemiology MAS is a rare disease and reliable data of prevalence are not available (the estimated prevalence ranges between 1/ 100,000 and 1/1,000,000). In contrast, the skeletal aspect of the disease, FD, especially monostotic disease, is not rare [16]. FD has been reported to account for up to 7% of all benign bone tumors. Café-au-lait skin pigmentation Figure 1 Café-au-lait skin pigmentation. A) A typical lesion on the face, chest, and arm of a 5-year-old girl with McCune- Albright syndrome which demonstrates jagged "coast of Maine" borders, and the tendency for the lesions to both respect the midline and follow the developmental lines of Blashko. B) Typical lesions that are often found on the nape of the neck and crease of the buttocks are shown (arrows). Definition O i i ll Originally, the McCune-Albright syndrome (MAS) was defined by the triad of polyostotic fibrous dysplasia of bone (FD), café-au-lait skin pigmentation, and precocious Page 1 of 12 (page number not for citation purposes) Page 1 of 12 (page number not for citation purposes) http://www.ojrd.com/content/3/1/12 Orphanet Journal of Rare Diseases 2008, 3:12 Café-au-lait skin pigmentation Figure 1 Café-au-lait skin pigmentation. A) A typical lesion on the face, chest, and arm of a 5-year-old girl with McCune- Albright syndrome which demonstrates jagged "coast of Maine" borders, and the tendency for the lesions to both respect the midline and follow the developmental lines of Blashko. B) Typical lesions that are often found on the nape of the neck and crease of the buttocks are shown (arrows). Café-au-lait skin pigmentation Figure 1 Cushing syndrome could be found in association with the original triad [7-9]. Rarely, other organ systems may be involved (liver, cardiac, parathyroid, pancreas) [10]. While MAS is rare, FD is not. FD can involve a single skel- etal site (monostotic FD, MFD), or multiple sites (polyos- totic FD, PFD) [11-14]. Very rarely PP can be found in association with café-au-lait skin pigmentation in the absence of FD (about 1% of the cases), but in general, FD seems to be the most common component of MAS. There- fore, a more clinically relevant definition of MAS, broader than the original triad of FD + PP + café-au-lait is: MAS = FD + at least one of the typical hyperfunctioning endo- crinopathies and/or café-au-lait spots, with almost any combination possible [13,15]. Clinical description Typically, the signs and symptoms of either PP or FD usu- ally account for the initial presentation. In girls with PP, it is usually vaginal bleeding or spotting, accompanied by development of breast tissue, usually without the devel- opment of pubic hair. In boys, it can be bilateral (or uni- lateral) testicular enlargement with penile enlargement, scrotal rugae, body odor, pubic and axillary hair, and pre- cocious sexual behavior. In retrospect, café-au-lait spots (Fig. 1), which are usually present at birth or shortly there- after, are the most common but unappreciated "present- ing" sign. skeleton, 90% were present by 15.5 yr. The appearance of new lesions later in life is a very uncommon occurrence in FD. The incidence of fractures is greatest in childhood, between the age of 6 and 10 yr, but due to the intrinsic abnormalities in FD bone, some fractures continue to occur into adulthood (Fig. 4)[18]. Other features of the presentation related to the specific aspect of the disease are outlined in Table 1. Fibrous dysplasia in the appendicular skeleton usually presents with a limp and/or pain (sometimes reported by children as being "tired"), but occasionally a pathologic fracture may be the presenting sign. Radiographs will demonstrate typical expansile lesions with endosteal scal- loping and thinning of the cortex with the matrix of the intramedullary tissue demonstrating a "ground glass" appearance (Fig. 2A &2B). FD in the craniofacial bones usually presents as a painless "lump" or facial asymmetry. Representative radiographic findings and the histological appearance of FD are shown in Figures 2 and 3. The areas most commonly involved are the proximal femora and skull base. The sites of FD involvement are established early; 90% of the total body skeletal disease burden is usu- ally established by age 15 [17]. Hart et al. found that lesions in the craniofacial region were established earliest, with 90% of the lesions present by 3.4 years of age. In the extremities, 90% were present by 13.7 yr, and in the axial Page 2 of 12 (page number not for citation purposes) Etiology Representative 99Tc-MDP bone scans which show tracer uptake at affected skeletal sites, and the associated skeletal disease burden score (see ref. Collins, 2005) are shown. E) A 50-year-old woman with monostotic FD confined to a single focus involving contiguous bones in the craniofacial region. F) A 42-year-old man with polyostotic FD shows the tendency for FD to be predominantly (but not exclusively) unilateral, and to involve the skull base and prox- imal femur. G) A 16-year-old boy with McCune-Albright syn- drome and involvement of virtually all skeletal sites (panostotic) is shown [65]. Radiographic appearance of fibrous dysplasia (FD) Figure 2 Radiographic appearance of fibrous dysplasia (FD). A) A proximal femur with typical ground glass appearance and shepherd's crook deformity in a 10-year-old child is shown. B) The appearance of FD in the femur of an untreated 40- year-old man demonstrates the tendency for FD to appear more sclerotic with time C) The typical ground glass appear- ance of FD in the craniofacial region on a CT image of a 10- year-old child is shown. The white arrows indicate the optic nerves, which are typically encased with FD. D) A CT image in a 40-year-old woman demonstrates the typical appearance of craniofacial FD in an older person, with mixed solid and "cystic" lesions. The Hounsfield Unit measurements of "cystic" lesions are quite useful in distinguishing soft tissue "cystic" lesions from true fluid-filled cysts, which are much more uncommon and tend to behave aggressively with rapid expansion and compression of vital structures. E-G) Bone Scintigraphy in FD. Representative 99Tc-MDP bone scans which show tracer uptake at affected skeletal sites, and the associated skeletal disease burden score (see ref. Collins, 2005) are shown. E) A 50-year-old woman with monostotic FD confined to a single focus involving contiguous bones in the craniofacial region. F) A 42-year-old man with polyostotic FD shows the tendency for FD to be predominantly (but not exclusively) unilateral, and to involve the skull base and prox- imal femur. G) A 16-year-old boy with McCune-Albright syn- drome and involvement of virtually all skeletal sites (panostotic) is shown [65]. Malignancies in MAS While malignancies associated with MAS are distinctly rare occurrences, they warrant mentioning due to their importance. Malignant transformation of FD lesions is probably the most common and best described malig- nancy that occurs in association with MAS [12,16,19]. This occurs in probably less than 1% of the cases of FD/ MAS. High dose external beam radiation is a risk factor for sarcomatous transformation [19]. There may be a greater tendency for malignant transformation to occur in patients who have concomitant GH excess [20]. While some have suggested that sarcomatous transformation of skeletal lesions may occur more commonly in patients with Mazabraud's syndrome (benign intramuscular myxomas in association with long standing FD) [21], this may represent selection bias. Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) http://www.ojrd.com/content/3/1/12 http://www.ojrd.com/content/3/1/12 Orphanet Journal of Rare Diseases 2008, 3:12 Radiographic appearance of fibrous dysplasia (FD) Figure 2 Radiographic appearance of fibrous dysplasia (FD). A) A proximal femur with typical ground glass appearance and shepherd's crook deformity in a 10-year-old child is shown. B) The appearance of FD in the femur of an untreated 40- year-old man demonstrates the tendency for FD to appear more sclerotic with time C) The typical ground glass appear- ance of FD in the craniofacial region on a CT image of a 10- year-old child is shown. The white arrows indicate the optic nerves, which are typically encased with FD. D) A CT image in a 40-year-old woman demonstrates the typical appearance of craniofacial FD in an older person, with mixed solid and "cystic" lesions. The Hounsfield Unit measurements of "cystic" lesions are quite useful in distinguishing soft tissue "cystic" lesions from true fluid-filled cysts, which are much more uncommon and tend to behave aggressively with rapid expansion and compression of vital structures. E-G) Bone Scintigraphy in FD. Representative 99Tc-MDP bone scans which show tracer uptake at affected skeletal sites, and the associated skeletal disease burden score (see ref. Collins, 2005) are shown. E) A 50-year-old woman with monostotic FD confined to a single focus involving contiguous bones in the craniofacial region. F) A 42-year-old man with polyostotic FD shows the tendency for FD to be predominantly (but not exclusively) unilateral, and to involve the skull base and prox- imal femur. Malignancies in MAS G) A 16-year-old boy with McCune-Albright syn- drome and involvement of virtually all skeletal sites (panostotic) is shown [65]. Etiology gy The observation that the G protein/cAMP/adenylate cyclase signaling pathway was central to all of the tissues involved in MAS eventually led to the discovery that mutations in the regulatory Gsα protein (encoded by the GNAS gene) were the underlying molecular etiology of MAS [25,26] (Fig. 5). In all published cases of MAS, PFD, and even MFD, activating mutations of Gsα at the R201 position have been identified [27]. More recently, muta- tions at the Q227 position have been found in association with FD [28]. The lack of vertical transmission of the disease, along with the observation that skin and bone lesions tend to respect the midline and be on one or the other side of the body, has led to the unproven, but accepted, concept that the disease is the result of postzygotic mutations, and that patients are therefore somatic mosaics. The point in time in development at which the mutation occurs, the specific cell in which it occurs, and to where its progeny migrate, determines what tissues will be affected, and thus the phe- notype. Therefore, in cases in which tissues of endoder- mal, mesodermal, and ectodermal origin are involved, it would appear that the mutation occurred at the inner cell mass stage (Fig. 6) [29]. Radiographic appearance of fibrous dysplasia (FD) Figure 2 Radiographic appearance of fibrous dysplasia (FD). A) Radiographic appearance of fibrous dysplasia (FD) Figure 2 Radiographic appearance of fibrous dysplasia (FD). A) A proximal femur with typical ground glass appearance and shepherd's crook deformity in a 10-year-old child is shown. B) The appearance of FD in the femur of an untreated 40- year-old man demonstrates the tendency for FD to appear more sclerotic with time C) The typical ground glass appear- ance of FD in the craniofacial region on a CT image of a 10- year-old child is shown. The white arrows indicate the optic nerves, which are typically encased with FD. D) A CT image in a 40-year-old woman demonstrates the typical appearance of craniofacial FD in an older person, with mixed solid and "cystic" lesions. The Hounsfield Unit measurements of "cystic" lesions are quite useful in distinguishing soft tissue "cystic" lesions from true fluid-filled cysts, which are much more uncommon and tend to behave aggressively with rapid expansion and compression of vital structures. E-G) Bone Scintigraphy in FD. Diagnosis, diagnostic criteria, diagnostic methods, differential diagnosis , g Diagnosis of MAS is usually established on clinical grounds. Plain radiographs are often sufficient to make the diagnosis of FD (Fig. 2). Isotopic bone scans are the most sensitive tool for detecting the presence of FD lesions, and are often useful, especially at the initial eval- uation, for determining the extent of the disease and pre- dicting functional outcome (Fig. 4E,F, &4G) [17,30]. FD has a typical appearance on radiographs described as "ground glass." In general, lesions in the long bones have a "lytic" appearance. The lesions usually arise in the med- ullary cavity and expand outward replacing normal bone, which results in thinning of the cortex (Fig. 2A &2B). It is usually the metaphysis and/or the diaphysis that are involved, with sparing of the epiphysis. It is possible for any bone to be involved, but the skull base and the prox- imal femur are the sites most commonly involved [16,31,32]. Due to the fact that these lesions are under- mineralized [27], the bones are "soft" and prone to defor- mation, as exemplified by the classic "shepherd's crook" deformity of the proximal femur (Fig. 2A). In addition, the risk of breast cancer may be elevated in patients with MAS [22,23]. Besides the published reports, in the series of approximately 120 patients seen at the National Institutes of Health (NIH), the prevalence of breast cancer was approximately 2.5% (n = 3). In this group of three patients, there also seems to be an effect of GH excess, in that all patients who had breast cancer also had GH excess (unpublished data). FD in the craniofacial bones tends to have a "sclerotic" appearance on plain radiographs. This is due to the rela- tively greater degree of mineralization of FD tissue in the craniofacial bones (Fig. 3) [33]. Computed tomography (CT) scanning is the best technique for imaging FD lesions in the skull, revealing a "ground glass" appear- Thyroid cancer [24] and testicular cancer (unpublished data) are also rare occurrences. Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:12 http://www.ojrd.com/content/3/1/12 Representative histological images of FD Figure 3 Representative histological images of FD. A) Calvarial FD lesions are characterized by uninterrupted networks of bone trabeculae (b) embedded in the fibrous tissue (ft). Diagnosis, diagnostic criteria, diagnostic methods, differential diagnosis B) In FD lesions from gnathic bones, newly formed bone trabeculae (b) are deposited within the fibrous tissue (ft) in a typical discontinuous and parallel pattern. C) Collagen fibers perpendicularly ori- ented to forming bone surfaces (Sharpey fibers, arrows) represent a recurrent histological feature of FD at all skeletal sites. D- E) Osteomalacic changes and FGF23 production in FD. D) In many cases of FD, processing for undecalcified embedding reveals excess osteoid (asterisks) and severe undermineralization of the fibrous dysplastic bone. E) The mineralization defect of the FD tissue is related to elevated levels of FGF23 produced by activated FD osteogenic cells (arrows), as shown by in situ hybridiza- tion. Represent Figure 3 Representative histological images of FD Figure 3 Representative histological images of FD. A) Calvarial FD lesions are characterized by uninterrupted networks of bone trabeculae (b) embedded in the fibrous tissue (ft). B) In FD lesions from gnathic bones, newly formed bone trabeculae (b) are deposited within the fibrous tissue (ft) in a typical discontinuous and parallel pattern. C) Collagen fibers perpendicularly ori- ented to forming bone surfaces (Sharpey fibers, arrows) represent a recurrent histological feature of FD at all skeletal sites. D- E) Osteomalacic changes and FGF23 production in FD. D) In many cases of FD, processing for undecalcified embedding reveals excess osteoid (asterisks) and severe undermineralization of the fibrous dysplastic bone. E) The mineralization defect of the FD tissue is related to elevated levels of FGF23 produced by activated FD osteogenic cells (arrows), as shown by in situ hybridiza- tion. Representative histological images of FD Figure 3 Representative histological images of FD. A) Calvarial FD lesions are characterized by uninterrupted networks of bone trabeculae (b) embedded in the fibrous tissue (ft). B) In FD lesions from gnathic bones, newly formed bone trabeculae (b) are deposited within the fibrous tissue (ft) in a typical discontinuous and parallel pattern. C) Collagen fibers perpendicularly ori- ented to forming bone surfaces (Sharpey fibers, arrows) represent a recurrent histological feature of FD at all skeletal sites. D- E) Osteomalacic changes and FGF23 production in FD. D) In many cases of FD, processing for undecalcified embedding reveals excess osteoid (asterisks) and severe undermineralization of the fibrous dysplastic bone. E) The mineralization defect of the FD tissue is related to elevated levels of FGF23 produced by activated FD osteogenic cells (arrows), as shown by in situ hybridiza- tion. Diagnosis, diagnostic criteria, diagnostic methods, differential diagnosis Page 4 of 12 (page number not for citation purposes) Fracture rates in FD Figure 4 Fracture rates in FD. Fractures rates (reported as mean number of fractures per patient per year) are shown. Frac- tures are more frequent in childhood, with the highest rate occurring between 6–10 years of age. While fractures do lessen after childhood, there is a persistent rate into adult- hood [18]. ance. In children and young adults, the lesions appear homogeneous on CT, but in older patients the appearance is mixed, with the development of "cystic" lesions in some areas. The density of these areas is that of soft tissue, so while they may have a cystic appearance they are not true cysts. That said, it is possible for true cysts to develop in FD, both in the long bones, but more often in the cranio- facial bones (Fig. 7). This has occurred in about 5% of the patients with FD in the NIH cohort (unpublished data). If needed, bone cysts may be diagnosed using magnetic res- onance imaging (MRI). The cysts tend to have a more aggressive course. They can expand rapidly and produce symptoms which vary, depending on the location. One of the complications can be the fracture through a cyst. This usually requires surgical intervention. Page 4 of 12 (page number not for citation purposes) adults, the lesions appear der patients the appearance of "cystic" lesions in some as is that of soft tissue, so pearance they are not true or true cysts to develop in t more often in the cranio- ccurred in about 5% of the hort (unpublished data). If gnosed using magnetic res- ysts tend to have a more pand rapidly and produce ng on the location. One of acture through a cyst. This ntion. rm the diagnosis if doubt ographs. One characteristic e lamellation pattern seen d light. This indicates that Fracture rates in FD Figure 4 Fracture rates in FD. Fractures rates (reported as mean number of fractures per patient per year) are shown. Frac- tures are more frequent in childhood, with the highest rate occurring between 6–10 years of age. While fractures do lessen after childhood, there is a persistent rate into adult- hood [18]. Genetic testing Molecular defect and phenotype in McCune-Albright syn- drome (MAS) Figure 5 Molecular defect and phenotype in McCune-Albright syndrome (MAS). The hormones MSH (melanocyte stimu- lating hormone), LH (luteinizing hormone), TSH (thyroid stimulating hormone), GHRH (growth hormone stimulating hormone), and ACTH (adrenocrotical stimulating hormone) all signal through the G protein (alpha, beta, gamma subunits) pathway. In MAS, the alpha subunit is mutated in such a way as to induce constitutive activation of adenylate cyclase, and thus produce high levels of intracellular cAMP. This results in increased production of melanin, estradiol (E2), testosterone (T), thyroxine (T4), growth hormone (GH), and cortisol. Dysregulated production of these hormones results in café- au-lait spots, precocious puberty, fibrous dysplasia, acromeg- aly, hyperthyroidism, and Cushing's disease, depending on the tissue harboring the somatic mutation. Genetic testing is possible, but is not routinely available (see below). Because of the somatic mosaic nature of the disease, a negative result from readily available (but unaf- fected) tissue does not exclude the presence of the muta- tion. Testing on leukocyte DNA is possible [35], but it is unreliable. In most cases, genetic testing contributes little Molecular and developmental defect in McCune-Albright syn- drome (MAS) Figure 6 Molecular and developmental defect in McCune- Albright syndrome (MAS). A sporadic mutation occurs in a single cell (bright spot) at some point early in development. If this occurs at the inner call mass stage (embryonic stem cell stage), tissues from all 3 germ layers will be affected. As the cells derived from this mutated clone are dispersed throughout the organism, the final phenotype emerges, MAS. Molecular defect and phenotype in McCune-Albright syn- drome (MAS) Figure 5 Molecular defect and phenotype in McCune-Albright syndrome (MAS). The hormones MSH (melanocyte stimu- lating hormone), LH (luteinizing hormone), TSH (thyroid stimulating hormone), GHRH (growth hormone stimulating hormone), and ACTH (adrenocrotical stimulating hormone) all signal through the G protein (alpha, beta, gamma subunits) pathway. In MAS, the alpha subunit is mutated in such a way as to induce constitutive activation of adenylate cyclase, and thus produce high levels of intracellular cAMP. This results in increased production of melanin, estradiol (E2), testosterone (T), thyroxine (T4), growth hormone (GH), and cortisol. Dysregulated production of these hormones results in café- au-lait spots, precocious puberty, fibrous dysplasia, acromeg- aly, hyperthyroidism, and Cushing's disease, depending on the tissue harboring the somatic mutation. Fracture ra Figure 4 [16, 18] nerve decompression is contraindicated [40, 41] usually asymptomatic, scoliosis common and possibly progressive [56] rib pain develops late often painful in adults, but pain is quite variable [17, 18] Vaginal bleeding, breast development with little or no pubic hair, secondary central PP often develops, GnRH test [66] common, detectable by ultrasound only, cancer rare, but possible [67] ultrasound and biopsy large solid or changing lesions [3], cancer can occur [24] worsens pain [6]; etiology is FGF23 of FD bone origin [43] suggests GH excess, worsens craniofacial FD [5, 41] quite ill, spontaneous resolution possible [9] alopecia [70], myxomas and others cutaneous abnormalities [71] Gonadal – Male Thyroid abnormalities Phosphaturia Growth hormone (GH) excess Cushing syndrome Additional clinical features the matrix produced in the lesions is of the woven type. The histopathological description of FD is often described as a "Chinese writing" pattern, and with special prepara- tion and stains used to detect mineralized and unmineral- ized tissue, extensive areas of unmineralized osteoid are evident (Fig. 3) [27]. For an extensive description of the histopathological changes that can be observed in FD, the reader is referred to Riminucci et al. and Corsi et al. [33,34]. Fracture ra Figure 4 g Fracture rates in FD. Fractures rates (reported as mean number of fractures per patient per year) are shown. Frac- tures are more frequent in childhood, with the highest rate occurring between 6–10 years of age. While fractures do lessen after childhood, there is a persistent rate into adult- hood [18]. Biopsy of FD lesions can confirm the diagnosis if doubt remains after review of the radiographs. One characteristic of FD bone is the absence of the lamellation pattern seen in normal bone under polarized light. This indicates that Page 4 of 12 (page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:12 http://www.ojrd.com/content/3/1/12 Table 1: Clinical presentation Clinical feature Presentation/Feature Café-au-lait spots appearance: "coast-of-Maine", location: nape of neck, base of spine; usually respect midline; trunk & face more commonly involved than limbs (Figure 1) Fibrous dysplasia (FD) phosphatase, medullary cavity of the bone. [16, 18] Craniofacial FD nerve decompression is contraindicated [40, 41] Axial FD usually asymptomatic, scoliosis common and possibly progressive [56] rib pain develops late Appendicular FD often painful in adults, but pain is quite variable [17, 18] Gonadal – Female Vaginal bleeding, breast development with little or no pubic hair, secondary central PP often develops, GnRH test [66] Gonadal – Male common, detectable by ultrasound only, cancer rare, but possible [67] Thyroid abnormalities ultrasound and biopsy large solid or changing lesions [3], cancer can occur [24] Phosphaturia worsens pain [6]; etiology is FGF23 of FD bone origin [43] Growth hormone (GH) excess suggests GH excess, worsens craniofacial FD [5, 41] Cushing syndrome quite ill, spontaneous resolution possible [9] Additional clinical features alopecia [70], myxomas and others cutaneous abnormalities [71] Presentation/Feature appearance: "coast-of-Maine", location: nape of neck, base of spine; usually respect midline; trunk & face more commonly involved than limbs (Figure 1) phosphatase, medullary cavity of the bone. Genetic testing The fluid-filled nature of the lesion is demonstrated by the fluid-fluid level (arrow). These lesions are more frequent in the craniofacial bones and can be aggressive. They usually require prompt surgical intervention. to the diagnosis and not at all to management. There is no known genotype/phenotype correlation, so knowledge of the specific mutation does not affect management. For this reason, when the diagnosis of MAS is suspected or established, it is important to be cognizant of the spec- trum of tissues that possibly can be involved, and to screen for involvement. Screening, at a minimum, requires a medical history and physical examination, and usually involves specific imaging and biochemical testing. 1. Craniofacial FD f CT scan of the skull is the most useful test for diagnosis of craniofacial FD (Fig. 4). CT should be repeated periodi- cally, annually then less frequently once stability has been demonstrated. Vision should be evaluated, ideally by a Genetic testing These lesions are almost exclusively found in the tibia and fib- ula, and are histologically distinct from FD [12]. Non- ossifying fibromas (NOF) may also share radiological and histological similarities with FD in the long bones. Lack of multiple skeletal foci and absence of extraskeletal findings may help to distinguish FD from NOF. FD in the jaw may share several histological features with cemento ossifying fibromas, which can be confused with FD. The cemento ossifying fibroma lesions tend to behave more aggres- sively than do FD lesions. Testing for the GNAS mutation, if tissues and assays are available, may be helpful in distin- guishing cemento ossifying fibroma lesions from FD. Isolated skeletal lesions in the absence of skin or endo- crine findings represent FD (MFD or PFD). Osteofibrous dysplasia (ossifying fibroma of long bones, so-called Campanacci's lesion) may be confused with FD. These lesions are almost exclusively found in the tibia and fib- ula, and are histologically distinct from FD [12]. Non- ossifying fibromas (NOF) may also share radiological and histological similarities with FD in the long bones. Lack of multiple skeletal foci and absence of extraskeletal findings may help to distinguish FD from NOF. FD in the jaw may share several histological features with cemento ossifying fibromas, which can be confused with FD. The cemento ossifying fibroma lesions tend to behave more aggres- sively than do FD lesions. Testing for the GNAS mutation, if tissues and assays are available, may be helpful in distin- guishing cemento ossifying fibroma lesions from FD. Fluid-filled cyst in FD Figure 7 Fluid-filled cyst in FD. True fluid-filled cysts can occur in FD lesions. Shown is the CT scan of fluid-filled cyst that arose in a 12 year old boy with MAS who presented with facial nerve parasthesias and displacement of the orbit that occurred over approximately one week. The fluid-filled nature of the lesion is demonstrated by the fluid-fluid level (arrow). These lesions are more frequent in the craniofacial bones and can be aggressive. They usually require prompt surgical intervention. Fluid-filled cyst in FD Figure 7 Fluid-filled cyst in FD. True fluid-filled cysts can occur in FD lesions. Shown is the CT scan of fluid-filled cyst that arose in a 12 year old boy with MAS who presented with facial nerve parasthesias and displacement of the orbit that occurred over approximately one week. Genetic testing Molecular and developmental defect in McCune-Albright syn- drome (MAS) Figure 6 Molecular and developmental defect in McCune- Albright syndrome (MAS). A sporadic mutation occurs in a single cell (bright spot) at some point early in development. If this occurs at the inner call mass stage (embryonic stem cell stage), tissues from all 3 germ layers will be affected. As the cells derived from this mutated clone are dispersed throughout the organism, the final phenotype emerges, MAS. Page 5 of 12 (page number not for citation purposes) http://www.ojrd.com/content/3/1/12 http://www.ojrd.com/content/3/1/12 Orphanet Journal of Rare Diseases 2008, 3:12 Fluid-filled cyst in FD Figure 7 Fluid-filled cyst in FD. True fluid-filled cysts can occur in FD lesions. Shown is the CT scan of fluid-filled cyst that arose in a 12 year old boy with MAS who presented with facial nerve parasthesias and displacement of the orbit that occurred over approximately one week. The fluid-filled nature of the lesion is demonstrated by the fluid-fluid level (arrow). These lesions are more frequent in the craniofacial bones and can be aggressive. They usually require prompt surgical intervention. lait spot. The location and shape of the spots usually can help to distinguish between the MAS and NF. The spots in MAS have jagged borders (coast of Maine), whereas those in NF are smooth (coast of California). Although the spots can cross the midline, more often, they demonstrate a "respect" of the midline. Frequent locations are the nape of the neck and the crease at the apex of the buttocks (Fig. 1). In MAS, the skeletal disease (PFD) almost always involves one or both proximal femurs and/or the skull base, as well as other locations. Skeletal involvement in NF is uncommon and usually involves the diaphyses of the long bones, especially the tibiae, often leading to pseudoarthrosis [38,39]. When precocious puberty is the presenting sign, the differ- ential diagnosis includes idiopathic central precocious puberty, and an ovarian neoplasm. Suppressed gonado- tropins exclude central PP. Other components of the MAS (skin pigmentation, skeletal changes on x-ray or bone scan, etc.) can help to assure the clinician that the ovarian cyst is not neoplastic. Isolated skeletal lesions in the absence of skin or endo- crine findings represent FD (MFD or PFD). Osteofibrous dysplasia (ossifying fibroma of long bones, so-called Campanacci's lesion) may be confused with FD. Specific diagnostic considerations and follow-up A. Skeletal Skeletal disease, especially involving the skull base, is very common. Vision and/or hearing loss are uncommon, and sarcomatous transformation is rare (<1%). FD in the appendicular skeleton tends to quiet with age, but the course of craniofacial disease is less clear [18,40]. Nuclear medicine bone scans are useful for not only detecting the extent of the disease, but quantifying the skeletal disease burden of FD and predicting functional outcome [30]. The following are listed as potential sources for genetic testing: Center for Genetic Testing at Saint Francis [36], Genome Diagnostics [37]. In addition, the research labo- ratories of Professors Francis Glorieux, Shriners Hospital for Children (Montreal, Canada), Paolo Bianco (Rome, Italy) and Charles Sultan (Montpellier, France) can per- form genetic testing on a research basis. 2. Axial and appendicular skeleton Plain radiographs, demonstrating the classic ground glass appearance, are usually sufficient for the diagnosis of FD in the axial and appendicular skeleton (the axial skeleton is comprised of bones of the skull, hyoid bone, vertebra, sternum and ribs; the appendicular skeleton includes the hip, pelvic bone and the shoulder girdle (clavicle and scapula) (Fig. 2). However, x-rays are often unable to detect new, small microfractures. When new focal pain develops in an FD lesion and no fracture is evident on plain radiograph, CT and or MRI can be useful for detect- ing subtle fractures (Fig. 8). The classic lesion of the prox- imal femur in FD, the shepherd's crook deformity is common (Fig. 2). B. Endocrine 1. Gonads neuro-ophthalmologist initially, then periodically; annu- ally or less frequently once stability has been demon- strated. Hearing evaluation is recommended at baseline to assess involvement and should also be repeated periodi- cally. All endocrinopathies which adversely affect bone should be screened for and treated. GH excess in particu- lar may adversely affect craniofacial FD [5,40,41]. 99Tc- methyl diphosphonate (99Tc-MDP) bone scan at baseline and periodically is recommended. PP is more common in girls then boys. However, small testicular masses of Leydig and/or Sertoli cell hyperplasia are common in boys. These are usually never detected on physically examination. Therefore, ultrasound of the tes- tes is recommended in all males with FD and MAS. It is probably safe to monitor testicular masses without surgi- cal intervention as cancer is probably quite rare. The PP of MAS in girls is due to high levels of serum estradiol due to intermittent autonomous activation of ovarian tissue. Some of them will progress to central PP, secondary to activation of the hypothalamic-pituitary-gonadal (HPG) axis. Since central PP usually develops in children who have had PP for some years, children with PP should be evaluated for secondary central PP. 2. Thyroid Hyperthyroidism is common (38%) [3]. Evidence of thy- roid involvement without frank hyperthyroidism is even more common (63% in the NIH series, unpublished data). This is manifested as a relatively suppressed thyroid stimulating hormone (TSH) with elevated Triiodothyro- nine (T3+) and an abnormal thyroid gland on ultrasound. Some of these patients may go on to develop frank hyper- thyroidism, therefore, follow-up with measurement of TSH, free thyroxine (FT4), T3, and thyroxine (T4), and periodic ultrasound is recommended to detect those patients that progress to frank hyperthyroidism. While the development of thyroid cancer in patients with MAS is rare [24], patients should be monitored for this possibil- ity. This is accomplished by annual ultrasound of the thy- roid. The ultrasonographic appearance of the thyroid in MAS is complicated [42]. There are usually admixed areas of relatively normal appearing thyroid with adjacent cystic areas. Suspicion for cancer should be raised if a large or expanding solid lesion is noted. This should prompt fine needle aspiration with cytopathological evaluation. CT for detecting subtle fractures Figure 8 CT for detecting subtle fractures. A) This radiograph was from a 9-year-old boy who complained of new-onset, focal pain in the groin. No discernable fracture is apparent. B) Reformatted CT images of the lesion revealed a fracture in the medial proximal femur (arrows). Differential diagnosis MAS is most commonly confused with neurofibromatosis (NF), usually when a child presents with a large café-au- Page 6 of 12 (page number not for citation purposes) Page 6 of 12 (page number not for citation purposes) http://www.ojrd.com/content/3/1/12 http://www.ojrd.com/content/3/1/12 Orphanet Journal of Rare Diseases 2008, 3:12 Antenatal diagnosis Not relevant. Appearanc Figure 9 Appearanc Figure 9 Appearance of Cushing in a neonate Figure 9 Appearance of Cushing in a neonate. Rounded (moon) facies with plethora and facial hirsutism are present. This is usually accompanied by the presence of typical café-au-lait spots elsewhere on the examination. pp g g Appearance of Cushing in a neonate. Rounded (moon) facies with plethora and facial hirsutism are present. This is usually accompanied by the presence of typical café-au-lait spots elsewhere on the examination. Genetic counseling The most important aspect of counseling families is to assure the patients or families that there is no vertical transmission of the disease, nor are there any known envi- ronmental associations or predilection for ethnic groups. Therefore, parents need not feel "responsible," and patients can be assured they will not transmit the disease to their offspring. 3. Renal phosphate wasting l h h Renal phosphate wasting, as part of a proximal tubulopa- thy, with or without hypophosphatemia, and/or rickets/ osteomalacia is common [6]. The etiology is likely due to elaboration of the phosphaturic factor, fibroblast growth factor-23 (FGF23), by FD tissue [43]. Measurement of serum phosphate and calculation of renal phosphate han- dling by either the tubular reabsorption of phosphate (TRP) or maximal tubular reabsorption of phosphate per glomerular filtration rate (TmP/GFR) are recommended. While it is debatable whether or not hypophosphatemia, per se, should be treated, or if treatment should be reserved for only patients with rickets or biopsy-proven rickets [44,45], it is our feeling that frank hypophosphatemia should be treated (see Additional file 1). CT for det Figure 8 g g CT for detecting subtle fractures. A) This radiograph was from a 9-year-old boy who complained of new-onset, focal pain in the groin. No discernable fracture is apparent. B) Reformatted CT images of the lesion revealed a fracture in the medial proximal femur (arrows). g g CT for detecting subtle fractures. A) This radiograph was from a 9-year-old boy who complained of new-onset, focal pain in the groin. No discernable fracture is apparent. B) Reformatted CT images of the lesion revealed a fracture in the medial proximal femur (arrows). Page 7 of 12 (page number not for citation purposes) Page 7 of 12 (page number not for citation purposes) http://www.ojrd.com/content/3/1/12 Orphanet Journal of Rare Diseases 2008, 3:12 Appearance of Cushing in a neonate Figure 9 Appearance of Cushing in a neonate. Rounded (moon) facies with plethora and facial hirsutism are present. This is usually accompanied by the presence of typical café-au-lait spots elsewhere on the examination. cated. Adrenal reserve should be checked in patients in whom Cushing resolved spontaneously (personal com- ment, in the NIH series 2 out of 9 patients with Cushing had spontaneously cure and inadequate adrenal reserve later in life). 4. Pituitary GH and prolactin excess are common (21%), and the signs and symptoms can be very subtle. Since GH excess can worsen craniofacial bone disease [5,40,41], it is important to make the diagnosis and treat. All patients should have an oral glucose tolerance test (OGTT) to look for non-suppressible GH at least once. Most GH secreting tumors are co-secretors of GH and prolactin, so prolactin levels should also be assessed, as hyperprolactinemia can independently have an adverse affect on gonadal func- tion. 1. Craniofacial FD In the vast majority of cases of craniofacial FD, surgery is not needed, and observation is the correct approach. Observation involves annual vision and hearing testing, and imaging. Vision should be checked by a neuro-oph- thalmologist. CT of skull and/or mandible is the imaging test of choice for the cranial structures in general, but MRI will give better resolution of the optic nerves. Cranial nerves are often surrounded by FD bone, but remain unaf- fected for years to decades [41]. Indications for surgery include documented progressive visual disturbance, severe pain, or severe disfigurement. Surgery should be avoided in the absence of visual or hearing impairment. However, when surgery is indicated, it is very important to find a craniofacial and neurosurgical team experienced in treating craniofacial FD. It is possible the craniofacial dis- ease may continue to progress slowly into adulthood, but this subject has not been well studied in a prospective manner [40]. 5. Parathyroid Primary hyperparathyroidism in MAS is rare and is prob- ably not a part of the syndrome [46]. Secondary hyperpar- athyroidism, usually due to vitamin D deficiency is common in the general population as well as in FD/MAS [47-50]. Hyperparathyroidism can worsen FD and should be treated [34]. Total or ionized serum calcium and par- athyroid hormone (PTH) need to be assessed periodically. There is little evidence that bisphosphonates are effective in craniofacial FD, even for pain, but in the case of pain that is difficult to manage, bisphosphonate treatment should be considered (Additional file 2). Page 8 of 12 (page number not for citation purposes) 2. Thyroid Bisphosphonates are frequently used in the treatment of FD (reviewed in [45,52]. The original thinking in the use of bisphosphonates in FD was that they would prevent the progression of disease [53]. However, more recent work concludes that bisphosphonates have no effect on the nat- ural history of the disease [54,55]. It is clear, however, that bisphosphonates are effective in relieving bone pain asso- ciated with FD [32,45,52]. Many regimens have been sug- gested. The long term safety of bisphosphonates, especially in children, and the effects of relatively high doses of bisphosphonates on normal bone not affected by FD, are unknown. Therefore, to minimize exposure, our approach has been to use the minimum dose with the longest interval between doses possible needed to achieve and maintain pain relief (Additional file 2). Antithyroidal medications, such as thionamides, are usu- ally effective in controlling hyperthyroidism in MAS [60]. Spontaneous resolution of hyperthyroidism in MAS almost never occurs, so some form of definitive treatment, either in the form of surgery or radiation, is eventually indicated. Periodic ultrasounds of the thyroid to follow lesions should be performed annually. If a dominant or changing lesion is identified, fine needle aspiration is indicated to exclude cancer, given that thyroid cancer can be seen in the setting of MAS [24]. 2. Axial and appendicular skeleton Cushing syndrome can occur in the neonatal period, but has not been reported past the first year. Some cases of neonatal Cushing resolve spontaneously [9]. Cushing syndrome must be considered and screened for in very young patients. The physical examination is usually sug- gestive and shows moon facies with plethora, hirsutism, and lack of linear growth (Fig. 9). If suspected, laboratory evaluation with some combination of a 24-hour urine for urinary free cortisol (if possible), midnight, or salivary cortisol, and/or dexamethasone suppression test are indi- The surgical management of FD has evolved over the years. While curettage and bone grafting were once con- sidered the treatment of choice, experts in the care of FD no longer feel this is effective [31,51]. Intramedullary devices are preferable, in general [31,51]. Bracing may be helpful, but has not been well-studied. Shepherd's crook deformity can develop, sometimes very rapid, and the management in growing children is very challenging. Generally, some form of surgical intervention is recom- Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:12 http://www.ojrd.com/content/3/1/12 mended. Screening for and treatment of all endocrinopa- thies which adversely affect the skeleton should be performed [34]. This includes PP, hyperthyroidism, Cush- ing's syndrome, GH excess, and secondary hyperparathy- roidism. may be observation with annual testicular ultrasounds, and CT of the chest abdomen and pelvis if there is any sus- picion for malignancy. Lesions should be followed annu- ally with ultrasound. There are no reported cases of gynecological malignancy in females with MAS. 3. Renal Renal involvement, in the form of renal phosphate wast- ing, is seen in approximately 50% of the patients with MAS [6]. However, only some of those patients will have a degree of phosphate wasting that results in hypophos- phatemia (18% of the total population seen at NIH). It is debatable whether the hypophosphatemia warrants treat- ment with oral phosphorus supplementation if signs of rickets are absent [44]. However, we recommend that patients with frankly low serum phosphate should be treated. Treatment is the same as that for patients with X- linked hypophosphatemia and tumor induced osteoma- lacia: high dose of oral phosphate with high dose of calci- triol (Additional file 1). Maintaining the musculature around the FD bone is important for protecting the bone. Therefore, strengthen- ing exercises and strength maintenance is important. Swimming and cycling are excellent modes of exercise for patients with FD that will promote strengthening and minimize the risk for fracture. Scoliosis is common, and may be progressive [56]. There- fore, it is important to screen for the presence of scoliosis and monitor for any progression. Significant progression can occur within a short period of time. 4. Pituitary GH GH excess is seen in approximately 20% of the patients with MAS [5]. GH excess has been shown to be associated with vision and hearing loss, and macrocephaly in patients with MAS [41]. For that reason, patients with GH excess and an elevated insulin-like growth factor-1 (IGF- 1) should be treated. Treatment options include medica- tions, surgery, and radiation. Effective medical treatment includes long-acting somatostatin analogues [4,5] and the GH receptor antagonist, pegvisomant [61-63]. There is greater long-term efficacy and safety with the somatosta- tin analogues, especially in children. Pegvisomant is sig- nificantly more expensive than the somatostatin analogues and may be associated with more side effects [61]. The goal of treatment in a growing child is to reduce the calculated serum IGF-1 Z-score to 0. B. Endocrine 1. Gonads The management of PP in boys and girls is detailed in Additional file 3. The class of drugs with the longest his- tory of safety and efficacy are the aromatase inhibitors [57,58]. More recently, the estrogen antagonist/agonist, tamoxifen, has also shown promise [59]. Secondary cen- tral PP in children with MAS frequently develops after a period of PP. Its manifestation is often pubertal progres- sion in a child that had previously been well-controlled on medical therapy. The treatment of secondary central PP in children with MAS is the same as in children with idio- pathic PP, and involves the use of long-acting gonadotro- pin releasing hormone analogues. Ultrasound is used to screen and follow boys and men with testicular masses. These are usually Leydig tumors, but testes can demonstrate a mixture of Leydig and Sertoli cell masses. Microlithiasis is also commonly seen. The development of malignancy in the testes of men with MAS appears to be very rare. Therefore, prudent management Surgery is an option, but is sometimes not possible due to the massive thickening of the skull bones. A trans-sphe- noidal approach is the preferred method in pituitary sur- gery, but the skull base is virtually always involved with FD in patients with MAS and GH excess, so this approach Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:12 http://www.ojrd.com/content/3/1/12 http://www.ojrd.com/content/3/1/12 http://www.ojrd.com/content/3/1/12 bisphosphonates changes the natural history of the dis- ease remains an open question. The most recent, and strongest data to date, suggests that they do not [54]. Ongoing placebo controlled trials in the US and Europe, should help to resolve this open question. More effective treatment for FD is needed. The development of cell based and/or Gsα directed therapies may hold promise. is often impossible, or difficult, at best. A trans-frontal approach is a less desirable approach to the pituitary, in general, and in MAS it is also usually complicated by FD. Furthermore, in patients with MAS and GH excess the pituitary gland is almost always diffusely involved with areas of hyperplasia and adenoma (Edward Oldfield, per- sonal communication). Therefore, the only hope for "cure" of the GH excess is a total hypophysectomy. Support A list of groups that support patients, families, and clini- cians caring for patients with MAS is supplied in Addi- tional file 4. 5. Parathyroid While primary hyperparathyroidism has been reported to be part of the syndrome in the past [64], this may not be the case. When the mutations that cause MAS were sought in parathyroid tissue, they were not present [46]. Addi- tionally, parathyroid hormone secretion and hyperplasia is likely not a cAMP, Gsα mediated phenomenon. Second- ary hyperparathyroidism due to vitamin D deficiency is common and can worsen FD [34]. Therefore, it should be screened for and treated. B. Endocrine 1. Gonads This may be the best treatment in some cases, but physicians, surgeons, and patients should embark upon this option knowing that the patient will be rendered panhypopitui- tary at the end of the operation. The best treatment for PP is also not clear. Letrozole, a potent third generation aromatase inhibitor, has recently been shown to be an effective therapy in some girls with MAS [57]. Studies suggest tamoxifen, the estrogen ago- nist/antagonist, may also be effective [59]. However, all trials for the treatment of PP have been uncontrolled, and this fact, combined with the extreme variability in the clinical course of the disease, makes conclusions about relative efficacy very difficult. A trial with the pure anti- estrogen, fulvestrant, in girls with MAS is underway. Con- trolled and head-to-head comparison trials are needed to establish the best treatment for PP in MAS. Radiotherapy is an option when surgery is not possible and medicinal treatment fails. However, there may be a higher risk of sarcomatous transformation [19,62]. Since most of the GH secreting tumors co-secrete prolac- tin [5], dopamine agonists (cabergoline, etc.) are usually necessary, and are effective at normalizing the serum pro- lactin. Abbreviations FD: fibrous dysplasia of bone; PP: precocious puberty; GNAS: G protein binding adenylyl cyclase stimulatory; cAMP: cyclic adenosine monophosphate; Gs alpha: G pro- tein α-subunit; MAS: McCune-Albright syndrome; GH: growth hormone; NF: neurofibromatosis; NOF: Non-ossi- fying fibromas; CF: craniofacial; TSH: thyroid stimulating hormone; FT4: free thyroxine; T3: triiodothyronine; T4: thyroxine; FGF-23: fibroblast growth factor-23; TRP: tubu- lar reabsorption of phosphate; TmP/GFR: maximal tubu- lar reabsorption of phosphate per glomerular filtration rate; OGTT: oral glucose tolerance test; MRI: magnetic res- onance imaging; CT: computed tomography; IGF-1: insu- lin-like growth factor-1; PTH: parathyroid hormone; MSH: melanocyte stimulating hormone; LH: luteinizing hormone; GHRH: growth hormone stimulating hor- mone; ACTH: adrenocrotical stimulating hormone; E2: estradiol; T: testosterone; 99 Tc-MDP: Technetium 99 methylene diphosphonate Consent Written informed consent was obtained from the parents of the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. p g The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 6. Adrenal Cushing syndrome is one of the rarest, but most profound manifestations of MAS. In some cases, it can resolve spon- taneously [9], but in most cases treatment is required, and in some cases, in spite of the best of care, it can be fatal. Adrenalectomy is probably the treatment of choice. If the child is too ill, adrenal blockade may provide stabilization of the disease and buy time until a point in time when sur- gery can be performed. Liver function abnormalities usu- ally accompany neonatal Cushing syndrome, so ketoconazole as a medicinal therapy is usually not an option. Metryrapone at an initial dose of 300 mg/m2/day is recommended. The dose may be escalated to as high as 1200 mg/m2/day. In patients with a history of neonatal Cushing, adrenal reserve should be checked. Cushing syn- drome in MAS is usually accompanied by hyperthy- roidism, which, if not treated, can complicate and worsen the clinical course. References y 22. Scanlon EF, Burkett FE, Sener SF, Green OC, Traisman HS, Marr TJ, Victor TA, Crist ML: Breast carcinoma in a 11-year-old girl with Albright's syndrome. Breast 1980, 6:. 1. McCune DJ: Osteitis fibrosa cystica: the case of a nine-year-old girl who also exhibits precocious puberty, multiple pigmen- tation of the skin and hyperthyroidism. Am J Dis Child 1936, 52:743-744. g y 23. Tanabeu Y, Nakahara S, Mitsuyama S, Ono M, Toyoshima S: Breast Cancer in a Patient with McCune-Albright Syndrome. Breast Cancer 1998, 5:175-178. 2. Albright F, Butler AM, Hampton AO, Smith P: Syndrome charac- terized by osteitis fibrosa disseminata, areas, of pigmenta- tion, and endocrine dysfunction, with precocious puberty in females: report of 5 cases. N Engl J Med 1937, 216:727-746. 24. Collins MT, Sarlis NJ, Merino MJ, Monroe J, Crawford SE, Krakoff JA, Guthrie LC, Bonat S, Robey PG, Shenker A: Thyroid carcinoma in the McCune-Albright syndrome: contributory role of acti- vating Gs alpha mutations. J Clin Endocrinol Metab 2003, 88:4413-4417. p g J 3. Mastorakos G, Mitsiades NS, Doufas AG, Koutras DA: Hyperthy- roidism in McCune-Albright syndrome with a review of thy- roid abnormalities sixty years after the first report. Thyroid 1997, 7:433-439. 25. Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM: Activating mutations of the stimulatory G protein in the McCune – Albright syndrome [see comments]. N Engl J Med 1991, 325:1688-1695. 4. Sherman SI, Ladenson PW: Octreotide therapy of growth hor- mone excess in the McCune-Albright syndrome. Journal of endocrinological investigation 1992, 15:185-190. 26. Schwindinger WF, Francomano CA, Levine MA: Identification of a mutation in the gene encoding the alpha subunit of the stim- ulatory G protein of adenylyl cyclase in McCune-Albright syndrome. Proc Natl Acad Sci USA 1992, 89:5152-5156. 5. Akintoye SO, Chebli C, Booher S, Feuillan P, Kushner H, Leroith D, Cherman N, Bianco P, Wientroub S, Robey PG, Collins MT: Charac- terization of gsp-mediated growth hormone excess in the context of McCune-Albright syndrome. J Clin Endocrinol Metab 2002, 87(11):5104-5112. 27. Bianco P, Riminucci M, Majolagbe A, Kuznetsov SA, Collins MT, Mankani MH, Corsi A, Bone HG, Wientroub S, Spiegel AM, Fisher LW, Robey PG: Mutations of the GNAS1 gene, stromal cell dysfunction, and osteomalacic changes in non-McCune- Albright fibrous dysplasia of bone. J Bone Miner Res 2000, 15:120-128. ( ) 6. http://www.ojrd.com/content/3/1/12 http://www.ojrd.com/content/3/1/12 Orphanet Journal of Rare Diseases 2008, 3:12 Additional file 4 18. Leet AI, Chebli C, Kushner H, Chen CC, Kelly MH, Brillante BA, Robey PG, Bianco P, Wientroub S, Collins MT: Fracture incidence in polyostotic fibrous dysplasia and the McCune-Albright syndrome. J Bone Miner Res 2004, 19:571-577. Support. This file lists groups that support patients, families, and clini- cians caring for patients with MAS. Click here for file J 19. Ruggieri P, Sim FH, Bond JR, Unni KK: Malignancies in fibrous dys- plasia. Cancer 1994, 73:1411-1424. [http://www.biomedcentral.com/content/supplementary/1750- 1172-3-12-S4.doc] p 20. Blanco P, Schaeverbeke T, Baillet L, Lequen L, Bannwarth B, Dehais J: Chondrosarcoma in a patient with McCune-Albright syn- drome. Report of a case. Rev Rhum Engl Ed 1999, 66:177-179. p g 21. Lopez-Ben R, Pitt MJ, Jaffe KA, Siegal GP: Osteosarcoma in a patient with McCune-Albright syndrome and Mazabraud's syndrome. Skeletal Radiol 1999, 28:522-526. Additional material 10. Shenker A, Weinstein LS, Moran A, Pescovitz OH, Charest NJ, Boney CM, Van Wyk JJ, Merino MJ, Feuillan PP, Spiegel AM: Severe endo- crine and nonendocrine manifestations of the McCune- Albright syndrome associated with activating mutations of stimulatory G protein GS. J Pediatr 1993, 123:509-518. Additional file 1 Recommendations for treatment of hypophosphatemia in MAS. This file describes the goal, treatment regimen and possible complications. Click here for file [http://www.biomedcentral.com/content/supplementary/1750- 1172-3-12-S1.doc] Additional file 2 Bisphosphonate treatment. This file describes the goal, regimen and possi- ble complications of bisphosphonate treatment. Click here for file [http://www.biomedcentral.com/content/supplementary/1750- 1172-3-12-S2.doc] Additional file 3 Treatment of precocious puberty. This file describes treatment of preco- cious puberty in girls and boys. Click here for file [http://www.biomedcentral.com/content/supplementary/1750- 1172-3-12-S3.doc] Additional file 4 Support. This file lists groups that support patients, families, and clini- cians caring for patients with MAS. Click here for file [http://www.biomedcentral.com/content/supplementary/1750- 1172-3-12-S4.doc] Additional file 2 13. Collins MT: Spectrum and natural history of fibrous dysplasia of bone. J Bone Miner Res 2006, 21(Suppl 2):P99-P104. Bisphosphonate treatment. This file describes the goal, regimen and possi- ble complications of bisphosphonate treatment. Click here for file [http://www.biomedcentral.com/content/supplementary/1750- 1172-3-12-S2.doc] 14. Collins MT, Bianco P: Fibrous dysplasia. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism 6th edition. Edited by: Favus MJ. Washington, D.C.: American Society for Bone and Min- eral Research; 2006:415-418. 15. Collins MT, Shenker A: McCune-Albright syndrome: new insights. Curr Opin in Endocrinol and Diabetes 1999, 6:119-125. Additional file 1 11. Lichtenstein LJH: Fibrous dysplasia of bone: a condition affect- ing one, several or many bones, graver cases of which may present abnormal pigmentation of skin, premature sexual development, hyperthyroidism or still other extraskeletal abnormalities. Arch Path 1942, 33:777-816. 12. Bianco P, Robey PG, Wientroub S: Fibrous dysplasia. In Pediatric Bone: Biology and Disease Edited by: Glorieux FH, Pettifor J, Juppner H. New York, NY: Academic Press, Elsevier; 2003:509-539. Additional file 3 16. Dorfman HD, Czerniak B: Fibroosseous Lesions. In Bone Tumors Edited by: Dorfman HD, Czerniak B. St. Louis, MO: Mosby; 1998:441-491. 17. Hart ES, Kelly MH, Brillante B, Chen CC, Ziran N, Lee JS, Feuillan P, Leet AI, Kushner H, Robey PG, Collins MT: Onset, progression, and plateau of skeletal lesions in fibrous dysplasia and the relationship to functional outcome. J Bone Miner Res 2007, 22:1468-1474. Unresolved questions q While bisphosphonates are usually effective in relieving FD-related pain, whether or not the treatment of FD with Page 10 of 12 (page number not for citation purposes) Page 10 of 12 (page number not for citation purposes) References Collins MT, Chebli C, Jones J, Kushner H, Consugar M, Rinaldo P, Wientroub S, Bianco P, Robey PG: Renal phosphate wasting in fibrous dysplasia of bone is part of a generalized renal tubu- lar dysfunction similar to that seen in tumor-induced osteo- malacia. J Bone Miner Res 2001, 16:806-813. 28. Idowu BD, Al-Adnani M, O'Donnell P, Yu L, Odell E, Diss T, Gale RE, Flanagan AM: A sensitive mutation-specific screening tech- nique for GNAS1 mutations in cases of fibrous dysplasia: the first report of a codon 227 mutation in bone. Histopathology 2007, 50:691-704. J 7. Danon M, Crawford JD: The McCune-Albright syndrome. Ergeb Inn Med Kinderheilkd 1987, 55:81-115. 8. Diaz A, Danon M, Crawford J: McCune-Albright syndrome and disorders due to activating mutations of GNAS1. J Pediatr Endocrinol Metab 2007, 20:853-880. 29. Riminucci M, Saggio I, Robey PG, Bianco P: Fibrous dysplasia as a stem cell disease. J Bone Miner Res 2006, 21(Suppl 2):P125-131. 9. Kirk JM, Brain CE, Carson DJ, Hyde JC, Grant DB: Cushing's syn- drome caused by nodular adrenal hyperplasia in children with McCune-Albright syndrome. The Journal of pediatrics 1999, 134:789-792. stem cell disease. J Bone Miner Res 2006, 21(Suppl 2):P125-131. 30. Collins MT, Kushner H, Reynolds JC, Chebli C, Kelly MH, Gupta A, Brillante B, Leet AI, Riminucci M, Robey PG, Bianco P, Wientroub S, Chen CC: An instrument to measure skeletal burden and pre- J ( pp ) 30. Collins MT, Kushner H, Reynolds JC, Chebli C, Kelly MH, Gupta A, Brillante B, Leet AI, Riminucci M, Robey PG, Bianco P, Wientroub S, Chen CC: An instrument to measure skeletal burden and pre- Page 11 of 12 (page number not for citation purposes) Page 11 of 12 (page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:12 http://www.ojrd.com/content/3/1/12 dict functional outcome in fibrous dysplasia of bone. J Bone Miner Res 2005, 20:219-226. 51. Stanton RP: Surgery for fibrous dysplasia. J Bone Miner Res 2006, 21(Suppl 2):P105-109. dict functional outcome in fibrous dysplasia of bone. J Bone Miner Res 2005, 20:219-226. ( pp ) 52. Chapurlat RD: Medical therapy in adults with fibrous dysplasia of bone. J Bone Miner Res 2006, 21(Suppl 2):P114-119. 31. References Terpstra L, Rauch F, Plotkin H, Travers R, Glorieux FH: Bone min- eralization in polyostotic fibrous dysplasia: histomorpho- metric analysis. J Bone Miner Res 2002, 17:1949-1953. 64. Ehrig U, Wilson DR: Fibrous dysplasia of bone and primary hyperparathyroidism. Ann Intern Med 1972, 77:234-238. p p 65. Leslie WD, Reinhold C, Rosenthall L, Tau C, Glorieux FH: Panos- totic fibrous dysplasia. A new craniotubular dysplasia. Clinical nuclear medicine 1992, 17:556-560. ship to renal phosphate wasting. J Clin Invest 2003, 112:683 692. 44. Terpstra L, Rauch F, Plotkin H, Travers R, Glorieux FH: Bone min- eralization in polyostotic fibrous dysplasia: histomorpho- metric analysis. J Bone Miner Res 2002, 17:1949-1953. 66. Feuillan PP: McCune-Albright syndrome. Curr Ther Endocrinol Metab 1997, 6:235-239. y J 45. Glorieux FH, Rauch F: Medical therapy of children with fibrous dysplasia. J Bone Miner Res 2006, 21(Suppl 2):P110-113. 67. Defilippi C, Chiappetta D, Marzari D, Mussa A, Lala R: Image diag- nosis in McCune-Albright syndrome. J Pediatr Endocrinol Metab 2006, 19(Suppl 2):561-570. y p J ( pp ) 46. Hammami MM, Hussain SS, Vencer LJ, Butt A, al-Zahrani A: Primary hyperparathyroidism-associated polyostotic fibrous dyspla- sia: absence of McCune-Albright syndrome mutations. J Endocrinol Invest 1997, 20:552-558. ( pp ) 68. Silva ES, Lumbroso S, Medina M, Gillerot Y, Sultan C, Sokal EM: Dem- onstration of McCune-Albright mutations in the liver of chil- dren with high gammaGT progressive cholestasis. Journal of hepatology 2000, 32:154-158. 47. Barone A, Giusti A, Pioli G, Girasole G, Razzano M, Pizzonia M, Palummeri E, Bianchi G: Secondary hyperparathyroidism due to hypovitaminosis D affects bone mineral density response to alendronate in elderly women with osteoporosis: a rand- omized controlled trial. Journal of the American Geriatrics Society 2007, 55:752-757. p gy 69. Zimmerman D: Fetal and neonatal hyperthyroidism. Thyroid 1999, 9:727-733. 70. Schwartz RA, Spicer MS, Leevy CB, Ticker JB, Lambert WC: Cuta- neous fibrous dysplasia: an incomplete form of the McCune- Albright syndrome. Dermatology (Basel, Switzerland) 1996, 192:258-261. 48. Giusti A, Barone A, Razzano M, Pizzonia M, Oliveri M, Palummeri E, Pioli G: High prevalence of secondary hyperparathyroidism due to hypovitaminosis D in hospitalized elderly with and without hip fracture. Journal of endocrinological investigation 2006, 29:809-813. 71. Pierini AM, Ortonne JP, Floret D: [Cutaneous manifestations of McCune-Albright syndrome: report of a case (author's transl)]. Annales de dermatologie et de venereologie 1981, 108:969-976. 49. References Byard RW: Forensic considerations in cases of neurofibroma- tosis – an overview. Journal of forensic sciences 2007, 52:1164-1170. 59. Eugster EA, Rubin SD, Reiter EO, Plourde P, Jou HC, Pescovitz OH: Tamoxifen treatment for precocious puberty in McCune- Albright syndrome: a multicenter trial. The Journal of pediatrics 2003, 143:60-66. J 39. Theos A, Korf BR: Pathophysiology of neurofibromatosis type 1. Annals of internal medicine 2006, 144:842-849. 60. Congedo V, Celi FS: Thyroid disease in patients with McCune- Albright syndrome. Pediatr Endocrinol Rev 2007, 4(Suppl 4):429-433. 40. Lee JS, FitzGibbon E, Butman JA, Dufresne CR, Kushner H, Wien- troub S, Robey PG, Collins MT: Normal vision despite narrowing of the optic canal in fibrous dysplasia. N Engl J Med 2002, 347:1670-1676. ) 61. Akintoye SO, Kelly MH, Brillante B, Cherman N, Turner S, Butman JA, Robey PG, Collins MT: Pegvisomant for the treatment of gsp-mediated growth hormone excess in patients with McCune-Albright syndrome. J Clin Endocrinol Metab 2006, 91:2960-2966. 41. Cutler CM, Lee JS, Butman JA, FitzGibbon EJ, Kelly MH, Brillante BA, Feuillan P, Robey PG, DuFresne CR, Collins MT: Long-term out- come of optic nerve encasement and optic nerve decom- pression in patients with fibrous dysplasia: risk factors for blindness and safety of observation. Neurosurgery 2006, 59:1011-1017. discussion 1017–1018 62. Chanson P, Salenave S, Orcel P: McCune-Albright syndrome in adulthood. Pediatr Endocrinol Rev 2007, 4(Suppl 4):453-462. 42. Feuillan PP, Shawker T, Rose SR, Jones J, Jeevanram RK, Nisula BC: Thyroid abnormalities in the McCune-Albright syndrome: ultrasonography and hormone studies. J Clin Endocrinol Metab 1990, 71:1596-1601. 63. Galland F, Kamenicky P, Affres H, Reznik Y, Pontvert D, Le Bouc Y, Young J, Chanson P: McCune-Albright syndrome and acrome- galy: effects of hypothalamopituitary radiotherapy and/or pegvisomant in somatostatin analog-resistant patients. J Clin Endocrinol Metab 2006, 91:4957-4961. 43. Riminucci M, Collins MT, Fedarko NS, Cherman N, Corsi A, White KE, Waguespack S, Gupta A, Hannon T, Econs MJ, Bianco P, Gehron Robey P: FGF-23 in fibrous dysplasia of bone and its relation- ship to renal phosphate wasting. J Clin Invest 2003, 112:683-692. 43. Riminucci M, Collins MT, Fedarko NS, Cherman N, Corsi A, White KE, Waguespack S, Gupta A, Hannon T, Econs MJ, Bianco P, Gehron Robey P: FGF-23 in fibrous dysplasia of bone and its relation- ship to renal phosphate wasting. J Clin Invest 2003, 112:683-692. 44. References Ippolito E, Bray EW, Corsi A, De Maio F, Exner UG, Robey PG, Grill F, Lala R, Massobrio M, Pinggera O, Riminucci M, Snela S, Zambakidis C, Bianco P: Natural history and treatment of fibrous dysplasia of bone: a multicenter clinicopathologic study promoted by the European Pediatric Orthopaedic Society. Journal of pediat- ric orthopaedics 2003, 12:155-177. 53. Liens D, Delmas PD, Meunier PJ: Long-term effects of intrave- nous pamidronate in fibrous dysplasia of bone. Lancet 1994, 343:953-954. 54. Plotkin H, Rauch F, Zeitlin L, Munns C, Travers R, Glorieux FH: Effect of pamidronate treatment in children with polyostotic fibrous dysplasia of bone. J Clin Endocrinol Metab 2003, 88:4569-4575. 32. Kelly MH, Brillante B, Collins MT: Pain in fibrous dysplasia of bone: age-related changes and the anatomical distribution of skeletal lesions. Osteoporos Int 2007. 55. Chan B, Zacharin M: Pamidronate treatment of polyostotic fibrous dysplasia: failure to prevent expansion of dysplastic lesions during childhood. J Pediatr Endocrinol Metab 2006, 19:75-80. 33. Riminucci M, Liu B, Corsi A, Shenker A, Spiegel AM, Robey PG, Bianco P: The histopathology of fibrous dysplasia of bone in patients with activating mutations of the Gs alpha gene: site- specific patterns and recurrent histological hallmarks. The Journal of pathology 1999, 187:249-258. 56. Leet AI, Magur E, Lee JS, Wientroub S, Robey PG, Collins MT: Fibrous dysplasia in the spine: prevalence of lesions and asso- ciation with scoliosis. J Bone Joint Surg Am 2004, 86-A(3):531-537. J f p gy 34. Corsi A, Collins MT, Riminucci M, Howell PG, Boyde A, Robey PG, Bianco P: Osteomalacic and hyperparathyroid changes in fibrous dysplasia of bone: core biopsy studies and clinical cor- relations. J Bone Miner Res 2003, 18:1235-1246. 57. Feuillan P, Calis K, Hill S, Shawker T, Robey PG, Collins MT: Letro- zole Treatment of Precocious Puberty in Girls with the McCune-Albright Syndrome: A Pilot Study. J Clin Endocrinol Metab 2007, 92:2100-2106. J 35. Hannon TS, Noonan K, Steinmetz R, Eugster EA, Levine MA, Pesco- vitz OH: Is McCune-Albright syndrome overlooked in sub- jects with fibrous dysplasia of bone? J Pediatr 2003, 142:532-538. 58. Feuillan PP: Treatment of sexual precocity in girls with the McCune-Albright syndrome. In Sexual precocity: etiology, diagnosis and management Edited by: Grave GD, Cutler GB. New York: Raven Press; 1993:243-251. j y p J , 36. Center for Genetic Testing [http://www.sfh-lab.com] 37. Genome Diagnostics [http://www.umcutrecht.nl/subsite/ genome-diagnostics/DNA-diagnostics/Gene-Tests.htm] g g g ] 38. Page 12 of 12 (page number not for citation purposes) References Reginster JY: The high prevalence of inadequate serum vita- min D levels and implications for bone health. Current medical research and opinion 2005, 21:579-586. 50. Hashemipour S, Larijani B, Adibi H, Sedaghat M, Pajouhi M, Bastan- Hagh MH, Soltani A, Javadi E, Shafaei AR, Baradar-Jalili R, Hossein- Nezhad A: The status of biochemical parameters in varying degrees of vitamin D deficiency. Journal of bone and mineral metabolism 2006, 24:213-218. Page 12 of 12 (page number not for citation purposes) Page 12 of 12 (page number not for citation purposes)

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Figure S3 from Nanoparticle STING Agonist Reprograms the Bone Marrow to an Antitumor Phenotype and Protects Against Bone Destruction

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Supplementary Figure 8: Concentration of FOXP3+ cells in healthy STING NP-treated BM over time. (A) IHC staining of FOXP3+ Tregs in healthy BM from untreated mice (control) and from mice that received the standard treatment regimen. Mice were not tumor-bearing. (B) FOXP3+ cells were counted individually and normalized to total marrow area. One-way ANOVA with Holm-Šídák's multiple-comparisons test. *: p < 0.05, **: p < 0.01, ***: p < 0.001 Supplementary Figure 8: Concentration of FOXP3+ cells in healthy STING NP-treated Supplementary Figure 8: Concentration of FOXP3+ cells in healthy STING NP-treated BM over time. (A) IHC staining of FOXP3+ Tregs in healthy BM from untreated mice (control) and from mice that received the standard treatment regimen. Mice were not tumor-bearing. (B) FOXP3+ cells were counted individually and normalized to total marrow area. One-way ANOVA with Holm-Šídák's multiple-comparisons test. *: p < 0.05, **: p < 0.01, ***: p < 0.001 Supplementary Figure 8: Concentration of FOXP3+ cells in healthy STING NP-treated BM over time. (A) IHC staining of FOXP3+ Tregs in healthy BM from untreated mice (control) and from mice that received the standard treatment regimen. Mice were not tumor-bearing. (B) FOXP3+ cells were counted individually and normalized to total marrow area. One-way ANOVA with Holm-Šídák's multiple-comparisons test. *: p < 0.05, **: p < 0.01, ***: p < 0.001

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A Case of Intra‐Uterine Amputation of the Right Humerus in the Lower Third.

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D a w n : Intra- Uhrint?Amputation 626 A Case of Intra-Uterine Amputation of the Right Humerus in the Lower Third. By C. E. DAWSON,L.R.C.P. THE mother, aged 30, had had one previous normal labour; that child, a female, being quite healthy and strong. The eecond labour took place in January, 1903, prematum1y a t 64 months as far as I could say. There was ante- and post-partum hemorrhage but not alarming in quantity. The pains were normal. I was sent for on account of ante-partum hsemorrhage and pain. Upon examination per vaginam I felt something quite sharp, stretching across the vagina, which pricked my h g e r . Feeling it was part of an arm I very carefully pushed it up, and released the sharp end which had caught in the vaginal wall, and as the pains were strong and frAquent the child, a female, was born without any further trouble. The placenta was expressed quite easily after which there was a Atle post-partum hsemorrhage, otherwise it wae a perfectly normal labour. Not a trace of the amputated limb could be found. The patient made a gooa recovery. There were a slight rise of temperature and offensive lochia which soon cleared up after a few douchings with mercury biniodide (1.4000) by the woman in charge. The child was ill-nourished at birth and cried feebly. It WEE wrapped in cotton-wool, after the usual bath, and took ita food, milk and water from a bottle, indifferently, but gradually lost flesh and died from asthenia at the end of two weeks. The amputated limb looked exactlylike the end of a sausage with the bone of the humerue protruding for 1+ inches; it was very sharp at the end and showed signs of absorption. In the early period of this pregnancy the patient was whitewashing a ceiling when she suddenly felt acute pain in the abdomen which howelrer soon passed off, and thinking it waa stomach-ache she thought nothing more of it. 

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<i>Escherichia coli</i> as a Tool for Disease Risk Assessment of Drinking Water Sources

International journal of microbiology

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Hindawi Hindawi Hindawi International Journal of Microbiology Volume 2020, Article ID 2534130, 7 pages https://doi.org/10.1155/2020/2534130 Stephen T. Odonkor 1 and Tahiru Mahami2 Stephen T. Odonkor and Tahiru Mahami 1School of Public Service and Governance, Ghana Institute of Management and Public Administration, Accra, Ghana 2Ghana Atomic Energy Commission, Kwabenya, Accra, Ghana 1School of Public Service and Governance, Ghana Institute of Management and Public Administration, Accra, Ghana 2Ghana Atomic Energy Commission, Kwabenya, Accra, Ghana Correspondence should be addressed to Stephen T. Odonkor; [email protected] Received 19 March 2020; Revised 25 May 2020; Accepted 28 May 2020; Published 15 June 2020 Academic Editor: Joseph Falkinham Copyright © 2020 Stephen T. Odonkor and Tahiru Mahami. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Many diseases have been associated with poor drinking water quality including diseases caused by diarrheagenic pathogens, especially in developing countries where access to a consistent water supply is a problem. Te objective of the study was to evaluate the health risks associated with the sources of drinking water in the Dangme West District of Ghana using E. coli as a measurement tool, aiming at ascertaining the paths leading to contamination of the water sources. A total of 464 water samples were obtained for analysis. Sampling covered a year across the dry and wet seasons in Ghana. Water samples were obtained from groundwater and surface water sources. E. coli counts were determined using the most probable number method (MPN). Disease risk assessment was carried out using the WHO drinking water risk assessment guidelines. Generally, the study revealed significantly higher E. coli counts in the wet season than in the dry season. Among the water samples analyzed, surface water, especially from the dams, was found to pose the highest disease risk as compared to the other water sources. Samples from groundwater sources, especially boreholes, posed the lowest disease risk. In conclusion, observations from the study implied that most water sources in the study district are highly polluted with bacteria pathogens beyond recommended safety guidelines. Te main causes of faecal con- tamination in these water sources were purported to be anthropogenic. Terefore, there is a need to formulate a policy aimed at managing and improving rural water sources. death of more than 2 million people per year worldwide, mostly children under the age of five. In fact, each day, nearly 1,000 children die due to preventable water and sanitation- related diarrhoeal diseases [6, 7]. 1. Introduction Water is today a scarce resource, particularly in the devel- oping world. Globally, 663 million people do not have access to safe water [1]. Rural communities in sub-Saharan Africa account for more than 50% of those lacking potable water [2, 3]. Most of these communities, therefore, rely on un- treated sources such as streams, dams, boreholes, wells, and rivers to meet fundamental needs such as drinking, sani- tation, and cooking and for their sustainable development [4]. However, these untreated sources are contributing significantly to the global burden of disease, as a result of water-related infectious diseases such as cholera, dysentery, and typhoid [5]. Every year, millions of lives are lost worldwide with a chunk in developing countries as a result of waterborne diseases. Diarrhoea is a major cause of the Good quality water is a vital deciding factor for human health, which directly relates to the socioeconomic progress of a country [8–10]. Tus, the importance of water cannot be overemphasized. In fact, sustainable socioeconomic devel- opment and productive livelihoods can be built on access to water. Productive uses of water are employed at the household levels, including a range of small-scale domestic activities, in agriculture and industrial purposes. Although the quality requirement for water varies from one sector to the other and from one form of usage to the other, many of these activities have implications for public health, because it can result in the transmission of waterborne diseases [11–13]. 2 International Journal of Microbiology rainfall increases from 762.5 milliliters on the coast to 1220 milliliters to the North and Northeast close to the foothills of Akwapim range and on the summit [26]. Tese water-related infections are a result of the pol- lution of the water sources, due to anthropogenic influences, population growth, and urbanization [14]. Te pollution thus leads to the development and propagation of patho- genic waterborne microorganisms. Escherichia coli is one of the major pathogens associated with waterborne diseases. Naturally, Escherichia coli (E. coli) is a facultative anaerobic bacterium that inhabits the large gastrointestinal tracts of warm-blooded animals and is a major normal flora asso- ciated with the human colon [15–17]. 2.3. Climate and Vegetation. Te southeastern coastal plain of Ghana, which encompasses the Dangme West District, is one of the hottest and driest parts of the country. 1. Introduction coli in water does not necessarily imply the presence of disease-causing mi- crobes. However, it gives an indication of the possible ex- istence of faecal-borne microorganisms such as Salmonella and hepatitis A [21, 22]. Tis accounts for the usage of E. coli as an indicator microbe to examine food and water samples aimed at detecting the levels of faecal contaminations [21]. Water safety management depends primarily on hazard identification through drinking water surveillance, which is “the continuous and vigilant public health assessment and review of the safety and acceptability of drinking water supplies” [23, 24]. Tis surveillance is critical to public health and safety, as it contributes and supports improvements in water quality and accessibility. Te present study evaluated the health risks associated with sources of drinking water in the Dangme district in Ghana using E. coli as a bacteria source tracking tool measurement tool, aimed at ascertaining the paths leading to contamination of the water sources. 2.4. Water Sources and Sanitation. Te supply of potable water in the district is woefully inadequate, and only a few sections of the district have a regular supply of pipe-borne water. Analysis of the current water and sanitation in the district shows that more effort is needed to meet the 85% water and sanitation coverage. On the basis of the National Community Water and Sanitation Standards of 600 people per standpipe, 350 persons per borehole, and 150 persons per hand-dug well, the district has achieved about 66% coverage with 34% of the population lacking access to a potable water supply. As it stands now, below 37% of the district population has access to pipe-borne water. Te remaining population in the district depends on untreated water sources. Visually, the water from the streams and dams are light brownish yellow caused by mostly decayed dead leaves. If turbulently disturbed, it turns to deep brownish yellow and some suspended soils can be seen [26]. 1. Introduction Tem- peratures are, however, subjected to occasional and minimal moderating influences along the coast and altitudinal in- fluences affected by the Akwapim range in the northwest. Temperatures are appreciably high for most parts of the year, with the highest during the main dry season (Novem- ber–March) and the lowest during the short dry season (July–August). Tey average a few degrees lower on the coast and close to the Akwapim range than they do over most of the plains. Te absolute maximum temperature is 40°C [26]. Along some stream courses, however, higher vegetation type ranging from thickets to light forest is common. Some light forest with tall trees is also found along the foothills of the Akwapim Ridge, especially around Dodowa, Ayikuma, and Agomeda areas. Tere is a Forest, Game, and Wildlife Re- serve around the Shai hills [26]. Terefore, the existence of E. coli in food or water normally signals recent faecal contamination or poor hy- gienic conditions in food processing facilities [18]. Tus, faecal contamination, poor sanitation measures, and poor storage conditions have a major influence on E. coli pop- ulation [19, 20]. Te mere existence of E. coli in water does not necessarily imply the presence of disease-causing mi- crobes. However, it gives an indication of the possible ex- istence of faecal-borne microorganisms such as Salmonella and hepatitis A [21, 22]. Tis accounts for the usage of E. coli as an indicator microbe to examine food and water samples aimed at detecting the levels of faecal contaminations [21]. Water safety management depends primarily on hazard identification through drinking water surveillance, which is “the continuous and vigilant public health assessment and review of the safety and acceptability of drinking water supplies” [23, 24]. Tis surveillance is critical to public health and safety, as it contributes and supports improvements in water quality and accessibility. Te present study evaluated the health risks associated with sources of drinking water in the Dangme district in Ghana using E. coli as a bacteria source tracking tool measurement tool, aimed at ascertaining the paths leading to contamination of the water sources. Terefore, the existence of E. coli in food or water normally signals recent faecal contamination or poor hy- gienic conditions in food processing facilities [18]. Tus, faecal contamination, poor sanitation measures, and poor storage conditions have a major influence on E. coli pop- ulation [19, 20]. Te mere existence of E. 2. Materials and Methods A total of 15 tubes comprising 5 tubes for each of three dilution factors (0.1 ml, 1 ml, and 10 ml) were used for International Journal of Microbiology 3 3 Dangme west district map-greater accra region Legend District capital 820000 670000 660000 650000 640000 670000 660000 650000 640000 830000 840000 850000 860000 870000 820000 830000 840000 850000 860000 870000 Settlements Trunk roads Feeder roads River/stream Forest reserve Game reserve District boundary Adjoining district Volta river Sea Figure 1: A map of the Dangme district of Ghana. Figure 1: A map of the Dangme district of Ghana. inoculation in the study. As a result, three sets, each con- taining five tubes, were used in the presumptive test. In the first set, each of the five tubes holding 10 ml of MacConkey Broth with double strength was inoculated with 10 ml of the water sample. Te tubes in the second set, which contain 10 ml of single strength MAC broth, were inoculated with 1 ml of subwater samples and the remaining five tubes containing 10 ml of single strength MAC broth were in- oculated with 0.1 ml of the sample. After that, the tubes were incubated at 37°C for 24 hours for total coliforms and 44°C for faecal coliforms. Te tubes were then examined visually for turbidity. Each tube contains an inverted Durham tube (this is a very small test-tube), which is examined for gas production (bubble) and the media is examined for acid production, i.e, change in colour from pink to yellow. Table 1: Te range of seasonal E. coli counts (MPN/100 ml) in water. Water sources Seasonal counts (MPN/100 ml) Dry season Wet season Borehole 0–0.2 ×101 0–0.2 ×101 Canal 2.3 ×101–1.1 × 102 3.1 × 101–6.3 ×101 Dam 0.7 ×101–1.1 × 102 1.2 ×101–7.9 ×101 Hand-dug well 0–0.2 ×101 0–0.4 ×101 River 0–0.1 × 101 0.2 ×101–0.7 ×101 Streams 0–3.3 ×101 0–3.3 ×101 hand-dug wells, boreholes, and stream all had a zero- minimum count across both seasons. Tere was a low count of E. coli in water samples obtained from river sources in the wet season. Te range of E. coli counts (1.2 × 101–7.9 ×101 MPN/100 ml) was observed in samples col- lected from dams throughout the wet season. Confirmation of E. coli was carried out by aseptically plating from tubes with acid and gas formation onto L-EMB agar in Petri dishes to obtain pure isolates. 2. Materials and Methods 2.1. Study Area. Te Dangme West District (Figure 1) is situated in the southeastern part of Ghana, lying between longitude 5° 45′ south and 6° 05′ north and longitude 0° 05′ east and 0° 20′ west. Te district has a total land area of 1,442 square kilometers, making it the largest in the Greater Accra Region. Te land size represents 41.5% of the regional land area. Te district forms part of sixteen (16) metropolitan, municipal, and districts in the Greater Accra Region of Ghana. Te district has a 37 km Coastline and a 17 km stretch of the Volta River [25]. 2.5.SampleCollectionandAnalysis. Four hundred sixty-four (464) water samples were obtained from the drinking sources at the study location for analysis. Sampling duration was a year, and sampling was carried out throughout the wet and dry seasons in Ghana. Te water samples were obtained from the surface (dams, river, canals, and streams) and underground (hand-dug wells and boreholes) water sources. 2.2. Topography and Drainage. Te district forms the central portions of the Accra plains. Te relief is generally gentle and undulating, a low plain with heights not exceeding 70 metres. Te plains are punctuated in isolated areas by a few prominent inselbergs, isolated hills, outliers, and knolls scattered errat- ically over the area. Prominent relief features include the Yongua inselberg (427 metres) which appears conical in the air with a number of outliers close to the north of the district around Asutsuare and Osuwem areas, the Krabote inselberg also to the north and the Shai hills (289 metres) found towards the western portions of the district [26]. Te mean annual Sampling and care of samples were done following guidelines of the WHO [27] while analyses of samples were carried out as described by APHA [28, 29]. Sterile bottles were used to collect the water samples from each of the sites. Samples were taken to the laboratory for analyses. Te most probable number method (MPN) illustrated by Prescott et al. [30] was used to determine E. coli counts. 2. Materials and Methods Te plates were incubated at 35°C with a standard error of 0.5°C for 18–24 hours. Te incubated plates were examined for centrally dark and flat colonies having or not having a metallic sheen. Tese morphological features are associated with E. coli. A maximum of 5 colonies from each L-EMB plate was inoc- ulated onto slants of PCA and incubated at 35°C for about 24 hours to prepare stork culture. API20E was used to identify a 24 hr culture of each purified isolate following the manu- facturer’s instructions. A summary of comparative analysis between E. coli counts measured in MPN/100 ml in samples from the water sources is presented in Table 2. Te highest counts for E. coli (59.67 ± 45.08) were identified in water samples collected from the canal sources in the wet season. On the other hand, the lowest counts (0–0.1 × 101 MNP/100 ml) were identified in the dry season from samples sourced from the rivers. Analysis of mean variations and confidence intervals for E. coli proportions throughout the wet and dry seasons is shown in Tables 3 and 4, respectively. Tere was a significant difference between mean proportions of E. coli from boreholes and canal water sources in the wet season. Mean differences of counts among boreholes, river, and canals in the wet season were also significant. 3. Results Table 1 shows the range of seasonal E. coli counts (MPN/ 100 ml) in water. E. coli populations in samples sourced from International Journal of Microbiology 4 Table 2: Comparison of E. coli counts (MPN/100 ml) in the various water sources (SD ˆ standard deviation, df ˆ degree of freedom, Min ˆ minimum, and Max ˆ maximum). Water sources Dry season Wet season P value Mean ± SD Min Max df Mean ± SD Min Max df Bore hole 0.63 ± 0.74 0 2 7 1 ± 0.76 0 2 7 0.090 Canal 47.67 ± 16.04 31 63 2 59.67 ± 45.08 23 110 2 0.280 Dam 38.07 ± 30.71 7 110 14 39.87 ± 19.76 12 79 14 0.350 Hand-dug well 0.93 ± 0.80 0 2 14 1.47 ± 1.06 0 4 14 0.001 River 0 ± 0 0 0 2 4.33 ± 2.52 2 7 2 0.050 Stream 15.06 ± 13.59 0 33 16 20.77 ± 11.05 0 31 16 0.020 Table 3: Analysis of mean differences and confidence intervals for E. coli levels in the dry season (test value ˆ 6; the mean difference is significant at P ≤0.05). Water sources T df Sis (2-tailed) Mean difference 95% confidence interval Upper limit Lower limit Borehole 2.376 7 0.049 0.625 0.00 1.25 Canal 5.147 2 0.036 47.667 7.82 87.52 Dam 4.800 14 0.001 38.067 21.06 55.08 Hand-dug well 4.525 14 0.001 0.933 0.49 1.38 River — 2 — — — — Stream 4.569 16 0.001 15.059 8.07 22.05 Table 3: Analysis of mean differences and confidence intervals for E. coli levels in the dry season (test significant at P ≤0.05). fferences and confidence intervals for E. coli levels in the dry season (test value ˆ 6; the mean difference is Table 3: Analysis of mean differences and confidence intervals for E. coli levels in the dry season (test value ˆ 6; the mean difference is significant at P ≤0.05). Table 4: Analysis of mean differences and confidence intervals for E. coli levels in the wet season (test value ˆ 6; the mean difference is significant at P ≤0.05). 4. Discussion Figure 3: WHO disease risk levels of water sources in the dry season. Te present study evaluated the health risks associated with sources of drinking water in the Dangme West District in Ghana using E. coli as a measurement tool, aimed at ascertaining the paths leading to contamination of the water sources. E. coli is best suited as an indicator for faecal co- liforms because there are fewer instances of encountering false positives [31, 32]. Also, E. coli enumerations are better indicators of faecal pollution than faecal coliform counts [33]. Te reason being that certain strains of faecal coliform bacteria are capable of multiplying in the environs which can lead to pseudoelevation of indicator levels [34]. Additionally, recent studies have identified a direct association between the density of E. coli microbes in water and the prevalence of water-related gastroenteritis [35]. Tis, among other rea- sons, accounts for the use of E. coli counts as an indicator of water quality and, by extension, a signal of human, domestic, and natural sources of faecal contamination [18, 36–39]. Findings of the investigation of the extent of disease risk associated with the drinking water sources in both seasons across the seasons (Figures 1 and 2) using WHO drinking water risk assessment guidelines. Water samples from the canals showed an intermediate risk level. An intermediate risk level of 59% was also observed in water obtained from streams. However, in the dry season, none of the water sources showed a high-risk level invariance to observations made in the wet season. Tis observation is consistent with several similar studies done in the developing world [43]. Undisputedly, anthropogenic activities and faecal re- leases as preeminent contributing determinants of faecal pollution could explain the high enumerations of E. coli in samples collected from various water sources. However, since the comparative significance of precise animals with regard to E. coli counts were not befittingly evaluated, it is difficult to confidently determine their contribution to the higher counts observed. Possibly, the higher counts could be associated with other related factors including animal populace density and exploitation of areas close to the water sampling sources or sites. 3. Results International Journal of Microbiology 5 Disease risk associated with the drinking water sources from hand-dug wells revealed a 67% low-risk level. Sources from boreholes showed 50% low risk and 50% conformity with standards set by the WHO. Stream water sources had a 59% intermediate risk level with 41% con- formity with the WHO guidelines. Disease risk associated with the drinking water sources in the wet season Water sources Bore hole Canal Dam Hand-dug well River Stream High risk Intermediate risk Low risk Conformity with WHO guidelines Percentage of risk (%) 100 90 80 70 60 50 40 30 20 10 0 0 6 15 12 13 2 0 0 0 0 0 0 0 0 1 3 3 3 Figure 3: WHO disease risk levels of water sources in the dry season. y g Te extent of disease risk of the various water sources during the dry season is presented in Figure 3. Data from Figure 3 show an important trend. All of the sources did not show a high-risk level in the dry season, just as observed in the wet season. All water sources from rivers, hand-dug wells, and canals had 100% intermediate risk. Boreholes had the lowest dis- ease risk, with a 75% low-risk level and 25% compliance with WHO standards. Tis was followed by hand-dug wells at 80% low-risk level. On the other hand, dams showed a high disease risk. 3. Results Water sources T df Sis (2-tailed) Mean difference 95% confidence interval Upper limit Lower limit Bore hole 3.742 7 0.007 1.000 0.37 1.63 Canal 2.292 2 0.149 59.667 −52.32 171.66 Dam 7.814 14 0.001 39.867 28.92 50.81 Hand-dug well 5.358 14 0.001 1.467 0.88 2.05 River 2.982 2 0.096 4.333 −1.92 10.58 Stream 7.745 16 0.001 20.765 15.08 26.45 fferences and confidence intervals for E. coli levels in the wet season (test value ˆ 6; the mean difference is : Analysis of mean differences and confidence intervals for E. coli levels in the wet season (test value ˆ 6; the ant at P ≤0.05). Percentage of risk (%) Water sources 0 4 3 13 4 0 0 5 3 7 0 1 0 1 0 10 10 0 0 0 100 90 80 70 60 50 40 30 20 10 0 Bore hole Canal Dam Hand-dug well River Stream Disease risk associated with the drinking water sources in the dry season High risk Intermediate risk Low risk Conformity with WHO guidelines Figure 2: WHO disease risk levels of water sources in the wet season. Findings of the investigation of the extent of disease risk associated with the drinking water sources in both seasons (wet and dry) are shown in Figures 1 and 2, respectively. Tis analysis was carried using WHO drinking water risk as- sessment guidelines. According to the World Health Or- ganization, a zero count of E. coli per 100 ml of water is considered safe for drinking. A count of 1–10 MPN/100 ml is regarded as low risk; 11–100 MPN/100 ml is medium risk. Finally, an E. coli count greater than 100 MPN/100 ml is adjudged high risk. With reference to Figure 2, it is evident that the dam and canal water sources revealed intermediate disease risk (100%) throughout the wet season. All water samples (100%) sourced from the canals showed an intermediate risk level followed by that of a dam with an 87% intermediate risk level. An intermediate risk level of 59% was observed in water obtained from streams. Water from rivers had 100% compliance with WHO standards. However, water from canals and dams showed no conformity with the guidelines, thereby implying the sub- stantial health risks they pose to human health. Water Figure 2: WHO disease risk levels of water sources in the wet season. International Journal of Microbiology International Journal of Microbiology 6 Second, we observed that their groundwater sources had similar characteristics, that is, the absence of efficient physical barriers such as concrete seals, well linings, hygienic covers, and secured aseptic lids among several others capable of avoiding terrestrial runoffcomprising anthropogenic and animal waste which will assuredly pollute the sources. Tis could be linked to the detection of E. coli counts in the groundwater sources in the present study. In view of the essence of physical barriers in wells, WHO [44] purported that groundwater is less susceptible to pollution as a result of the presence of barriers. Hence, contamination levels will rise if these barriers are compromised. Tis is particularly worrisome because, according to Chapman [45] when groundwater is polluted, it can remain polluted for many years. Te inference of this result is pivotal and, as such, cannot be overelaborated. and the Sustainable Development Goals, pp. 207–218, Springer, Berlin, Germany, 2020. [2] [2] C. Osunla and A. Okoh, “Vibrio pathogens: a public health concern in rural water resources in sub-saharan Africa,” International Journal of Environmental Research and Public Health, vol. 14, no. 10, p. 1188, 2017. [3] A. Furtatova and L. Kamenik, “Modeling features of sus- tainable urban development in modern conditions of water supply,” MATEC Web of Conferences, vol. 170, p. 04002, 2018. [4] S. T. Odonkor and K. K. Addo, “Prevalence of multidrug- resistant Escherichia coli isolated from drinking water sour- ces,” International Journal of Microbiology, vol. 2018, Article ID 7204013, 7 pages, 2018. [5] K. O. Odeku and E. L. Meyer, “Socioeconomic implications of energy poverty in South African poor rural households,” Academy of Entrepreneurship Journal, vol. 25, no. 3, pp. 1–12, 2019. [6] E. Bisung and S. Dickin, “Concept mapping: engaging stakeholders to identify factors that contribute to empow- erment in the water and sanitation sector in West Africa,” SSM-Population Health, vol. 9, Article ID 100490, 2019. Data Availability Te data used to support the findings of this study are in- cluded within the article. [13] J. N. Njeru, “Sanitation,” Te Wiley Blackwell Encyclopedia of Urban and Regional Studies, Wiley, Hoboken, NJ, USA, pp. 1–5, 2019. 5. Conclusions Te present study showed that most water sources analyzed were contaminated with bacterial pathogens surpassing rec- ommended standards thereby suggesting that residents living in the rural neighborhoods of the study area are exposed to heightened risks and susceptibility to waterborne diseases or health complications. A number of practices are associated with contamination of the water sources especially anthro- pogenic activities. Terefore, regular tests of water quality in rural vicinities are not only advantageous but necessary. [7] F. M. Gimelli, J. J. Bos, and B. C. Rogers, “Fostering equity and wellbeing through water: a reinterpretation of the goal of securing access,” World Development, vol. 104, pp. 1–9, 2018. [8] C. Anthonj, B. Diekkr¨uger, C. Borgemeister, and T. Kistemann, “Health risk perceptions and local knowledge of water-related infectious disease exposure among Kenyan wetland communities,” International Journal of Hygiene and Environmental Health, vol. 222, no. 1, pp. 34–48, 2019. [9] X. Xue, S. Cashman, A. Gaglione et al., “Holistic analysis of urban water systems in the greater cincinnati region: (1) life cycle assessment and cost implications,” Water Research X, vol. 2, Article ID 100015, 2019. Terefore, the government, in close collaboration with community chiefs and elders of the district, should develop a keen interest in monitoring water quality in those envi- ronments. Government and other stakeholders must also initiate education on environmental awareness including hygienic practices. Additionally, education on proper water storage practices, sanitary ways of handling water, among others, should be done to help to limit water pollution for safe consumption. [10] H. Yousefi, S. Zahedi, and M. H. Niksokhan, “Modifying the analysis made by water quality index using multi-criteria decision making methods,” Journal of African Earth Sciences, vol. 138, pp. 309–318, 2018. [11] M. Pal, Y. Ayele, M. Hadush, S. Panigrahi, and V. J. Jadhav, “Public health hazards due to unsafe drinking water,” Air and Water Borne Diseases, vol. 7, no. 1, p. 1, 2018. [12] A. Efstratiou, J. E. Ongerth, and P. Karanis, “Waterborne transmission of protozoan parasites: review of worldwide outbreaks-an update 2011–2016,” Water Research, vol. 114, pp. 14–22, 2017. Conflicts of Interest [14] M. V. Yates, “Drinking water microbiology,” in Manual of Environmental Microbiology, Wiley, Hoboken, NJ, USA, 4th edition, 2015. Te authors declare that they have no conflicts of interest. [15] H. T. M. Nguyen, Q. T. P. Le, J. Garnier, J.-L. J. Janeau, and E. Rochelle-Newall, “Seasonal variability of faecal indicator bacteria numbers and die-offrates in the red river basin, North Viet Nam,” Scientific Reports, vol. 6, no. 1, Article ID 21644, 2016. Acknowledgments Te authors’ sincere appreciation goes to laboratory tech- nicians who assisted with the water sampling. Te authors also want to acknowledge the enormous resources obtained from the thesis “Radiation Sensitivity and Molecular Characterization of Waterborne Multidrug Resistant Escherichia coli” towards this work. [16] E. Tursby and N. Juge, “Introduction to the human gut microbiota,” Biochemical Journal, vol. 474, no. 11, pp. 1823–1836, 2017. [17] N. R. Krieg and J. G. Holt, Bergey’s Manual of Systematic Bacteriology, p. 964, Williams and Wilkins, Balrimore, MD, USA, 1984. 4. Discussion Tough the current study did not reveal anthropogenic input as a critical cause of the presence of high faecal coliforms in most water samples investigated, these results hint that human faecal contaminations could be camouflaged by other sources (such as wild and domestic animals) especially when high counts are observed. In this study, we found E. coli counts to be predomi- nantly higher in the wet season than the dry season (Table 1). Terrestrial wastes usually budge into most water sources in periods of excessive hailstorms or downpours, which could cause higher levels of bacteria counts during wet seasons than dry seasons [40, 41]. Tis could account for the higher counts observed in the water samples collected during the wet season than in the dry season. Although there were higher counts in the wet season than in the dry season, several reasons could also explain the counts observed in the dry season. First of all, water shortage and brisk multipli- cation of bacteria as a result of suitable conditions such as temperature throughout the dry season [42] could account for such a revelation. It is worth noting, rainfalls provided higher water volumes and large surface area for rapid mi- crobial growth, leading to higher microbial counts and subsequently, higher extents of pollution in the wet season. Furthermore, bacterial pollutants in dumpsites, human waste, and heating systems could be drained into the dif- ferent water sources during the wet season, after which rapid multiplication occurs. Tese sources are thus, probable sources of transmitting pathogens resulting in more critical health problems in rural neighborhoods. It is important to note that a number of observations were made concerning the environmental conditions in the district during the research period, which gives insights of the pollution of the water sources. First, there were limited dumpsites for waste disposal in the district, which could account for the use of streams as alternative waste dumping sites. Moreover, it was observed that inhabitants in some communities in the district adopt unhygienic activities particularly, defecation and urination in the open and in drain ways, which eventually gets into water bodies. International Journal of Microbiology Ampofo, “Seasonal analysis of bacteriological quality of drinking water sources in communities surrounding Lake Bosomtwe in the Ashanti Region of Ghana,” Applied Water Science, vol. 9, no. 4, p. 82, 2019. [24] R. Hope, P. Tomson, J. Koehler, and T. 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Bartram, “Seasonal variation of fecal contamination in drinking water sources in developing countries: a systematic review,” Science of the Total Environment, vol. 514, pp. 333–343, 2015. [28] APHA, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, American Water Works Association and Water Environment Federa- tion, Washington, DC, USA, 19th edition, 1995. pp [44] WHO, Guidelines for Drinking-Water Quality, WHO, Geneva, Switzerland, 3rd edition, 2006. g [29] APHA, Standard Methods for the Examination of Water and Wastewater, United Book Press, Inc., Baltimore, MD, USA, 20th edition, 1998. [45] D. Chapman, Water Quality Assessments. A Guide to Use of Biota, Sediments and Water in Environmental Monitoring, E. & F. N. Spon, New York, NY, USA, 2nd edition, 1996. [30] L. M. Prescott, J. P. Harley, and A. K. Donald, Microbiology, W. C. Brown Publishers, McGraw-Hill Companies Inc., New York, NY, USA, 5th edition, 1996. [31] K. L. Quiroz, N. G. Rodriguez, S. Murinda, and M. Ibekwe, “Determination of the water quality of a constructed wetland monitoring fecal indicator bacteria,” 2018. [32] C. Rodrigues and M. ˆA. Cunha, “Assessment of the micro- biological quality of recreational waters: indicators and methods,” Euro-Mediterranean Journal for Environmental Integration, vol. 2, no. 1, p. 25, 2017. [33] E. M. Hachich, M. D. Bari, A. P. G. Christ, C. C. Lamparelli, S. S. Ramos, and M. I. Z. International Journal of Microbiology International Journal of Microbiology virulence genes along a rural aquatic continuum,” Frontiers in Microbiology, vol. 8, p. 609, 2017. [19] A. Agensi, J. Tibyangye, A. Tamale, E. Agwu, and C. Amongi, “Contamination potentials of household water handling and storage practices in kirundo subcounty, kisoro district, Uganda,” Journal of Environmental and Public Health, vol. 2019, Article ID 7932193, 8 pages, 2019. [36] S. L. McLellan and A. M. Eren, “Discovering new indicators of fecal pollution,” Trends in Microbiology, vol. 22, no. 12, pp. 697–706, 2014. [37] M. A. Nkansah, N. O. Boadi, and M. Badu, “Assessment of the quality of water from hand-dug wells in Ghana,” Environ- mental Health Insights, vol. 4, no. 2, pp. 7–12, 2010. g [20] J. M. Kayembe, F. Tevenon, A. Laffite et al., “High levels of faecal contamination in drinking groundwater and recrea- tional water due to poor sanitation, in the sub-rural neigh- bourhoods of Kinshasa, Democratic Republic of the Congo,” International Journal of Hygiene and Environmental Health, vol. 221, no. 3, pp. 400–408, 2018. [38] M. Odagiri, A. Schriewer, M. E. Daniels et al., “Human fecal and pathogen exposure pathways in rural Indian villages and the effect of increased latrine coverage,” Water Research, vol. 100, pp. 232–244, 2016. [21] R. G. Price and D. Wildeboer, “E. coli as an indicator of contamination and health risk in environmental waters,” in Escherichia coli-Recent Advances on Physiology, Pathogenesis and Biotechnological Applications, IntechOpen, London, UK, 2017. [39] M. Pritchard, A. Edmondson, T. Craven, and T. Mkandawire, “Development of sustainable drinking water quality solutions for rural communities in the developing world,” in Sustain- able Ecological Engineering Design, pp. 259–277, Springer, Berlin, Germany, 2016. [22] H. Br¨ussow, “Phage therapy: the Escherichia coli experience,” Microbiology, vol. 151, no. 7, pp. 2133–2140, 2005. [40] N. Ferrer, A. Folch, G. Mas´o, S. Sanchez, and X. Sanchez-Vila, “What are the main factors influencing the presence of faecal bacteria pollution in groundwater systems in developing countries?” Journal of Contaminant Hydrology, vol. 228, Article ID 103556, 2020. [23] T. Gara, L. Fengting, I. Nhapi, C. Makate, and W. Gumindoga, “Health safety of drinking water supplied in Africa: a closer look using applicable water-quality standards as a measure,” Exposure and Health, vol. 10, no. 2, pp. 117–128, 2018. [41] M. O. Akrong, F. K. Amu-Mensah, M. A. Amu-Mensah, H. Darko, G. N. D. Addico, and J. A. References [18] S. T. Odonkor and J. K. Ampofo, “Escherichia coli as an indicator of bacteriological quality of water: an overview,” Microbiology Research, vol. 4, no. 1, p. 2, 2013. [1] H. Chitonge, A. Mokoena, and M. Kongo, “Water and san- itation inequality in Africa: challenges for SDG 6,” in Africa 7 International Journal of Microbiology Sato, “Comparison of thermotolerant coliforms and Escherichia coli densities in freshwater bodies,” Brazilian Journal of Microbiology, vol. 43, no. 2, pp. 675–681, 2012. [34] S. L. McLellan, A. D. Daniels, and A. K. Salmore, “Clonal populations of thermotolerant enterobacteriaceae in recrea- tional water and their potential interference with faecal Escherichia coli counts,” Applied and Environmental Micro- biology, vol. 67, no. 10, pp. 4934–4938, 2001. gy [35] F. Petit, O. Clermont, S. Delannoy et al., “Change in the structure of Escherichia coli population and the pattern of

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Analysis of Small Group and Individual Teaching Skills Students of Microteaching Learning

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Citation (APA Style): Saidah, I. N. A., & Ngazizah, N. (2022). Analysis of Small Group and Individual Teaching Skills Students of Microteaching Learning. Islamic Journal of Integrated Science Education (IJISE), 1(3), 143–151. https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE) Program Studi Tadris IPA Institut Agama Islam Negeri Kediri e-ISSN : 2986-0865 https://jurnalfaktarbiyah.iainkediri.ac.id/index.php/ijise Analysis of Small Group and Individual Teaching Skills Students of Microteaching Learning Ika Nurul Afifatus Saidah1*, Nur Ngazizah2 1Universitas Muhammadiyah Purworejo, Indonesia 2Universitas Muhammadiyah Purworejo, Indonesia *Correspondence: E-mail: [email protected] Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE) Program Studi Tadris IPA Institut Agama Islam Negeri Kediri e-ISSN : 2986-0865 https://jurnalfaktarbiyah.iainkediri.ac.id/index.php/ijise Analysis of Small Group and Individual Teaching Skills Students of Microteaching Learning Ika Nurul Afifatus Saidah1*, Nur Ngazizah2 1Universitas Muhammadiyah Purworejo, Indonesia 2Universitas Muhammadiyah Purworejo, Indonesia *Correspondence: E-mail: [email protected] Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 slamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE) Program Studi Tadris IPA Institut Agama Islam Negeri Kediri e-ISSN : 2986-0865 https://jurnalfaktarbiyah.iainkediri.ac.id/index.php/ijise Program Studi Tadris IPA Institut Agama Islam Negeri Kediri e-ISSN : 2986-0865 ://jurnalfaktarbiyah iainkediri ac id/index php Analysis of Small Group and Individual Teaching Skills Students of Microteaching Learning Ika Nurul Afifatus Saidah1*, Nur Ngazizah2 1Universitas Muhammadiyah Purworejo, Indonesia 2Universitas Muhammadiyah Purworejo, Indonesia *Correspondence: E-mail: [email protected] Abstract: Small group and individual teaching is a form of learning that allows teachers to pay attention to each student, and establish a closer relationship between teachers and students as well as between students and students. This study aims to determine the ability of small group and individual teaching skills possessed by semester VI students of Primary School Teacher Education Study Program for the 2021/2022 academic year in the microteaching course. The basic teaching skills that must exist in a teacher or educator can be divided into eight types of skills. These skills play a very important role and determine the quality of learning, namely: explaining skills, opening and closing skills, questioning skills, skills to provide reinforcement, skills to carry out variations, skills to guide discussions and teach small groups and individuals, and skills to manage classes. To achieve the research objectives, the researcher used the type of research used was descriptive qualitative research to describe descriptively the basic teaching skills of prospective elementary school teacher students in microteaching courses involving 10 students. Data collection was done by direct observation in the form of a questionnaire given to students. Data were analyzed quantitatively and presented in tables. INTRODUCTION Elementary school is the first school that gets a big focus and hope to be able to provide basic concepts for children. Therefore, there should be a correlation between people's expectations and the goals of basic education. The general purpose of basic education is to lay the foundation for intelligence, knowledge, personality, noble character, and skills to live independently and participate in further education. Education is the main link in the formation of the nation's next generation. The more sophisticated and advanced the quality of education, the more advanced the country is. School is a conscious and appropriate exertion to stimulate, nurture, assist, and guide an individual to grow all his latent capacities to achieve superior self-quality. Education is not just a matter of information processing techniques, even the application of "learning theory" in the classroom or using the results of subject-centered "achievement exams". Education is a complex endeavor to adapt culture to the needs of its members and to adapt its members to the way they know the needs of the culture. According to Ki Hajar Dewantoro, education generally means efforts to promote the growth of character (inner strength, character), mind (intellect), and the child's body. Some experts formulate the notion of learning including according to Syaiful Sagala in Ramayulis, learning is teaching students to use educational principles and learning theories which are the main determinants of educational success. Learning is a two-way communication process. Teaching is done by the teacher as an educator, while learning is done by students. Learning is a process in which a person's environment is intentionally managed to allow him to participate in behavior that can produce a response to a particular situation (Putu Cahyani, 2021). According to Oemar Hamalik, learning is a structured combination that includes human elements, material facilities, equipment, and procedures that influence each other to achieve learning objectives. The success of a learning process and being accepted by students as a teacher requires a Learning Implementation Plan and skills in teaching. The lesson plan is one of the tools in a teaching and learning process that must be prepared by the teacher. Teachers are required to have competence in preparing lesson plans in accordance with government regulations. Lesson plan describes the procedure and organization of learning to achieve a basic competency. Without planning, you will experience difficulties and even failure in achieving the desired goals. Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE) Program Studi Tadris IPA Institut Agama Islam Negeri Kediri e-ISSN : 2986-0865 https://jurnalfaktarbiyah.iainkediri.ac.id/index.php/ijise Analysis of Small Group and Individual Teaching Skills Students of Microteaching Learning Ika Nurul Afifatus Saidah1*, Nur Ngazizah2 1Universitas Muhammadiyah Purworejo, Indonesia 2Universitas Muhammadiyah Purworejo, Indonesia *Correspondence: E-mail: [email protected] Keywords: analysis, individual teaching skills, small group teaching skills y ved: 26 June 2022; Revised: 05 August 2022; Accepted: 11 September 2022; Published: 30 November Copyright : © 2022 Program Studi Tadris IPA, Fakultas Tarbiyah, IAIN Kediri. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution - ShareAlike 4.0 International License (CC BY SA) license (https://creativecommons.org/licenses/by-sa/4.0/). Saidah & Ngazizah., 2022 143 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 INTRODUCTION Lesson plans are an attempt to predict the actions to be taken in learning activities. Lesson plan needs to be 144 Saidah & Ngazizah., 2022 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 developed to coordinate learning components including core competencies, basic competencies, learning objectives, learning materials, methods, learning activities, learning tools and resources, and assessment (Putri, 2016; Rashad Ali Bin-Hady & Abdulsafi, 2018). developed to coordinate learning components including core competencies, basic competencies, learning objectives, learning materials, methods, learning activities, learning tools and resources, and assessment (Putri, 2016; Rashad Ali Bin-Hady & Abdulsafi, 2018). Based on the problems related to the duties of an educator, it becomes a discussion in the community so that the aspects of competence that educators must have become the public's assessment. The low quality of learning caused by the demands for an educator, the lack of facilities and infrastructure in schools, and the low competence possessed by educators also cause the learning process to not run optimally (Telaumbanua, 2020). In other aspects, educators are required to be able to provide the best for their students. Whereas achieving educational goals does not happen as easily as turning the palm of the hand, but it takes a long process and a strong will from an educator himself. In preparing for this, Primary School Teacher Education Study Program, Universitas Muhammadiyah Purworejo, Indonesia, prepared several courses to provide students with a set of knowledge and skills regarding the teaching and learning process and other activities in theory and practice courses. One of the practical courses is a microteaching course. In this course, students will gain experience related to basic teaching skills (Fernández, 2005). Micro Learning or also called Micro Teaching is an integrated process formed from several elements whose main task is to carry out learning. Learning and teaching are components that cannot be separated; teaching contains simultaneously in the form of elements such as technology, knowledge, art, and also taste (Azis et al., 2021). In addition, experts in Fatwa Arifah et al. (2020) define microteaching as follows, Fakhrah & Unaida (2021) said microteaching is one model of teaching practice training in a limited scope (micro) to develop basic teaching skills (base teaching skills) that are carried out in isolation and in simplified situations. INTRODUCTION Microteaching is a teacher training technique through the practice of various teaching skills in specific situations with the help of feedback in the form of images to increase student engagement (Kula Ünver et al., 2020; Suryana, 2018). Regarding teaching ability, (Chrisvianty et al., 2020) states that there are elements in microteaching: (1) Skill goals or objectives; (2) Structure and organization; (3) Planning and schedule; (4) Coaching; (5) Feedback; (6) Students for microteaching; and (7) Activity suggestions. The function of micro learning is that it is hoped that students who have participated in micro teaching are no longer awkward and shy in teaching, students are expected to be able to practice what they have learned well in front of the class, students must also be creative in 145 Saidah & Ngazizah., 2022 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 their lesson plans, can master the material, combine methods used, learning media, and evaluation that will be used in teaching (Apriani et al., 2020; Higgins & Nicholl, 2003). their lesson plans, can master the material, combine methods used, learning media, and evaluation that will be used in teaching (Apriani et al., 2020; Higgins & Nicholl, 2003). In general, micro learning aims to form and develop basic teaching competencies as a provision for teaching practice in schools (Hazmi, 2019). In particular, the objectives of micro learning are as follows: (1) Forming and improving basic teaching competencies for limited; (2) Establishing and improving integrated and intact basic teaching competencies; (3) Forming personality competencies; and 4) Forming social competencies (Nurwahidah, 2020). The benefits of micro learning that are trained intensively will provide benefits for students (Suryana, 2018), especially in the following ways: (1) Students become sensitive to phenomena that occur in the learning process; (2) Students become more prepared to carry out practical learning activities in schools/institutions education; (3) Students can self-reflect on their competence in teaching; and (4) Students become more acquainted with and understand the competence of teachers so that they can appear as teachers. Characteristics of microteaching is a teaching situation that is carried out in a limited time and number, namely for approximately 15 minutes with a total of approximately 20 students practicing. The micro teaching procedures which were originally developed at Stanford are: planning, micro-teaching practice, observation, discussion, re-planning, microteaching re- practice, re-observation, and re-discussion. INTRODUCTION Microteaching procedures at The New University of Ulster, namely: planning, microteaching, observation, and discussion (Berangka, 2018). The basic teaching skills that must exist in a teacher or educator can be divided into eight types of skills. These skills play a very important role and determine the quality of learning, namely: explaining skills, opening and closing skills, questioning skills, skills to provide reinforcement, skills to carry out variations, skills to guide discussions and teach small groups and individuals, and skills to manage classes (Rahmi & Rahman, 2018). Small group and individual teaching is a form of learning that allows teachers to pay attention to each student, and establish closer relationships between teachers and students as well as between students and students (Hidayah, 2018). This activity includes the teacher's ability to give more attention to students and to approach students personally, the intensity of guiding and facilitating participants in learning activities so that more familiar learning conditions arise between teachers and students (Azis et al., 2021; Yuhandika et al., 2021). The number of students in this form of teaching ranges from 3 to 8 people for each small group, and 1 person for an individual. The limited number of students in this form of teaching allows the teacher to give optimal attention to each student. The relationship between teachers and students became more intimate, as well as the relationship between students. 146 Saidah & Ngazizah., 2022 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 The components and principles of this skill are: personal approach skills, organizing skills, guiding and facilitating learning skills, planning and implementing teaching and learning activities skills, and designing and implementing learning activities skills. The indicators for assessing the basic skills of teaching small groups and individuals are: (1) Personal approach skills; (2) Organizational skills; (3) Guiding skills and facilitating lessons; and (4) Planning and implementation skills for teaching and learning activities. METHOD The type of research used is descriptive qualitative research to describe descriptively the ability of basic teaching skills of prospective elementary school teacher students in microteaching courses. Data collection was carried out in the even semester of the 2021-2022 academic year in the microteaching learning of the Primary School Teacher Education Study Program at Purworejo Muhammadiyah University, Indonesia. This study involved 10 students. The target of this research is the ability to use appropriate teaching skills in classroom learning. Data collection was done by direct observation in the form of a questionnaire given to students. In addition, to confirm the data, an observation sheet was used by looking at the results of the teaching practice assessment in the microteaching course. Data were analyzed quantitatively and presented in tables. INTRODUCTION The principle of the researcher uses the title of teaching skills analysis for small groups and individual students of Primary School Teacher Education Study Program in Purworejo Muhammadiyah University, Indonesia semester VI based on the results of the microteaching learning evaluation conducted by assessing student performance, especially in terms of the ability to prepare lessons and carry out learning. Therefore, this study aims to describe the performance of students of the Primary School Teacher Education Study Program at the Purworejo Muhammadiyah University when carrying out microteaching learning. Student performance is measured based on the ability of students to develop lesson plans and carry out classroom learning. Furthermore, it was analyzed to determine the correlation between the two abilities, and to determine the student's response to feedback from fellow microteaching participants after their performance. FINDING AND DISCUSSION Based on problems related to the duties of an educator, it becomes a discussion in the community so that the aspects of competence that educators must have become the public's assessment. The low quality of learning caused by the demands for an educator, the lack of 147 Saidah & Ngazizah., 2022 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 facilities and infrastructure in schools, and the low competence possessed by educators also cause the learning process to not run optimally. In other aspects, educators are required to be able to provide the best for their students. Whereas achieving educational goals does not happen as easily as turning the palm of the hand, but it takes a long process and a strong will from an educator himself. facilities and infrastructure in schools, and the low competence possessed by educators also cause the learning process to not run optimally. In other aspects, educators are required to be able to provide the best for their students. Whereas achieving educational goals does not happen as easily as turning the palm of the hand, but it takes a long process and a strong will from an educator himself. In preparing for this, Primary School Teacher Education Study Program, Purworejo Muhammadiyah University prepared several courses to provide students with a set of knowledge and skills regarding the teaching and learning process and other activities in theory and practice courses. One of the practical courses is a microteaching course. In this course, students will gain experience related to basic teaching skills. Microteaching is one of the teaching practice training models in a limited scope (micro) to develop basic teaching skills (base teaching skills) which is carried out in isolation and in simplified situations. Regarding teaching ability, Chrisvianty et al. (2020) states that there are elements in microteaching: (1) Skill goals or objectives; (2) Structure and organization; (3) Planning and schedule; (4) Coaching; (5) Feedback; (6) Students for microteaching; and (7) Activity suggestions. In general, micro learning aims to form and develop basic teaching competencies as a provision for teaching practice in schools (Lubis et al., 2019). FINDING AND DISCUSSION In particular, the objectives of micro learning are as follows: (1) Forming and improving basic teaching competencies for limited; (2) Establishing and improving integrated and intact basic teaching competencies; (3) Forming personality competencies; and (4) Forming social competencies. The basic teaching skills that must exist in a teacher or educator can be divided into eight types of skills. These skills play a very important role and determine the quality of learning, namely: explaining skills, opening and closing skills, questioning skills, skills to provide reinforcement, skills to carry out variations, skills to guide discussions and teach small groups and individuals, skills to manage classes. Small group and individual teaching is a form of learning that allows teachers to pay attention to each student, and establish closer relationships between teachers and students as well as between students and students. Individual/individual teaching skills. The indicators for the assessment of basic small group and individual teaching skills can be seen in Table 1. Saidah & Ngazizah., 2022 148 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Table 1. Basic teaching skills assessment indicators Basic Teaching Skills Indicator Small group and individual teaching skills Personal approach skills Organizing skills Skills to guide and facilitate lessons Skills in planning and carrying out teaching and learning activities slamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 The research was conducted in 2021-2022 microteaching learning semester VI, students of the Primary School Teacher Education Study Program, Purworejo Muhammadiyah University. The research was conducted by direct observation to students. In addition, to confirm the data, observation sheets were used by looking at the results of the teaching practice assessment in microteaching courses that focused on small group and individual teaching skills. Small group and individual teaching skills can be seen in Table 2. Table 2. Basic small group and individual teaching skills Norm Category Frequency Percentage 85-100 Very Good 5 50% 75-84 Good 3 30% 60-74 Enough 2 20% 50-59 Less 0 0% <50 Fail 0 0% Total 10 100% Based on the Table 2 can it was analyzed that the small group and individual teaching skills of semester VI students of Primary School Teacher Education Study Program obtained the results of data analysis that the percentage was categorized as good. FINDING AND DISCUSSION Teachers can take a personal approach, organize, guide and facilitate lessons, as well as plan and carry out teaching and learning activities properly. Overall, 10 students of semester VI Primary School Teacher Education Study Program students have performed their small group and individual teaching skills well. CONCLUSION Based on the description of the findings of the research and discussion, it can be concluded that the average basic teaching ability of prospective teachers of the semester VI Primary School Teacher Education Study Program is in the good category. Even so, there are still shortcomings in the use of basic teaching skills in microteaching. This can be corrected by increasing the number of teaching exercises so that it is hoped that students who have participated in macro teaching are no longer awkward and shy in teaching, students are expected to be able to practice what they have learned well in front of the class, students must 149 Saidah & Ngazizah., 2022 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 Islamic Journal of Integrated Science Education (IJISE), Vol. 1 No. 3, November 2022, pp. 143-151 DOI: https://doi.org/10.30762/ijise.v1i3.357 also be creative in their lesson plans, can master the material, combine the methods used, learning media, and evaluations that will be used in teaching. Suggestions for further research, further research is needed to improve the basic teaching skills of prospective teacher students. ACKNOWLEDGMENTS The author would like to thank and express appreciation to various parties who have provided support and assistance so that this research can be carried out properly and the results can be published in this paper. In particular, the author expresses his gratitude to the semester VI students of Primary School Teacher Education Study Program at Purworejo Muhammadiyah University, Indonesia who have become the research subjects. REFERENCES Apriani, L., Alpen, J., & Arismon, A. (2020). Tingkat percaya Diri dan Keterampilan Micro teaching. Edu Sportivo: Indonesian Journal of Physical Education, 1(1), 42–49. https://doi.org/10.25299/es:ijope.2020.vol1(1).5155 Azis, A. C. K., Mesra, M., & Sugito, S. (2021). Pengembangan Bahan Ajar Micro Teaching Bagi Mahasiswa Seni Rupa Universitas Negeri Medan. Gorga : Jurnal Seni Rupa, 10(1), 223–229. https://doi.org/10.24114/gr.v10i1.26011 Berangka, D. (2018). 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Peran Mutual Legal Assistance dalam Pemberantasan Tindak Pidana Korupsi di Negara-Negara ASEAN: Perspektif Tantangan Kedepan

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Peran Mutual Legal Assistance dalam Pemberantasan Tindak Pidana Korupsi di Negara-Negara ASEAN: Perspektif Tantangan Kedepan Fatika Azzahra Ainiyyah Hartono Universitas Jember, Indonesia Fatika Azzahra Ainiyyah Hartono Universitas Jember, Indonesia Ihdini Salimah Kaafah Universitas Jember, Indonesia Martha Hasibuan Universitas Jember, Indonesia Yunita Lestari Universitas Jember, Indonesia Jurnal Anti Korupsi Volume 13 Issue 1 (2023), pp. 28-45 Publised online Mei 2023 ISSN 2986-0741 | DOI: 10.19184/jak. v13i1. 38815 Peran Mutual Legal Assistance dalam Pemberantasan Tindak Pidana Korupsi di Negara-Negara ASEAN: Perspektif Tantangan Kedepan Fatika Azzahra Ainiyyah Hartono Universitas Jember, Indonesia Ihdini Salimah Kaafah Universitas Jember, Indonesia Martha Hasibuan Universitas Jember, Indonesia Yunita Lestari Universitas Jember, Indonesia Jurnal Anti Korupsi Volume 13 Issue 1 (2023), pp. 28-45 Publised online Mei 2023 ISSN 2986-0741 | DOI: 10.19184/jak. v13i1. 38815 Peran Mutual Legal Assistance dalam Pemberantasan Tindak Pidana Korupsi di Negara-Negara ASEAN: Perspektif Tantangan Kedepan KATA KUNCI: Mutual Legal Assistance (MLA), Pencucian Uang, Pengembangan. I. PENDAHULUAN Tindak pidana korupsi merupakan permasalahan yang sangat serius di seluruh dunia terkhusus di wilayah Association of Southeast Asian Nation (ASEAN). Hal ini dibuktikan berdasarkan data Corruption Perceptions Index (CPI) yang diterbitkan di laman Transparency International pada tahun 2021, negara-negara di wilayah ASEAN berada di urutan negara paling korup karena memiliki skor CPI yang rendah (Faizi, 2022). Sampai saat ini, negara- negara di ASEAN masih kesulitan untuk memberantas korupsi, banyak kasus koruptor yang melarikan diri ke luar negeri sehingga negara-negara di ASEAN perlu bekerja sama untuk memberantas hal tersebut. Upaya yang bisa menjadi pilihan untuk memberantas korupsi di wilayah ASEAN yaitu Mutual Legal Assistance (MLA). MLA merupakan kerja sama dalam rangka Komunitas Politik Keamanan ASEAN atau ASEAN Political Security Community (APSC) di bidang hukum, bertujuan untuk memfasilitasi proses penyelidikan dan penuntutan tindak pidana di antara negara-negara yang bekerja sama. Melalui MLA, segala upaya pemberantasan tindak pidana korupsi di ASEAN sudah dilakukan sejak tahun 2004 setelah disahkannya Treaty on Mutual Legal Assistance in Criminal Matters (MLAT). Berdasarkan penjelasan diatas, maka permasalahan yang akan dibahas adalah bagaimana peran MLA dalam pemberantasan tindak pidana korupsi di ASEAN dan apa saja tantangan yang dihadapi dalam mengimplementasikan MLA dalam pemberantasan tindak pidana korupsi di ASEAN serta bagaimana prospek pengembangan MLA di masa depan untuk mendukung pemberantasan tindak pidana korupsi di ASEAN. Copyright © 2021 by Author(s) This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. All writings published in this journal are personal views of the authors and do not represent the views of this journal and the author's affiliated institutions. HOW TO CITE: Hartono, et al. " Peran Mutual Legal Assistance dalam Pemberantasan Tindak Pidana Korupsi di Negara- Negara ASEAN: Perspektif Tantangan Kedepan" (2023) 13:1 Jurnal Anti Korupsi 28-45 ABSTRAK Kejahatan Korupsi yang semakin merebak kini tergolong dalam kejahatan transnasional yang terorganisir. Kejahatan Korupsi yang terstruktur dan cukup sistematis menjadikannya polemik serius. Kejahatan korupsi yang diimbangi kemajuan teknologi mampu melahirkan tindak pidana yang lain yaitu pencucian uang (Money Laundering). Pelaku korupsi melarikan diri ke luar negeri untuk menyamarkan hartanya supaya tidak terjangkit jeratan hukum. Fenomena sosial ini mendorong negara- negara di wilayah Association of Southeast Asian Nation (ASEAN) untuk memberantas yang tidak mengenal batas-batas negara tersebut (Borderless Crime). Upaya khusus dalam menangani kejahatan korupsi tersebut diformulasikan dalam suatu perangkat yaitu Treaty on Mutual Legal Assistance in Criminal Matters (MLAT) atau dikenal sebagai Bantuan Timbal Balik dalam Masalah Pidana. Treaty on Mutual Legal Assistance (MLAT) merupakan salah satu instrumen hukum berupa kerjasama regional yang tergolong cukup efektif dalam menangani pelaku korupsi terkait pencucian uang (Money Laundering). Pemberantasan kejahatan korupsi ini juga meliputi prospek pengembangan teknologi untuk masa depan baik, pembaharuan perjanjian Treaty on Mutual Legal Assistance (MLAT) dan perjanjian eksekutif. Tujuan penelitian ini untuk mengetahui bagaimana peran, hambatan dan prospek pengembangan Treaty on Mutual Legal Assistance (MLAT) dalam ruang lingkup ASEAN. Metode yang digunakan dalam penelitian ini adalah hukum normatif dengan pendekatan doktrinal. Hasil yang diperoleh dari penelitian adalah meskipun Treaty on Mutual Legal Assistance (MLAT) dikenal sebagai instrumen hukum yang efektif dalam menangani pencucian uang (Money Laundering) di ASEAN, akan tetapi masih ditemukan beberapa hambatan yang cukup kompleks dalam pengembalian aset (Asset Recovery) yaitu adanya perbedaan sistem hukum dan diberlakukannya asas non retroaktif. Meskipun demikian, terdapat beberapa penawaran dalam pengembangan Treaty on Mutual Legal Assistance (MLAT) berbasis elektronik yang memberikan output berupa kemudahan bagi negara-negara di Kawasan ASEAN dalam mengakses data dan sistem pelacakan. Selain itu juga, aspek pengembangan tersebut juga meliputi pembahasan perjanjian dan ketentuan Treaty on Mutual Legal Assistance (MLAT) supaya mampu menghadirkan instrumen yang lebih baik di masa depan. KATA KUNCI: Mutual Legal Assistance (MLA), Pencucian Uang, Pengembangan. 29 | J u r n a l A n t i K o r u p s i 29 Copyright © 2021 by Author(s) II. METODE PENULISAN Metode penelitian yang digunakan dalam penelitian ini adalah metode penelitian hukum normatif dengan pendekatan doktrinal. Penelitian ini dilakukan dengan memanfaatkan data sekunder berupa buku-buku, artikel dalam jurnal-jurnal hukum serta dokumen-dokumen hukum lainnya yang relevan dengan pokok pembahasan. Tahapan awal pengumpulan data dilakukan dengan mencari sumber informasi yang berkaitan dengan peran MLA dalam memberantas tindak pidana korupsi di negara-negara ASEAN. Setelah itu, penelitian dilanjutkan dengan menentukan berbagai tantangan dalam penerapan MLA dan prospek MLA di masa depan. | J u r n a l A n t i K o r u p s i 30 A. Faktor-Faktor Penyebab Terjadinya Korupsi Korupsi dalam bahasa latin disebut juga corruptio yang memiliki beragam makna yakni tindakan menghancurkan, dapat disuap, penyimpangan dan lainya. Dalam Bahasa inggris, kata corruptio berubah menjadi corruption, sedangkan dalam bahasa Belanda disebut dengan corruptie. Kata corruptie ini kemudian diadaptasi kedalam Bahasa Indonesia dan kemudian lahirlah kata korupsi. Berdasarkan Kamus Besar Bahasa Indonesia (KBBI), korupsi adalah penyelewengan atau penyalahgunaan uang negara (perusahaan, organisasi, yayasan, dan sebagainya) untuk keuntungan pribadi atau orang lain. Definisi lain dari korupsi adalah penyalahgunaan kekuasaan publik untuk keuntungan pribadi (World Bank, 2000). Korupsi dapat terjadi karena beberapa aspek, antara lain: Korupsi dalam bahasa latin disebut juga corruptio yang memiliki beragam makna yakni tindakan menghancurkan, dapat disuap, penyimpangan dan lainya. Dalam Bahasa inggris, kata corruptio berubah menjadi corruption, sedangkan dalam bahasa Belanda disebut dengan corruptie. Kata corruptie ini kemudian diadaptasi kedalam Bahasa Indonesia dan kemudian lahirlah kata korupsi. Berdasarkan Kamus Besar Bahasa Indonesia (KBBI), korupsi adalah penyelewengan atau penyalahgunaan uang negara (perusahaan, organisasi, yayasan, dan sebagainya) untuk keuntungan pribadi atau orang lain. Definisi lain dari korupsi adalah penyalahgunaan kekuasaan publik untuk keuntungan pribadi (World Bank, 2000). Korupsi dapat terjadi karena beberapa aspek, antara lain: 1. Aspek pribadi manusia Aspek penyebab utama yang melatarbelakangi seseorangan untuk melakukan korupsi adalah perilaku materialistik, konsumtif dan sifat serakah manusia. Nursyam (2000) mengatakan pemicu seseorang melakukan korupsi adalah karena terhasut dengan kekayaan materialistik yang sulit ditahan. Ketika Hasrat keinginan untuk memiliki kekayaan materialistic tidak bisa ditahan, sementara jalan untuk memperoleh kekayaan itu bisa didapatkan melalui korupsi, maka seseorang dapat tanpa sulit melakukan korupsi. Selain itu, gaya hidup konsumtif yang tidak sebanding dengan gaji yang dihasilkan dapat menjadi penyebab seseorang melakukan korupsi supaya dapat memenuhi gaya hidup konsumtif tersebut. III. MUTUAL LEGAL ASSISTANCE DAN PERANNYA DALAM PEMBERANTASAN TINDAK PIDANA KORUPSI DI KAWASAN ASEAN Mengingat letak Asia Tenggara berada di wilayah yang strategis menjadikan kawasan ini rentan terhadap berbagai macam transnasional crime. Transnasional crime adalah kejahatan yang dapat mengganggu kemakmuran dan keutuhan suatu negara bahkan lebih dari satu negara contohnya seperti people smuggling; corruption and money laundering; perdagangan ilegal dan kejahatan narkotika. Salah satu kasus kejahatan yang sering terjadi di Kawasan Asia Tenggara adalah korupsi dan pencucian uang. Tindakan korupsi dapat dikategorikan sebagai transnational crime apabila korupsi yang dilakukan merupakan korupsi yang terjadi di lingkungan pemerintah dengan jumlah yang cukup besar. B. Klasifikasi Tindak Pidana Korupsi Dalam Undang Undang Nomor 20 Tahun 2001 terkait Perubahan Atas Undang-Undang No. 31 Tahun 1999 tentang Pemberantasan Tindak Pidana Korupsi yang kemudian disederhanakan dari 30 klasifikasi menjadi 7 klasifikasi tindak pidana korupsi yaitu: korupsi yang berkaitan dengan kerugian keuangan negara, suap-menyuap, penggelapan dalam jabatan, pemerasan, perbuatan curang, benturan kepentingan dalam pengadaan, dan gratifikasi (KPK, 2006). 1. Korupsi yang berkaitan dengan kerugian Keuangan negara Merugikan keuangan negara masuk kedalam unsur dari delik korupsi dan perbuatan melawan hukum yang menyebabkan aset negara berkurang. Perbuatan ini dapat berbentuk uang, surat berharga, maupun barang. Meskipun telah dilakukan pengembalian kerugian negara, hal ini tetap tidak membuat perbuatan pidana yang dilakukan oleh pelaku tindak pidana terhapus. Sanksi pidana untuk pelaku tindak pidana korupsi ini diatur dalam Undang Undang Nomor 20 Tahun 2001 Pasal 2 ayat 1 dan pasal 3. 3. Aspek ekonomi dan politik Jika dikaitkan dengan aspek politik, kontrol sosial dari masyarakat perlu dilakukan untuk mengawasi setiap orang agar tidak melakukan korupsi (Karsono, 2011; Indah Sri Utari. 2011). Kontrol sosial dapat dilakukan dengan menjalankan berbagai kegiatan yang terkontrol secara politis lewat lembaga-lembaga negara dan lembaga swadaya masyarakat. Tidak kuatnya kontrol sosial terhadap korupsi pada akhirnya menyebabkan praktik korupsi terus meningkat di lingkungan masyarakat (Karsono, 2011; Indah Sri Utari. 2011). 2. Aspek keluarga Selain aspek pribadi, terdapat juga aspek dari luar pribadi seseorang contohnya seperti faktor keluarga dan masyarakat. Faktor keluarga pada umumnya seringkali memberi keyakinan yang kuat kepada seseorang untuk melakukan tindak kejahatan korupsi. Karena pada faktanya, lingkungan keluarga seringkali tidak memberikan sanksi pada anggota keluarga yang melakukan penyelewengan kekuasaan terkait korupsi justru malah mendukung serta melindunginya (Karsono, 2011; Indah Sri Utari. 2011). 31 | J u r n a l A n t i K o r u p s i 31 2. Suap menyuap Tindakan suap adalah memberi atau mendapatkan uang serta hadiah yang dilakukan oleh pejabat pemerintah dalam melaksanakan atau tidak melaksanakan sesuatu yang berlawanan dengan kewajibannya. Pasal 12 huruf e menyatakan, seseorang yang melaksanakan tindak pidana suap dijatuhi pidana penjara seumur hidup atau pidana penjara paling sedikit 4 tahun dan paling lama 20 tahun serta pidana denda paling minim dua ratus juta rupiah dan paling banyak satu milyar rupiah. Dalam hal ini pegawai negeri yang memiliki maksud mendapat keuntungan untuk diri sendiri atau orang lain dengan menyalahgunakan kekuasaannya meminta seseorang memberikan sesuatu, membayar, atau menerima pembayaran dengan potongan, atau untuk mengerjakan sesuatu bagi dirinya sendiri. 5. Tindakan curang Tindakan curang yang dimaksud disini adalah kecurangan yang dilakukan pemborong ataupun pengawas proyek yang melakukan tindakan curang dalam pengadaan atau pembelian barang yang menyebabkan kerugian bagi orang lain atau terhadap keuangan negara dan bisa membahayakan keselamatan negara. Sanksi atau hukuman bagi pelaku yang melakukan Tindakan curang diatur dalam Undang Undang Nomor 20 Tahun 2001 Pasal 7 ayat 1 huruf a, b, c, d serta pasal 7 ayat 2 dan pasal 12 huruf h. 6. Benturan kepentingan dalam pengadaan Benturan kepentingan dalam pengadaan dihukum dengan penjara seumur hidup atau pidana penjara paling sedikit empat tahun dan paling lama dua puluh tahun serta pidana denda paling minim dua ratus juta rupiah dan maksimal satu milyar rupiah. Hal ini berlaku bagi pegawai negeri atau penyelenggara negara secara langsung maupun tidak yang dengan sengaja ikut dalam pemborongan, pengadaan, atau persewaan, yang saat dilakukannya Tindakan tersebut sedang bertugas untuk mengawasi 7. Gratifikasi Gratifikasi adalah pemberian barang dalam berbagai bentuk yang berkaitan dengan pekerjaan, jabatan, atau tugas secara cuma cuma. Pasal 12b Undang-Undang Nomor 20 Tahun 2001 menjelaskan yang dimaksud gratifikasi adalah memberikan uang, barang, komisi, pinjaman tanpa bunga, tiket perjalanan, fasilitas penginapan, perjalanan wisata, pengobatan gratis, dan fasilitas lainnya. Termasuk juga gratifikasi yang dilakukan di luar negeri ataupun di dalam negeri juga yang dilakukan melalui sarana elektronik atau nonelektronik. 4. Pemerasan Pemerasan yang dimaksud adalah apabila seorang petugas layanan menawarkan jasa atau meminta balasan kepada pengguna layanan dan bermaksud supaya bisa mempercepat tercapainya tujuan konsumen jasa, walaupun petugas layanan tahu hal tersebut melanggar prosedur yang berlaku. Pemerasan yang dimaksud adalah apabila seorang petugas layanan menawarkan jasa atau meminta balasan kepada pengguna layanan dan bermaksud supaya bisa mempercepat tercapainya tujuan konsumen jasa, walaupun petugas layanan tahu hal tersebut melanggar prosedur yang berlaku. Pemerasan yang dimaksud adalah apabila seorang petugas layanan menawarkan jasa atau meminta balasan kepada pengguna layanan dan bermaksud supaya bisa mempercepat tercapainya tujuan konsumen jasa, walaupun petugas layanan tahu hal tersebut melanggar prosedur yang berlaku. 3. Penggelapan dalam jabatan 3. Penggelapan dalam jabatan 3. Penggelapan dalam jabatan ini maksudnya adalah seorang pejabat pemerintah yang memiliki kewenangan melakukan penggelapan terkait laporan keuangan, menghancurkan barang bukti atau membiarkan orang lain memusnahkan barang bukti 32 | J u r n a l A n t i K o r u p s i 32 yang bertujuan untuk keuntungan pribadi dengan jalan merugikan Negara. Dalam Undang Undang Nomor 20 Tahun 2001, sanksi yang dimaksud hanya berlaku untuk seorang pegawai negeri. Maka dari itu selain penggelapan yang dilakukan pejabat publik/ pegawai negeri, dibutuhkan perluasan peraturan untuk mengatur penggelapan dalam jabatan swasta terkait dengan kepentingan khalayak umum karena perbuatan yang dilakukan merugikan kepentingan umum juga orang lain 7. Gratifikasi Tindakan pidana korupsi ini sangat merugikan suatu negara yang pada akhirnya dapat berdampak pada jalannya pemerintahan suatu negara. Lembaga Transparency International melalui CPI melakukan survey di 180 negara. Nilai 0 menunjukkan negara dengan tingkat 33 | J u r n a l A n t i K o r u p s i 33 korupsi paling tinggi, sedangkan nilai 100 artinya negara tersebut sangat minim dari korupsi. Di Kawasan Asia Tenggara Adapun negara dengan tingkat korupsi nomor satu adalah Myanmar, kemudian disusul oleh Kamboja, Laos, dan Filipina. Sementara Singapura menjadi negara yang memiliki tingkat korupsi paling minim di Asia Tenggara, dengan nilai IPK 83. Nilai ini juga menempatkan Singapura di peringkat ke-5 terbaik global pada 2022. Berikut rincian nilai indeks korupsi negara di kawasan Asia Tenggara pada 2022, diurutkan dari yang memiliki tingkat korupsi paling tinggi dan tingkat korupsi paling minim: No Nama Nilai / Skor Indeks (Skala 0-100) 1 Myanmar 23 2 Kamboja 24 3 Laos 31 4 Filipina 33 5 Indonesia 34 6 Thailand 36 7 Timor Leste 42 8 Vietnam 42 9 Malaysia 47 10 Singapura 83 *skor Brunei Darussalam tidak tersedia *skor Brunei Darussalam tidak tersedia Di era perkembangan teknologi khususnya teknologi informasi serta kemudahan untuk berpindah antar berbagai negara karena batas teritorial suatu negara yang tipis, kerjasama antar negara perlu ditingkatkan untuk mengamankan aset negara yang diambil oleh koruptor yang kabur dari suatu negara ke negara lain untuk bersembunyi. Dalam perkembangannya, ASEAN harus menguatkan metode yang sudah ada ataupun membentuk metode baru terkait dengan pembentukan komunitas keamanan dalam mencegah atau mengurangi konflik yang berhubungan dengan perkembangan konflik di ASEAN. Dalam menangani Korupsi dan juga money laundering di kawasan ASEAN, terdapat tiga kebijakan mendasar yaitu: 1) ASEAN Political Security Community (APSC) ; 2) Pembentukan Mutual Legal Assistance Treaty (MLAT) dikhususkan untuk mengatasi kasus Korupsi dan Money Laundering; serta 3) Membentuk Badan kerja sama antar negara negara ASEAN yang khusus mengatasi kasus Korupsi dan Money Laundering dengan menggunakan MLAT dan APSC sebagai dasar hukum ASEAN (Setiawan, 2016). Selain perjanjian ekstradisi, Mutual Legal Assistance merupakan salah satu bentuk kerja sama Internasional menurut UNCAC 2003. MLA atau Bantuan Hukum Timbal Balik merupakan kerjasama antar negara negara di lingkup pencegahan dan pemberantasan kejahatan khususnya terhadap transnational crime yang bertujuan untuk mengumpulkan dan bertukar | J u r n a l A n t i K o r u p s i 34 informasi dalam rangka mengupayakan penegakkan hukum publik atau hukum kriminal (Dan E. Stigall, 2013) Banyaknya transnasional crime yang terjadi di kawasan ASEAN, memunculkan kesadaran negara anggota ASEAN untuk membentuk dasar hukum yang bersifat mengikat negara negara tersebut yakni dengan menandatangani Treaty on Mutual legal Assistance in Criminal Matters in Such Matters Among ASEAN Member Countries (MLAT). Perjanjian ini pada mulanya disepakati dan ditandatangani oleh negara Brunei Darussalam, Kamboja, Indonesia, Laos, Malaysia, Filipina, Singapura dan Vietnam dalam pertemuan Attorneys Generaldi Kuala Lumpur tanggal 29 November 2004. Kemudian pada tanggal 17 Januari 2006 dua negara ASEAN lainnya yaitu Thailand dan Myanmar menandatangani MLAT setelah menyelesaikan persyaratan domestiknya. Pembentukan MLA awalnya didasari oleh kondisi sebenarnya sebagai akibat dari dismilaritas sistem hukum pidana di beberapa negara yang menimbulkan kelambatan dalam memeriksa kejahatan. Umumnya tiap tiap negara ingin menggunakan metode hukumnya sendiri dalam menangani kejahatan, begitupun dengan negara lain, sehingga penindakan kejahatan menjadi lambat dan rumit. Agar terdapat dasar hukum dalam praktek pelaksanaan Mutual Legal Assistance, di tahun 2006 dibuatlah Undang Undang No. 1 Tahun 2006 tentang Bantuan Timbal Balik dalam perkara pidana. *skor Brunei Darussalam tidak tersedia Meskipun Undang Undang ini tidak secara spesifik dibuat untuk mengatasi masalah korupsi, namun undang-undang ini tetap bisa dijadikan sebagai dasar hukum. Meskipun latar belakang lahirnya Mutual Legal Assistance didasari oleh perbedaan sistem hukum pidana di beberapa negara ASEAN, namun MLA tidak selalu tentang penyesuaian sistem hukum yang berlaku, tetapi rasa saling memerlukan informasi lah yang perlu diutamakan oleh Negara-negara ASEAN. Lalu pertanyaannya, bagaimanakah peran MLA dalam memberantas kejahatan korupsi di Kawasan ASEAN? Asean Mutual Legal Assistance memiliki mekanisme tersendiri yang ditentukan oleh negara ASEAN yang tergabung dan menandatangani MLA. Jadi dengan adanya ASEAN Mutual Legal Assistance setiap negara negara di ASEAN yang telah menyetujui perjanjian tersebut harus bersedia untuk memberikan informasi untuk menyelidiki kasus kejahatan korupsi yang diperlukan negara ASEAN lainnya untuk penegakan hukum (Setiawan, 2016). Mutual Legal Assistance digunakan sebagai tahap awal dalam menegakkan hukum terutama terkait dengan pengembalian aset negara hasil Tindak Pidana Korupsi. Penerapan perjanjian MLA adalah salah satu cara untuk menyengsarakan koruptor. Maka dari itu sangat penting untuk memiliki Perjanjian MLA dengan Negara-negara yang acap kali menjadi sarang bagi para koruptor menyimpan aset korupsi mereka. MLA ASEAN bisa menjadi suatu kerjasama multilateral, dalam mencegah dan memberantas kejahatan lintas batas negara yang terorganisasi, termasuk perkara tindak pidana korupsi. Selain itu, MLA bisa dijadikan acuan 35 | J u r n a l A n t i K o r u p s i 35 dalam menuntaskan masalah hukum dan diplomatik yang umumnya masuk beriringan dengan dilaksanakannya pencegahan dan pemberantasan korupsi sebagai transnasional crime yang terorganisasi. dalam menuntaskan masalah hukum dan diplomatik yang umumnya masuk beriringan dengan dilaksanakannya pencegahan dan pemberantasan korupsi sebagai transnasional crime yang terorganisasi. Contoh penerapan Mutual Legal Assistance di kawasan ASEAN adalah penangkapan koruptor Indonesia yang kabur ke Singapura. Dalam Pasal 3 ayat (1) Undang Undang Nomor 1 Tahun 2006 yang menyatakan: Mutyual Legal Assistance di perkara pidana, yang kemudian disebut Bantuan, adalah permintaan Bantuan berkaitan dengan penyidikan, penuntutan, dan pemeriksaan di sidang pengadilan sesuai dengan ketentuan peraturan perundang-undangan Negara Diminta. Dalam pasal diatas menyatakan jika dua negara bisa melaksanakan treaty dengan tujuan untuk bergantian memberikan informasi dan saling membantu dalam usaha mempertahankan hukum pidana. Selain Undang Undang Nomor 1 Tahun 2006, Mutual Legal Assistance juga dijelaskan dalam Undang-Undang Tindak Pidana Pencucian Uang, yang intinya MLA bisa dilakukan antar dua negara atau lebih. MLA bilateral ini dilandaskan pada MLAT atau atas resiprositas dua negara. *skor Brunei Darussalam tidak tersedia Indonesia telah memiliki kerjasama Mutual Legal Assistance multilateral di kawasan Asia Tenggara lewat MLA in Criminal Matters yang telah ditandatangani hampir semua anggota ASEAN, termasuk Indonesia. MLA ini telah disesuaikan dengan peraturan yang berlaku di Indonesia yakni dengan Undang Undang Nomor 15 Tahun 2008. Jadi penangkapan koruptor Indonesia yang kabur ke Singapura, pihak Indonesia yakni KPK bisa meminta bantuan dari negara Singapura untuk mengenali ciri seseorang yang masuk dalam list koruptor lari atau menjadi warga negara Singapura dan memberikan informasi terkait lainnya untuk membantu penangkapan koruptor IV. TANTANGAN YANG DIHADAPI DALAM IMPLEMENTASI MUTUAL LEGAL ASSISTANCE DALAM PEMBERANTASAN TINDAK PIDANA KORUPSI DI NEGARA-NEGARA ASEAN Kejahatan Korupsi acap kali terjadi sulit sekali untuk dikendalikan. Perkembangan teknologi melahirkan pola-pola baru bagi pelaku korupsi untuk menciptakan terobosan baru dalam menggencarkan aksinya. Salah satu yang menjadi tantangan dalam polemik pemberantasan korupsi ialah semakin mutakhirnya suatu teknologi terkait pengembalian aset (Asset Recovery) harta kekayaan yang disembunyikan atau disamarkan melalui pencucian uang (Money Laundering). Munculnya Money Laundering ialah bukti bahwa kejahatan Korupsi mampu melahirkan tindak pidana baru yang cukup sistematis. Money Laundering bertujuan untuk mengelabui Aparat Penegak Hukum sehingga dana hasil korupsi yang tidak sah tersebut dapat dinikmati melalui pengalihan harta kepemilikan sebenarnya. Dana korupsi tersebut ditempatkan bank tertentu (placement), melakukan transfer antar rekening yang berbeda (layering), menempatkan atau membelanjakan hasil korupsi tersebut pada barang-barang tertentu (integration) dengan tujuan untuk mengelabui hasil-hasil/harta kekayaan yang diperoleh dari kejahatan korupsi (Nasution, 2008). Luasnya wilayah di berbagai negara-negara 6 | J u r n a l A n t i K o r u p s i 36 Kawasan ASEAN dalam memberantas kejahatan korupsi transnasional terorganisir cukup mustahil dilakukan sendiri sehingga Treaty on Mutual Legal Assistance in Criminal Matters (MLAT) atau yang biasa disebut sebagai Perjanjian tentang Bantuan Timbal Balik dalam Masalah Pidana merupakan salah satu instrumen yang sangat diperlukan dalam memberantas kejahatan korupsi lintas batas negara dalam lingkup ASEAN. MLAT ASEAN ialah bentuk dari kerjasama regional negara-negara di lingkup asia tenggara. MLAT ASEAN diyakini sebagai suatu instrumen hukum yang efektif apabila dibandingkan dengan ekstradisi, tetapi bukan berarti tidak ada tantangan maupun hambatan dalam penerapannya. Walaupun telah dilaksanakan kerja sama, akan tetapi masih terdapat hambatan dalam pengembalian aset yang menyebabkan tidak optimalnya perjanjian MLAT ASEAN A. Perbedaan Sistem Hukum Masing-Masing Negara yang Berbeda ASEAN Treaty on Mutual Legal Assistance (MLAT) ialah perjanjian regional dengan melibatkan negara-negara regional kawasan ASEAN. Dalam penerapannya, negara-negara yang terlibat dalam regional ASEAN telah menyetujui MLAT dan melakukan ratifikasi untuk diimplementasikan dalam hukum positif masing-masing negara. Terciptanya suatu perjanjian berarti melahirkan suatu kewajiban bagi subjek hukum yang terlibat di dalamnya. Suatu perjanjian, membutuhkan klausul yang rinci dalam penyusunannya terlebih lagi setiap negara memiliki latar belakang yang berbeda. Setiap detail dari penyusunan perjanjian merupakan hal yang esensial dalam terciptanya MLA yang optimal. Namun, perbedaan sistem hukum dan mekanisme negara-negara di kawasan ASEAN masih menjadi salah satu kategori penghambat penerapan MLAT. Penghambat penerapan MLAT yaitu, beberapa negara menganut sistem hukum yang berbeda baik common law maupun civil law. Selain itu, perbedaan sistem peradilan negara-negara kawasan regional ASEAN. IV. TANTANGAN YANG DIHADAPI DALAM IMPLEMENTASI MUTUAL LEGAL ASSISTANCE DALAM PEMBERANTASAN TINDAK PIDANA KORUPSI DI NEGARA-NEGARA ASEAN Perbedaan tersebut meliputi crime control model (CCM) dan due process model (DPM). Kedua sistem tersebut memiliki perbedaan yang mana CCM memprioritaskan efisiensi waktu dengan menerapkan asas praduga bersalah. Sedangkan, DPM lebih menitikberatkan pada Hak Asasi Manusia (HAM) untuk tersangka. Oleh karena itu, dalam menangani kejahatan kerapkali berbelit-belit dan memakan waktu yang lama lantaran setiap negara yang mengajukan bantuan menghadapi hambatan dikarenakan negara yang diminta bantuan menangani perkara tersebut ingin menggunakan sistem hukumnya masing- masing. Penghambat selanjutnya, terkait terminologi kejahatan setiap negara berbeda. Setiap negara memiliki sudut pandang yang berbeda dalam memberikan suatu interpretasi atas tindak pidana. Apabila meninjau pada article 3 MLAT yang pada intinya memuat asas kejahatan ganda (double criminality) mengenai pembatasan dalam pemberian bantuan MLA. Asas kejahatan ganda berkaitan dengan interpretasi kejahatan di setiap negara yang berbeda-beda. Gambaran dalam asas ini dalam permohonan Bantuan Timbal Balik yakni, suatu kejahatan di negara peminta bantuan (Requesting State) dapat dianggap telah memenuhi suatu kualifikasi kejahatan yang kemudian dianggap sebagai kejahatan sedangkan di negara yang diminta bantuan 37 | J u r n a l A n t i K o r u p s i 37 (Requested State) hal tertentu itu tidaklah tergolong dalam suatu kejahatan. Penafsiran atau pengelompokan kejahatan yang berbeda tersebut, memungkinkan bagi suatu negara yang dimintai bantuan untuk tidak mengabulkan permohonan bantuan tersebut. Contohnya, permohonan bantuan Indonesia kepada Singapura. Singapura yang kerap sekali menolak permohonan bantuan Indonesia lantaran menerapkan asas kejahatan ganda sebab bagi Singapura, suap tidak tergolong atas perbuatan korupsi. Kemudian, permasalahan selanjutnya yang dihadapi yaitu beberapa negara masih belum memiliki instrumen hukum untuk melakukan perjanjian MLA. Hal ini kemudian diatasi UNODC melalui dibentuknya Legislative Guide sebagai tuntunan untuk melaksanakan perjanjian timbal balik terkait kejahatan Korupsi. B. Pengaruh Politik dan Ekonomi Bantuan Hukum Timbal Balik adalah salah satu instrumen hukum yang efektif karena mempermudah negara yang mengajukan permohon bantuan untuk memberantas kejahatan Korupsi. Namun, dalam realitanya, kerap kali dijumpai permasalahan bahwa MLAT tidak diimplementasikan dikarenakan adanya pengaruh baik dalam aspek politik, ekonomi serta yang lainnya. Selain perbedaan sistem hukum yang dibahas pada poin A, penolakan MLA juga diliputi oleh pengaruh politik yang terdapat dalam UNCAC 2003 dan AMLAT, apabila permohonan bantuan tersebut berhubungan dengan tindak pidana politik, maka pihak yang diminta bantuan tersebut dapat menolak permintaan tersebut. Oleh karena itu, penolakan yang dilakukan oleh negara diminta dapat dipahami dikarenakan disetujui atau tidaknya suatu permintaan bantuan tergantung pada negara yang dimintai pertolongan. Selanjutnya, terdapat permasalahan terkait pengembalian aset yang diperoleh melalui korupsi sebab dalam bantuan timbal balik, untuk melakukan penelusuran aset pada negara yang dimohon bantuan cukup memakan biaya yang besar sehingga hal ini memicu dua pilihan yaitu membagi hasil dana korupsi tersebut kepada negara yang diminta permohonan bantuan atau dikembalikan kepada asal negara pelaku korupsi. C. Penerapan Asas Retroaktif Dalam ASEAN Treaty on Mutual Legal Assistance (MLAT) C. Penerapan Asas Retroaktif Dalam ASEAN Treaty on Mutual Legal Assistance (MLAT) Hambatan selanjutnya yakni ASEAN MLA yang menganut asas non retroaktif. Merujuk pada Pasal 22 ayat 3 ASEAN Mutual Legal Assistance Treaty, permintaan bantuan hanya berlaku setelah dibentuknya perjanjian MLA yang maka dari itu perintah penyitaan dan putusan pengadilan sebelum itu, tidak dapat berlaku apabila perjanjian MLA masih belum terbentuk. Apabila disandingkan dengan perjanjian MLA yang dibentuk oleh negara Indonesia dengan Konfederasi Swiss yang menganut prinsip retroaktif maka tentunya hal ini sangat bertolak belakang. Pentingnya asas retroaktif ialah supaya kejahatan yang terjadi sebelum dibentuknya perjanjian MLA dapat ditangani mengenang bahwa kejahatan korupsi tergolong dalam kejahatan luar biasa (Extraordinary Crime) yang mampu merusak seluruh aspek stabilitas suatu negara. Selain itu, kejahatan korupsi mampu memantik munculnya tindak pidana baru yaitu pencucian uang yang saat ini dibahas. Maka, diperlukannya formula penyelesaian mencakup substansi hukum tegas dan kuat dalam memberantas situasi genting ini mengenang kejahatan 8 | J u r n a l A n t i K o r u p s i 38 korupsi memiliki dampak yang cukup besar dalam berbagai aspek-aspek kehidupan negara. Prinsip non retroaktif yang dianut oleh MLA ASEAN memperlihatkan bahwa terdapat kurangnya upaya yang serius untuk mewujudkan hasil timbal balik yang bijak dalam memberantas kejahatan korupsi dalam ruang lingkup ASEAN. Dimuatnya prinsip ini merupakan bentuk hambatan untuk tercapainya penerapan MLA yang efektif karena hal tersebut justru tidak mencerminkan esensi dari kerja sama itu tersendiri. Hukum dan keadilan merupakan dua hal yang tidak bisa dipisahkan. Implementasi prinsip non retroaktif memberikan pelaku korupsi celah yang berkeberlanjutan untuk mengelabui Aparat Penegak Hukum dalam menyamarkan dana hasil korupsi. Padahal, dalam memberantas tindak pidana korupsi Asas retroaktif memiliki peran yang penting karena keberadaan asas ini mampu memberikan justifikasi dalam keadaan darurat mengenang keberlakuannya ialah temporer sehingga pelaku korupsi mampu bertanggung jawab atas kejahatan yang dilakukan baik sebelum diberlakukannya perjanjian MLA maupun sesudahnya. Negara memiliki tanggung jawab untuk mencegah dan menangani seluruh dampak yang diakibatkan oleh kejahatan korupsi. Dengan demikian, keadilan akan berhasil dicapai dan dirasakan oleh seluruh lapisan masyarakat sebab kepastian hukum tidak ada artinya apabila masyarakat tidak bisa merasakan makna keadilan. V. TINJAUAN KEDEPAN DALAM PENGEMBANGAN MUTUAL LEGAL ASSISTANCE UNTUK MENDUKUNG PEMBERANTASAN KORUPSI Pengembangan implementasi MLA sangatlah penting untuk memfasilitasi kerja sama internasional terlebih pemberantasan kasus korupsi seperti yang sudah dibahas. Akan tetapi, di berbagai negara masalahnya bukanlah terletak pada kurangnya perjanjian MLA melainkan kurangnya proses MLA. Negara dalam sebagian besar kasus, MLA sudah ada tetapi tidak memadai sudah ada namun tidak memadai dalam pengoperasiannya. Maka dari itu, pembahasan selanjutnya membahas tentang bagaimana seharusnya pengembangan MLA di masa depan guna memberantas kasus korupsi khususnya di negara-negara ASEAN. A. Mutual Legal Assistance Berbasis Elektronik Penerapan MLA berbasis elektronik, dirancang agar implementasi dan proses MLA boleh diakses dengan mudah sehingga memberikan kemudahan dalam proses penerapannya oleh seluruh negara ASEAN. Berikut beberapa metode bagian dari MLA berbasis elektronik. 1. Teknologi mutual legal assistance yang lebih baik 1. Teknologi mutual legal assistance yang lebih baik Proses MLA yang ada saat ini berjalan lambat sebagian karena sangat sedikit proses yang distandarisasi sehingga memungkinkan sertifikasi, transmisi, penerimaan, dan pemrosesan secara digital, dan pemrosesan (Anwar, 2019). Sebagai contoh, permintaan dari agen penegak hukum di satu negara mungkin dikirim ke kedutaan negara tersebut di negara lain negara lain- dengan kantong diplomatik atau komunikasi yang aman-di mana permintaan tersebut dapat dikirim ke diplomat lokal yang dapat mengubahnya menjadi instrumen hukum domestik 39 | J u r n a l A n t i K o r u p s i 39 hukum domestik yang dapat mengubahnya menjadi instrumen untuk memaksa data. Proses ini membutuhkan banyak lompatan: dari penegak hukum lokal di Negara A ke pemerintah pusat Negara A ke kantor asing dari Negara A ke kantor luar negeri dari Negara B ke pusat pemerintah pusat Negara B ke penegak hukum lokal dari Negara B. Pergerakan permintaan awal dan tanggapan harus dilakukan secara elektronik (seringkali tidak) dan permintaan seharusnya tidak perlu dievaluasi secara sedikit demi sedikit (seperti yang sering terjadi). Proses yang lebih baik adalah proses elektronik di setiap langkah setiap langkahnya. Sebagai langkah awal, dua negara dapat menjalankan proyek percontohan untuk menguji bagaimana pemrosesan MLA berbasis digital, pemrosesan MLA berbasis formulir dapat bekerja. Bayangkan, misalnya, bahwa Inggris menciptakan sebuah portal online untuk permintaan MLA. Portal ini dapat menentukan bahwa jika permohonan diajukan secara akurat, lengkap, lengkap dan bersertifikasi, permintaan MLA elektronik akan memiliki prioritas di atas permintaan yang tidak lengkap dan/atau permintaan dalam bentuk kertas dalam dalam rangka memberikan insentif kepada negara pemohon untuk menggunakan menggunakan formulir elektronik. Untuk lebih jelasnya, pemrosesan MLA elektronik bukanlah obat mujarab: tidak menggantikan pekerjaan yang pekerjaan yang memakan waktu dengan meminta pengacara untuk meninjau kecukupan permintaan MLA. Namun, sebuah portal permintaan yang terpusat dan portal permintaan elektronik tentu akan meringankan beberapa ketegangan pada rezim saat ini dan mengurangi waktu dan upaya yang diperlukan untuk memastikan bahwa permintaan tersebut berisi semua bukti yang diperlukan (Nababan, 2010). Selain membuat permintaan MLA menjadi elektronik, penyediaan bukti digital yang sesuai dengan MLAT harus sepenuhnya elektronik. A. Mutual Legal Assistance Berbasis Elektronik Artinya, perusahaan harus dapat memberikan bukti elektronik dengan aman bukti elektronik ke negara yang memiliki memaksa mereka untuk memproduksinya, dan bukti tersebut harus diserahkan kembali ke negara yang meminta secara elektronik, bukan dalam bentuk kertas atau disk. Hal ini akan mengharuskan negara- negara untuk membangun sarana yang aman untuk mengirimkan data tersebut menggunakan saluran diplomatik yang sudah ada-tetapi juga akan membutuhkan investasi dalam pelatihan dan sumber daya untuk memastikan bahwa data disediakan dengan cara yang memenuhi persyaratan hukum kedua negara. 2. Transparansi yang lebih besar MLA harus mewajibkan setiap penandatangan untuk mengidentifikasi satu titik kontak pusat untuk mengelola permintaan MLA, dan harus permintaan MLA, dan harus mengidentifikasi kantor-kantor pemerintah yang bertanggung jawab untuk mengelola MLA. Hal ini harus mencakup ketentuan untuk melaporkan secara teratur tentang sifat, jumlah, dan lokasi permintaan yang diterima dan diberikan. Pemerintah harus setuju untuk menerbitkan laporan transparansi yang menguraikan rincian agregat dari data apa yang diminta, oleh siapa, dan untuk tujuan apa yang diminta, oleh siapa, dan untuk tujuan apa. B. Ketentuan Perjanjian Baru Mutual Legal Assistance Model Perjanjian PBB tentang Bantuan Timbal Balik dalam masalah Pidana adalah tempat awal yang baik untuk memasukkan prinsip-prinsip MLA modern ke dalam hukum internasional. Perjanjian ini mengatur elemen-elemen tradisional yang ada dalam perjanjian MLA: para pihak, ruang lingkup, alasan penolakan, konten yang diminta, eksekusi, pembatasan penggunaan, kerahasiaan, dan sebagainya. Namun, perjanjian model PBB harus diperbarui untuk mencerminkan penyebaran global komunikasi elektronik dan dampak yang ditimbulkannya terhadap penegakan hukum di abad kedua puluh satu. Secara khusus, MLA yang ada saat ini yang ada harus direvisi untuk mengakomodasi ruang lingkup yang lebih luas dan lebih luas dan lebih jelas; transparansi yang lebih besar; peningkatan efisiensi; dan lebih banyak perlindungan hak asasi manusia. 2. Sistem pelacakan (tracking system) Negara juga harus membuat sistem pelacakan internal untuk mengelola permintaan MLA. Hal ini akan memberikan penegak hukum yang meminta untuk mengetahui sejauh mana permintaan mereka sedang dalam proses. Sistem ini tidak harus online: sebagai gantinya, sistem dapat dengan mudah merutekan aman kembali ke penegak hukum yang meminta bahwa permintaan mereka telah diproses dan diteruskan, atau ditolak (Nababan, 2010). Tentu saja, sistem harus aman. Jika sistem seperti itu berhasil, maka secara signifikan dapat menghilangkan perasaan yang dimiliki oleh banyak agen penegak hukum bahwa permintaan mereka hanya memasukkan kotak hitam. 0 | J u r n a l A n t i K o r u p s i 40 B. Ketentuan Perjanjian Baru Mutual Legal Assistance 1. Ruang lingkup yang Jelas Sebagian besar MLA yang ada saat ini dirancang untuk mencakup data telekomunikasi tradisional. Untuk menghilangkan keraguan tentang ruang lingkup dan penerapannya untuk layanan Internet berbasis cloud modern, MLA harus menyertakan bahasa yang dengan jelas menunjukkan cakupan komunikasi saat ini dan masa depan tidak hanya pesan suara dan teks, tetapi juga komunikasi mesin-ke-mesin, lokasi data, layanan cloud, dan lainnya (Sitorus, 2015). Ini harus mencakup bahasa untuk memasukkan standar komunikasi internasional yang ada. komunikasi internasional yang sudah ada, sehingga memberikan ruang bagi standar tersebut untuk berkembang tanpa perlu memperbarui perjanjian tersebut. 2. Transparansi yang lebih besar 3. Peningkatan efisiensi MLA harus mengidentifikasi jadwal yang jelas untuk yang jelas untuk menanggapi permintaan data atau, paling tidak, tolok ukur untuk memproses permintaan MLA. Sebagai contoh, negara-negara dapat menguraikan bahwa 50% dari permintaan MLA harus ditangani dalam waktu dua minggu, dan 95% permintaan MLA harus ditangani dalam waktu 30 hari. Akan ada kasus-kasus luar biasa yang membutuhkan waktu lebih yang lebih lama, namun sebagian besar permintaan MLA harus diproses dalam waktu kurang dari satu bulan. Setiap penandatangan harus setuju untuk menunjuk satu titik kontak pusat, seseorang yang bertanggung jawab kepada negara peminta negara yang meminta untuk pembaruan dan untuk memenuhi | J u r n a l A n t i K o r u p s i 41 | 41 kepatuhan tenggat waktu. Hal ini idealnya juga mencakup ketentuan untuk sistem yang aman untuk melacak kemajuan permintaan MLA. kepatuhan tenggat waktu. Hal ini idealnya juga mencakup ketentuan untuk sistem yang aman untuk melacak kemajuan permintaan MLA. C. Perjanjian eksekutif Ada hambatan serius untuk mereformasi perjanjian bilateral, sering kali termasuk kebutuhan untuk ratifikasi domestik oleh badan legislatif. Tetapi negara-negara dapat membuat perjanjian eksekutif atau surat-surat pertukaran seringkali tanpa persetujuan legislatif untuk menguraikan prinsip-prinsip ini dan prinsip-prinsip lainnya, baik sebagai tambahan untuk MLA yang sudah ada sebelumnya MLA yang sudah ada atau sebagai penampung untuk memandu penegakan hukum sementara perjanjian yang lengkap dinegosiasikan. Sementara eksekutif tidak memerlukan persetujuan legislatif, namun dianggap mengikat di bawah hukum internasional sehingga mereka menawarkan cara yang menarik bagi negara untuk berkomitmen memodernisasi prinsip-prinsip MLA bahkan atau membuat yang baru tanpa harus merundingkan dan meratifikasi perjanjian baru. 4. Perlindungan hak asasi manusia MLA harus mencakup ketentuan eksplisit untuk perlindungan hak asasi manusia perlindungan hak asasi manusia. Bahkan ketika dua negara sepakat setuju untuk berbagi informasi mengenai investigasi terhadap kegiatan yang jelas-jelas merupakan tindak pidana di kedua negara, MLAT mereka harus mencakup kewajiban afirmatif untuk tidak berbagi data yang dapat menyebabkan pelanggaran hak asasi manusia pelanggaran hak asasi manusia (Christopher, 2017). Secara khusus, perjanjian-perjanjian ini harus memungkinkan negara untuk menolak permintaan MLA jika mereka mencurigai bahwa memberikan data akan mengarah pada pelanggaran hak asasi manusia di bawah Kovenan Internasional untuk Hak Sipil dan Politik. Perjanjian itu, yang yang memiliki 74 penandatangan dan 168 pihak, menjamin hak fundamental atas privasi (Pasal 17) dan hak kebebasan berekspresi (Pasal 19), antara lain. VI. KESIMPULAN ASEAN MLA adalah hubungan timbal balik yang dilakukan oleh negara negara di kawasan Asia Tenggara untuk saling bertukar informasi terkait transnational crime. Salah satu contoh dari transnational crime adalah korupsi. ASEAN MLA memiliki mekanisme tersendiri yang ditentukan oleh negara ASEAN yang tergabung dan menandatangani MLA. Jadi dengan adanya ASEAN MLA setiap negara negara di ASEAN yang telah menyetujui perjanjian tersebut harus bersedia untuk memberikan informasi untuk menyelidiki kasus kejahatan korupsi yang dibutuhkan oleh negara-negara ASEAN yang lain untuk penegakan hukum. Meskipun MLA dikenal sebagai instrumen hukum yang solutif dan efektif dalam penerapannya. Namun, masih terdapat banyak hambatan yang menjadi tantangan dalam penerapannya. Yang pertama, perbedaan sistem hukum berbagai negara di kawasan ASEAN sehingga kerjasama multilateral tidak cukup dalam menangani penyaluran dan pengembalian 42 | J u r n a l A n t i K o r u p s i 42 aset (Asset Recovery). Kedua, diterapkannya prinsip non retroaktif yang cukup kontradiktif dan bukan pemecahan masalah yang solutif. Kejahatan Korupsi sudah tergolong dalam kejahatan luar biasa (Extraordinary Crimes) yang mampu membahayakan stabilitas negara. Maka dari itu, seharusnya prinsip Retroaktif perlu diterapkan dalam keadaan genting seperti ini. aset (Asset Recovery). Kedua, diterapkannya prinsip non retroaktif yang cukup kontradiktif dan bukan pemecahan masalah yang solutif. Kejahatan Korupsi sudah tergolong dalam kejahatan luar biasa (Extraordinary Crimes) yang mampu membahayakan stabilitas negara. Maka dari itu, seharusnya prinsip Retroaktif perlu diterapkan dalam keadaan genting seperti ini. Perkembangan MLA di masa depan dalam memberantas kasus korupsi khususnya di negara- negara ASEAN dapat diterapkan dalam MLA berbasis elektronik, yang dirancang agar implementasi dan proses MLA boleh diakses dengan mudah sehingga memberikan kemudahan dalam proses penerapannya oleh seluruh negara ASEAN. 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MLA Lebih Efektif Mencegah Kejahatan Transnasional. https://www.hukumonline.com/berita/a/mla-lebih-efektif-mencegah-kejahatan- transnasional-hol18919?page=1 45 | J u r n a l A n t i K o r u p s i Kementerian Hukum Dan Hak Asasi Manusia. (2019, April 24). Peran Penting MLA dalam Penegakan Hukum Internasional. https://www.kemenkumham.go.id/berita-utama/peran-penting-mla- dalam-penegakan-hukum-internasional Kementerian Hukum Dan Hak Asasi Manusia, (2019, April 25). Tekan Kejahatan Trans-Nasional lewat Dokumen Perjanjian Resmi ASEAN. https://www.kemenkumham.go.id/berita- utama/tekan-kejahatan-trans-nasional-lewat-dokumen-perjanjian-resmi-asean Kementerian Koordinator Bidang Politik, Hukum dan Keamanan Republik Indonesia. (2019, April 25). Menkopolhukam Ajak Negara ASEAN Tingkatkan Kerjasama MLA dalam Masalah Pidana. https://portal.ahu.go.id/id/detail/75-berita-lainnya/2234-menkopolhukam-ajak-negara-asean- tingkatkan-kerjasama-mla-dalam-masalah-pidana Kementerian Lur Negeri Republik Indonesia. (2021, September 15). Perjanjian tentang bantuan hukum timbal balik antara Indonesia dan Swiss resmi berlaku. https://kemlu.go.id/portal/id/read/2922/berita/perjanjian-tentang-bantuan-hukum-timbal- balik-antara-indonesia-dan-swiss-resmi-berlaku Pusat Pelaporan dan Analisis Transaksi Keuangan. (2019, November 13). Tantangan Pemberantasan Korupsi Masa Kini. https://www.ppatk.go.id/siaran_pers/read/1007/tantangan-pemberantasan- korupsi-masa-kini.html Rachmadsyah, S. (2010, September 17). Asas non-retroaktif. https://www.hukumonline.com/klinik/a/asas-non-retroaktif-lt4c80ae57a77f0 RED. (2019, Februari 5). Menganut Prinsip Retroaktif, Perjanjian MLA Indonesia-Swiss Sah Ditandatangani. https://www.hukumonline.com/berita/a/menganut-prinsip-retroaktif-- perjanjian-mla-indonesia-swiss-sah-ditandatangani-lt5c591d5376666/ Rizki, M. J. (2022, Oktober 5). APH Didorong Manfaatkan MLA dalam Penanganan Korupsi dan Pencucian Uang. https://www.hukumonline.com/berita/a/aph-didorong-manfaatkan-mla-dalam- penanganan-korupsi-dan-pencucian-uang-lt633d3fb7755d9/?page=2 Rizki, M. J. (2022, September 15). Simak! Ini 5 Cara Terhindar dari Tindak Pidana Pencucian Uang. https://www.hukumonline.com/berita/a/simak-ini-5-cara-terhindar-dari-tindak-pidana- pencucian-uang-lt6322d81153c4a/?page=1 Rizki, M. J. (2022, September 15). Simak! Ini 5 Cara Terhindar dari Tindak Pidana Pencucian Uang. https://www.hukumonline.com/berita/a/simak-ini-5-cara-terhindar-dari-tindak-pidana- pencucian-uang-lt6322d81153c4a/?page=1 Rochman, F. (2019, November, 18). Dirjen AHU: berantas kejahatan transnasional perlu komitmen bersama. https://www.antaranews.com/berita/1167940/dirjen-ahu-berantas-kejahatan- transnasional-perlu-komitmen-bersama Rochman, F. (2019, November, 18). Dirjen AHU: berantas kejahatan transnasional perlu komitmen bersama. https://www.antaranews.com/berita/1167940/dirjen-ahu-berantas-kejahatan- transnasional-perlu-komitmen-bersama Peraturan Perundang-Undangan : Undang-Undang Nomor 8 Tahun 2010 Pasal 89 ayat (2) tentang Pencegahan dan Pemberantasan Tindak Pidana Pencucian Uang jo. Pasal 5 UU 1/2006 Undang-Undang Nomor 8 Tahun 2010 Pasal 89 ayat (2) tentang Pencegahan dan Pemberantasan Tindak Pidana Pencucian Uang jo. Pasal 5 UU 1/2006 Undang-Undang Nomor 20 Tahun 2001 Tentang Perubahan Atas Undang-Undang Nomor 31 Tahun 1999 Tentang Pemberantasan Tindak Pidana Korupsi, (2001). Undang-Undang Nomor 20 Tahun 2001 Tentang Perubahan Atas Undang-Undang Nomor 31 Tahun 1999 Tentang Pemberantasan Tindak Pidana Korupsi, (2001).

https://openalex.org/W4312920267

https://religiesamenleving.nl/article/download/13198/14751

Dutch

Tjaard Barnard (2006), Van ‘verstoten kind’ tot belijdende kerk. De Remonstrantse Broederschap tussen 1850 en 1940

Religie & samenleving

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BOEKBESPREKINGEN BOEKBESPREKINGEN 224 het Comeniusinstituut uit Duitsland werpt een ander licht op de Nederlandse situatie door een Europees perspectief in te nemen. Hij is het eens met Westerman: objectiviteit is ongewenst, en ook onmogelijk. Het gaat in onderwijs en in levensbeschouwelijk onderwijs om commitment, niet exclusief met een religie, daarover zijn alle auteurs het wel eens, maar om commitment met levensbeschouwing als bijdrage aan de opbouw van de samenleving van mensen Dit inzicht, en de daarover gevoerde discussie, die niet polariseert – alleen Cliteur is wat uitgesprokener in zijn opvatting van objectiviteit van onderwijs en onderzoek – maakt het boek belangrijk voor een bredere kring dan alleen godsdienstpedagogen. Leo van der Tuin (lector Praktische Theologie Fontys Hogeschool Theologie en Levensbe- schouwing, en universitair docent godsdienstpedagogiek Universiteit van Tilburg) Tjaard Barnard (2006), Van ‘verstoten kind’ tot belijdende kerk. De Remonstrantse Broederschap tussen 1850 en 1940, De Bataafsche Leeuw: Amsterdam, ISBN 90 6707 608 2, pp. 510, € 29,00. In dit boek, de handelseditie van een aan de Universiteit van Leiden verdedigd proefschrift, wordt de geschiedenis van de Remonstrantse Broederschap tussen 1850-1940 beschreven. Hierin staat de vraag centraal: hoe was het zelfbeeld van de Broederschap in die jaren. Wat voor kerk of geloofgemeenschap wilde ze zijn en hoe wilde ze tussen andere kerken staan? Het gaat dus in feite om de vraag naar de Remonstrantse ecclesiologie. Voor de Broeder- schap is onderzoek daarnaar niet zonder problemen: in het grootste gedeelte van haar bestaan kende zij geen gezaghebbende dogmatische geschriften en er was bij haar ook steeds de gedachte van voorlopigheid, van herstel met de Nederlandse Hervormde Kerk. De vraag naar de ecclesiologie van de Broederschap wordt in dit proefschrift dan ook via de indirecte weg beantwoord, door te kijken naar hoe zij handelde in de onderhavige periode en hoe zij sprak over zichzelf. Barnard verdeelt het tijdvak 1850-1940 in drie perioden. Voor elke periode poogt hij een antwoord te geven op de gestelde vragen. De eerste periode loopt van 1850-1880 en wordt door hem geduid als het tijdvak van ‘Het verstoten kind’. De Remonstrantse Broederschap beschouwt zich als een kerk in ballingschap. De broeders, verdreven uit de Nederlandse Hervormde Kerk vanwege de dogmatische, calvinistische opvattingen, hopen op een terugkeer naar een tolerantere moederkerk. Naar uitbreiding wordt ook niet gestreefd. Qua religieuze opvattingen staat men rond 1850 nog dicht bij de Gro- ninger richting, maar rond 1880 is het modernisme de hoofdstroming geworden. Religie & Samenleving, Jrg. 2, nr. 3 (december 2007) BOEKBESPREKINGEN De Broederschap beschouwt zich als een modern genootschap, met een bestuur. Doop, avondmaal en liturgie worden van weinig belang geacht. De keuze voor moderniteit uit zich ook in de verplaatsing van het Seminarie van Amsterdam naar het moderne Leiden. Het genootschap groeit door de instroom van vele malcontente Hervormden, Religie & Samenleving, Jrg. 2, nr. 3 (december 2007) BOEKBESPREKINGEN 225 die zich plaatselijk verenigen en door de Broederschap als nieuwe gemeenten worden erkend. Met name in de tweede periode 1880-1905 – de bloeitijd van het modernisme in de Broederschap – is er een grote toestroom van vrijzinnigen uit andere kerken. Ook een aanzienlijk aantal Nederlands Hervormde predikanten gaat over naar de Broederschap. Deze nieuwe gemeenten moeten hun plaats vinden binnen de oude en in het verband van de Broederschap geïntegreerd worden. Uitgelegd moet worden waar de Broederschap voor staat, en – mede – daarom wordt in 1889 het tijdschrift Uit de Remonstrantse Broederschap opgericht, waarin veel aandacht voor haar geschiedenis; uiteraard vanuit het modernisme geïnterpreteerd. In deze periode worden ook vele nieuwe kerkgebouwen gebouwd. Ook wordt nu aandacht gegeven aan broeders die verhuizen naar gebieden waar geen Remon- strantse gemeente is: gepoogd wordt hen bij de Broederschap te houden. De terugkeer naar de moederkerk wordt niet meer benadrukt: in feite is de Broederschap een concurrent geworden op de markt van de vrijzinnigheid, waar ook de Nederlandse Protestantenbond en de Vrije Gemeente (Amsterdam) opereren. De Broederschap positioneert zich tussen het dogmatisme en de belijdenisdwang van de Nederlandse Hervormde Kerk aan de ene kant en de (relatieve) onkerkelijkheid van NPB en Vrije Gemeente aan de andere kant. Zij beschouwt zich als het redelijke vrijzinnige alternatief: zij staat in de traditie der hervor- ming, maar laat ruimte aan haar leden om het geloof in vrijheid te beleven. De derde periode, van 1905-1940, wordt door Barnard aangeduid als de overgang van modernisme (dan ook oud-modernisme genoemd) naar belijdende kerk. De groei stag- neert, ook al om dat de vrijzinnigen in de Nederlandse Hervormde Kerk zich zelf binnen die kerk organiseren. Wel is er samenwerking met vrijzinnige groepen: er ontstaat een kleine vrijzinnige zuil, waar met name de VPRO een belangrijke onderdeel van uitmaakt. De Broederschap accepteert als tweede kerkgenootschap in Nederland de vrouw in het ambt, zij het na veel wikken en wegen. Religie & Samenleving, Jrg. 2, nr. 3 (december 2007) Religie & Samenleving, Jrg. 2, nr. 3 (december 2007) Lewis, J.T. (ed.) (2004), The Oxford Handbook of New Religious Movements, Oxford/New York: Oxford University Press, ISBN 0 19 514986 6, pp. 544, £52.00/$85.00. BOEKBESPREKINGEN Steeds sterker worden de stemmen die zeggen dat de Broederschap zich moet uitspreken over maatschappelijke problemen en dat wordt ook steeds vaker gedaan. Ook sluit zij zich aan bij de opkomende oecumenische beweging. De Broederschap weet zich deel van de onzichtbare kerk van Christus. Door haar beginselen van verdraagzaamheid en vrijheid voelt zij zich een voorbeeld voor de wereldkerk. De vraag naar het Christelijke gehalte van de Broederschap wordt weer met nadruk gesteld. Er komt belangstelling voor de liturgie; doop en avondmaal krijgen opnieuw een vaste plaats. De dienst wordt minder uitsluitend preekgericht. Men wil – weer – kerk zijn. In 1940 wordt een geloofsbelijdenis aan de gemeenten aangeboden – zonder die (uiteraard) op te leggen, want een ieder staat het vrij zijn eigen belijdenis te schrijven –, waaruit blijkt dat de Broederschap zich ziet als belijdende kerk. Dit omvangrijke proefschrift is een boeiend werk, dat met aandacht voor veel details, de lezer op de hoogte wil brengen van de geschiedenis van de Remonstrantse Broeder- schap in de onderhavige periode. Het kan dan ook als een Fundgrube worden beschouwd voor een ieder die zich er in wil verdiepen. Hiermee is ook een probleem dat ik met dit boek heb aangegeven. Duidelijk wordt bij lezing dat het de schrijver moeite heeft gekost om de grote hoeveelheid stof op overzichtelijke wijze te presenteren. Hij zoekt dan ook – Religie & Samenleving, Jrg. 2, nr. 3 (december 2007) 226 BOEKBESPREKINGEN en naar mijn smaak iets te vaak – zijn toevlucht tot het geven van concluderende para- grafen en samenvattende alinea’s. De hoeveelheid citaten had bovendien wel iets ingeperkt kunnen worden. Barnards onderzoek leidt tot de conclusie dat de geschiedenis van het zelfbeeld van de Broederschap geschetst kan worden als een overgang van ‘verstoten kind’, via ‘modern genootschap’ naar ‘belijdende kerk’. Merkwaardig genoeg gebruikt hij dit onderzoeksre- sultaat van het begin af aan ook al als periode-indeling, een wijze van presenteren die vanuit methodologisch oogpunt – voorzichtig uitgedrukt – niet zeer elegant is. Het ‘on- derzoeksresultaat’ in de slotbeschouwing verrast dan ook net zo min als het konijn in de hoge hoed van een goochelaar. Ondanks de genoemde bezwaren ben ik over het boek in zijn geheel positief. Het is, afgezien van de vele details en citaten, wel goed leesbaar en geeft een helder inzicht in hoe een vrijzinnig kerkgenootschap reflecteerde op en zich handhaafde in de zich moderni- serende maatschappij van 1850-1940. BOEKBESPREKINGEN Lammert G. Jansma Veenwouden Lammert G. Jansma Veenwouden Lewis, J.T. (ed.) (2004), The Oxford Handbook of New Religious Movements, Oxford/New York: Oxford University Press, ISBN 0 19 514986 6, pp. 544, £52.00/$85.00. Werd in 1970 nog geschreven dat het aantal actieve onderzoekers van nieuwe religieuze bewegingen (hierna NRBs) op de vingers van een hand kon worden geteld (Gordon Mel- ton), daarna is hun getal sterk toegenomen en is de studie van NRBs een erkend acade-misch specialisme geworden. Een verklaring voor deze spectaculaire groei is niet gemak-kelijk te geven. Het publieke debat over het ‘gevaar’ van de nieuwe bewegingen (the cult controversy) heeft er de aanzet toe gegeven; NRBs werden o.a. verdacht van uitbuiting van de leden en van het gebruik van hersenspoeltechnieken. Verder waren er de tragedies, zoals Jonestown en Waco, die de belangstelling ook hebben aangewakkerd. Het handboek wil een overzicht geven van het wetenschappelijke debat op genoemd terrein anno 2004 en ook wijzen op veelbelovende nieuwe velden van onderzoek. Ver- schillende specialisten, sociologen en godsdienstwetenschappers, komen aan het woord in 22 hoofdstukken, die onderverdeeld zijn in vier rubrieken. Het spreekt voor zich dat een behandeling van al die bijdragen voor een bespreking te ver zal voeren. Ik zal mij daarom op de kernhoofdstukken binnen die rubrieken richten en de andere meer in het voorbij-gaan noemen, om ten slotte mijn oordeel over deze bundel te geven. In het eerste deel, dat als thema heeft ‘modernisering en nieuwe religies’ verdedigt Christopher Partridge de stelling dat, hoewel secularisatie/Entzauberung invloed heeft gehad op de Westerse maatschappij, het niet zo is dat religie verdwijnt. Mensen reageren op nieuwe ontwikkelingen niet door ongodsdienstig te worden, maar door op de ruïnes van

https://openalex.org/W2560863928

https://link.springer.com/content/pdf/10.1007%2Fs11273-016-9524-9.pdf

English

Impacts of rewetting on peat, hydrology and water chemical composition over 15 years in two finished peat extraction areas in Sweden

Wetlands ecology and management

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Wetlands Ecol Manage (2017) 25:405–419 DOI 10.1007/s11273-016-9524-9 ORIGINAL PAPER Impacts of rewetting on peat, hydrology and water chemical composition over 15 years in two finished peat extraction areas in Sweden Lars Lundin . Torbjo¨rn Nilsson . Sabine Jordan . Elve Lode . Monika Stro¨mgren Received: 18 July 2016 / Accepted: 8 December 2016 / Published online: 23 December 2016  The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Restoration of wetlands is a high priority world-wide. Peat extraction areas can be restored by rewetting, however affecting the environment. It could be expected to turn the drained peat-cutover area from a source to a sink of most elements. This study examined effects of such rewetting on peat, hydrology and water chemistry over 15 years at two sites in Sweden; the nutrient-poor Porla peatland and the nutrient-rich Va¨stka¨rr peatland. Rewetting caused minor changes to peat chemistry, but at the Va¨stka¨rr site ammonium concentrations increased in superficial peat layers while nitrate decreased. In terms of hydrology, rewetting of the Porla site decreased annual runoff and both high and low discharges. Water pH at the Porla site stayed fairly stable, but at the Va¨stka¨rr site pH, after an initial 4 years dip, gradually increased to higher values than before rewetting. Water colour and organic matter content were fairly stable, but slightly lower values were found after 15 years than in initial 4–5 years. The concen- trations of base cations and of inorganic N were lower after rewetting, while total P was higher. However, these impacts could change from an initial phase as the wetlands in the long-term perspective develop into mires. wetlands in the long-term perspective develop into mires. Keywords After-use  Hydrochemistry  Peatland  Restoration  Wetland Keywords After-use  Hydrochemistry  Peatland  Restoration  Wetland L. Lundin (&)  T. Nilsson  S. Jordan  E. Lode  M. Stro¨mgren Department of Soil and Environment, Swedish University of Agricultural Sciences, P.O. Box 7014, 750 07 Uppsala, Sweden e-mail: [email protected] Introduction In total, four million km2 of the Earth’s surface (circa 3% of the land area) are covered with peatlands, and peatlands are found in almost every country of the world (Schumann and Joosten 2008). It is especially abundant in vast areas of North Europe and Canada. The peatlands and peat resource is considered a societal asset and is used in agriculture, forestry, horticulture and as an energy source. These uses have resulted in numerous remnants of damaged mires, for which restoration is a high concern. Peatland restora- tion aims to improve environmental service values for biodiversity objectives, ecosystem functions, carbon storage, flood mitigation, deposition purification, etc. (Rochefort et al. 2003; Vasander et al. 2003; Ram- chunder et al. 2012). Peat extraction has been carried out in many countries for at least 200 years. In Europe, peat losses have been considerable and in more than 50% of the original natural mire area peat organic material is no longer accumulating (Joosten and Clarke 2002). For a number of decades, it has been mandatory to find a suitable use for the remaining land 12 3 123 Wetlands Ecol Manage (2017) 25:405–419 406 after extraction ceases. In 1998, a new environmental law was established in Sweden, giving increased support for restoration. but the concentrations of dissolved organic matter (DOC) and protons in the leaching decrease, providing higher pH. Nitrogen (N) content changes from being dominated by the organic fraction (Norg, 80%) to inorganic nitrogen (Ninorg) release that can reach a level of 60% of total N (Ntot) (Lundin 1988). In the case of rewetting, a change from release to retention could be expected. There are multiple possibilities for wise after-use of former peat extraction areas (Joosten and Clarke 2002). On sites with low potential for forests or crop production, rehabilitation activities can seek to improve especially biodiversity values. Ecologically, wetland restoration has the main goal of bringing back disturbed terrestrial ecosystems and it aims to restore natural wetland hydrology (water level, flow paths and water chemistry) and to re-establish characteristic peatland plant cover (Vasander et al. 2003). For success in such operations, the starting conditions, i.e. the state of the peatland after peat extraction, should be considered, since it provides the main potential for successful end-results (Blankenburg and Tonnis 2004). Suitable indicators of wetland generation success include stable hydrol- ogy, appropriate water quality and vegetation devel- opment (Lode 2001). Introduction In modern industrial peat harvesting, the total peat resource is often extracted, with only thin remnants of peat left on top of mineral soil. This creates conditions strongly deviating from those in areas where a rather thick ([1 m) layer of peat still remains (Eggelsmann et al. 1993). There is probably some benefit in complete peat removal at one site, instead of affecting many sites by removing the top peat layer only. Such upper layer extraction keeps remnant peat conditions favourable and resembling the natural wetland prop- erties, facilitating vegetation restoration. In the case of total peat extraction, the new, or actually very old, bottom peat layers and mineral soil, which have long been largely preserved from surface water and atmo- sphere interface processes, fall under the influence of new environmental processes. This situation gives rise to new hydro-chemical and biological conditions (Wheeler 1995). p Rewetting of cut-over peat would be the reverse of drainage; so many findings in forest drainage research could apply to conclusions for rewetting turning the effects of drainage back to the pre-drainage situation (Lundin 1988). Drainage also has effects on the water balance, providing space for precipitation in tempo- rary surface water storage to be used in prolonged evapotranspiration and runoff. Such storage mitigates fast discharge responses. However, the storage capac- ity could be limited in situations when groundwater levels are high. In such circumstances, the ditches provide rapid channel flow capacity and possibly enhanced discharge peaks (Iritz et al. 1994). However, in general the effects of drainage on hydrology are lower peak flows, but somewhat higher annual runoff (Braekke 1970), accompanied by higher low discharge because the ditches promote release of water (Lundin 1988). In the case of rewetting, the reverse influence on discharge could be expected. This study primarily examined the impacts of peatland rewetting on properties of the peat, hydrology and water chemical conditions. The hypotheses tested were that rewetting former peat extraction areas: • Elevates water levels but decreases discharge. • Increases storage of most elements, however decreases pH and inorganic nitrogen (Ninorg) but increase DOC and phosphorus (P) concentrations in surface water. 123 Site description Not only hydrology is affected by peatland drainage, but also hydrochemistry. In the peatland formation process, when peat is accumulating and peat organic matter storage is increasing, there is seques- tration of most chemical elements in the organic material. Drainage alters this process to decomposi- tion when peat oxidation occurs and the stored elements are released (Sallantaus 1989). In drained conditions, higher outflow of most elements occurs, The long-term rewetting investigations were carried out at two peat extraction sites c. one year before rewetting and in the initial 14 years after wetland establishment. The two sites were the Porla and Va¨stka¨rr peatlands, both located in the south-west of central Sweden (59 010N; 14 380E for the Porla site and 59 060N; 14 450E for the Va¨stka¨rr site) (Fig. 1). 123 123 Wetlands Ecol Manage (2017) 25:405–419 407 Fig. 1 Map of Sweden showing the location of the two rewetted areas Porla (A1 and B, middle picture) with the poor fen south of the wetlands and Va¨stka¨rr (VK1–VK3, right picture) wetland in the north with a central ongoing peat cutover area and south of this another restored area and the reference (LA¨, left picture) to Porla. Colours represent: blue-open water, dark green-forest, light green and yellow-wetland plants, blueish- green-wet peat mixed with plants and brown-bare peat south of this another restored area and the reference (LA¨, left picture) to Porla. Colours represent: blue-open water, dark green-forest, light green and yellow-wetland plants, blueish- green-wet peat mixed with plants and brown-bare peat south of this another restored area and the reference (LA¨, left picture) to Porla. Colours represent: blue-open water, dark green-forest, light green and yellow-wetland plants, blueish- green-wet peat mixed with plants and brown-bare peat Fig. 1 Map of Sweden showing the location of the two rewetted areas Porla (A1 and B, middle picture) with the poor fen south of the wetlands and Va¨stka¨rr (VK1–VK3, right picture) wetland in the north with a central ongoing peat cutover area and The Porla peatland was originally a raised bog where peat extraction started in 1889, with the level of activity varying over time. In the two last decades of the 20th century, peat was extracted down to the till soil of the morainic landscape. Residual peat thickness in the area prior to rewetting varied considerably, from 0 to 2 m depth, because of the stony till mineral soil underlying the peat. Site description Only very small areas had mineral soil and thick 1-2 m peat cover was found on an estimated area of 20%. In the remaining area the thickness was mainly 0.2–1 m. Boulders and stones could be observed in many places and ordinary till hillocks and pits formed an uneven surface. South of the peat cut-over area there is a natural poor sedge fen (27 ha) that was excluded from the harvesting oper- ations, from where discharging nutrient-poor water enters the Porla rewetting area. In 1999, rewetting was initiated by closing the drainage outlet with a water level-regulating device in the outlet ditch passing out in a culvert. A road divides the total restoration area into two parts, Porla A1 (5 ha) to the north and Porla B (12 ha) to the south. During rewetting operations, Porla A1 was surrounded by bunds built to store water and a discharge weir was installed at the outlet. The total catchment area at the outlet is 133 ha (Fig. 1). The total extraction area was about 55 ha, representing 48% of the Porla peatland. The catchment area at the site can be easily distinguished based on the drainage ditch network. Tree coverage, mainly spruce and pine, covered 34 ha, i.e. 29% of the previous mire exten- sion. Due to varying peatland surface water condi- tions, the surface land cover was fragmented and varying in detail (Lode et al. 2012). Currently about 35% (19 ha) of the cut-over peatland has been rewetted, including areas rewetted by beaver inunda- tion. Due to swelled and floating residual peat, the bare 12 3 3 Wetlands Ecol Manage (2017) 25:405–419 408 peat surface in the total wetland area constituted 25% for Lake A1 and 49% for Lake B, with half of these surface areas covered by plants (Fig. 1). with on average a 3.7 m thick peat layer remaining. From 1940 to 1985 peat was hand-block-cut on 30 ha. The abandoned drainage system was partly blocked in 1989 and varying intensity of spontaneous re-coloni- sation occurred in inundated open pits (Lode 2001). In Porla the original peat thickness, estimated from the southern untouched low sedge fen, was 3.5–4.5 m. In the Va¨stka¨rr lagg area, the original peat thickness is uncertain but values at start of peat cutting on 2 m was mentioned. Site description The Va¨stka¨rr wetland is located between the Skagerhultamossen raised bog in the west and mineral soil grassland in the east. Prior to peat extraction, a 80 ha lagg area of the large bog was cultivated for potatoes and cereal crops. At the end of peat extrac- tion, there was rather even 0.1–0.2 m remaining peat thickness with comparatively high ash content over- lying a level surface formed by postglacial clay soils. Inflowing groundwater, partly of minerogenic origin, furnished most of the rewetting water. In the extrac- tion phase water was pumped out to keep the groundwater level low. In 1999, rewetting was initi- ated by stopping pumping water out of the area and a consecutive natural rise in the water level in the area gradually occurred, resulting in three shallow lakes formed as subbasins (VK 1, VK 2 and VK 3; Fig. 1). The catchment area for the Va¨stka¨rr site is not easily determined because of undefined groundwater inflow from the surroundings and inflow from the large Skagerhultamossen bog and the nearby river O¨ rebro Svarta˚. Climate conditions for the Porla and Va¨stka¨rr sites were quite similar, as being rather close by located with long-term (1960–1990) mean annual precipita- tion of 800 mm, evapotranspiration of 470 mm and temperature of ?5.7 C (Table 1) (Raab and Vedin 1995). Precipitation in the period before rewetting (1997–1999) was slightly higher (797 mm) than in the period after rewetting (2000–2013; mean 752 mm). In the period after rewetting precipitation was 9% higher than the long-term average and temperature was 0.7 C higher. 123 Field measurements and sampling In 1999, before the start of rewetting, both sites were prepared by soil and ground work and the surfaces mainly constituted bare, black, well-decom- posed fen peat alternating with patches of mineral soil (Fig. 1). In Va¨stka¨rr, the drains were filled in and the surface flattened. In Porla, bounds were built along the road in Porla B area and downslope, around half of the Porla A1 area. Field measurements of water depth and discharge and soil and water sampling were carried out in the period 1997–2013. Soil sampling was performed using corers, discharge determinations were made in 90 V-notch weirs with water level recorders at the notch and surface water sampling was carried out in wetlands and streams. Routine fortnightly field obser- vations of water depth and monthly water sampling at the Porla and LA¨ sites were carried out by a local In Va¨stka¨rr, vegetation colonisation took place during the first growing season after rewetting started, but in Porla the first plant colonisation took longer time, partly owing to problems with keeping the water on-site. A few spots of Eriophorum vaginatum and Politrichum commune could be seen close to old ditches already after one season, but not until 2004–2005 did wider colonisation occur. Table 1 Geographical, climate and hydrological (1961–1990) characteristics of the Porla and Va¨stka¨rr peatland restoration areas (Raab and Vedin 1995) Table 1 Geographical, climate and hydrological (1961–1990) characteristics of the Porla and Va¨stka¨rr peatland restoration areas (Raab and Vedin 1995) Table 1 Geographical, climate and hydrological (1961–1990) characteristics of the Porla and Va¨stka¨rr peatland restoration areas (Raab and Vedin 1995) Variable Porla Va¨stka¨rr Altitude, m.a.s.l. 85 65 Rewetted peatland area, ha 17 80 Annual mean temperature, C 5.7 5.7 Vegetation period (T [ 5 C), days 200 200 Annual precipitation, mm 800 800 Annual evapotranspiration, mm 470 470 Annual runoff, mm 330 330 Vegetation period is defined by daily average temperature exceeding 5 C for four consecutive days (Odin et al. 1983) At both sites, the soil hydrology and water chem- istry were studied, as well as vegetation development and greenhouse gas emissions from different ecotopes (Kozlov et al. 2016; Jordan et al. 2016). As an unchanged reference area for the hydrology and hydrochemistry measurements at Porla, a nearby earlier self-restored cut-over area, La¨sarmossen (LA¨ ) peatland was used. Field measurements and sampling The LA¨ peatland is located 2 km east of Porla wetland and was originally a 50 ha bog 123 Wetlands Ecol Manage (2017) 25:405–419 409 observer. Control visits for following instrument performance were made by the research team 2–6 times per year. Daily precipitation and temperature values were obtained from the Swedish Meteorolog- ical and Hydrological Institute. Chemical analysis The pH in soil samples was determined in deionized water suspension (2 g dry peat in 25 mL water). Peat total carbon (C), Ntot and total sulphur (S) were analysed by dry combustion according to ISO 13,878 (CN2000, Leco). Calcium (Ca) and magnesium (Mg) were extracted by NH4Ac at pH 7, while 2 M HCl was used to extract potassium (K) and P (KHCl and PHCl). After extraction, these elements were analysed by ICP-OES. Nitrate (NO3) and ammonium (NH4) were analysed in 100 g fresh samples extracted with 250 mL 2 M KCl and analysed with autoanalyzer. Continuous discharge measurements at Porla outlet started in summer 1998 and at LA¨ in 1996. For climate and technical reasons, discharge recording was inter- rupted for shorter or longer periods during 1997–2013. There are no full records for the period 2003–2006 for the Porla and LA¨ sites. Comparisons between the rewetted Porla and the reference LA¨ site were carried out using the control area and calibration period technique (Grip 1982) and was applicable for both hydrology and water chem- istry. The Porla rewet area was calibrated against the reference site for the period before rewetting and discharge for the following years could be calculated as non-rewetted. Measured discharge after rewetting was then compared against the calculated values without rewetting and the difference showed the change because of rewetting. The calibration period for discharge and water chemistry prior to rewetting was August 1998–August 1999. Water samples were analysed for pH, colour, electrical conductivity (EC), alkalinity, DOC, Na, K, Ca, Mg, manganese (Mn), iron (Fe), aluminium (Al), silicon (Si), chloride (Cl), sulphate (SO4), NO3-N, NH4-N, Norg, Ntot, PO4-P and Ptot according to methods at the SLU accredited laboratory (SLU 2016). For P the effect of water colour was eliminated by reduction for values in a blanc sample. For calculation of element runoff, daily discharge values and linear interpolated daily water chemical concen- trations were used. Effects of rewetting on transport of chemical compounds were calculated using the cali- bration period and control area technique. Field measurements and sampling The refer- ence LA¨ catchment values were correlated to the Porla catchment values for the period before rewetting. The relationship was then used together with the measured values at the reference for the rewetting period. These values were compared with values for the Porla catchment and the difference showed the effect of rewetting. At the Va¨stka¨rr site, 10 soil samples (0–10 cm depth) were taken before rewetting (1997) and after rewetting (in 2003 and 2007; 3–9 samples). The soil samples were taken using a steel auger (4 cm diam- eter) from the superficial peat layer (0–10 cm). In rewetted conditions, the lake peat bottom and a little new formed sediment were sampled from the bottom layers (0–10 cm). At site Porla similar sampling of lake bottoms sediment and peat was conducted, but at 0-20 depth. Prior to rewetting 5 samples were included and in 2003 there were 3 samples and in 2007, 8 samples. Sampling locations were distributed over the areas and resampling made in approximately the same locations. All these soil samples were placed in plastic bags, transported to the laboratory in Uppsala and kept refrigerated at 4 C until analyses. Before analysis, the samples were thawed, air-dried at 35 C and har- monised, grind through a 2 mm sieve. Nitrate (NO3) and ammonium (NH4) were analysed in fresh samples extracted with 2 M KCl. Results Peat soil properties In the Va¨stka¨rr area, the remaining peat layer after extraction was a fen sedge peat with thickness between 0 and 70 cm, but over large areas mainly 10–20 cm. Ash content was 10% higher than in former extracted layers. The remaining peat layer bulk density varied mainly between 0.2 g cm-3 and 0.3 g cm-3, overly- ing 5 cm gyttja with bulk density 0.4–1.0 g cm-3. Under this, postglacial clay occurred. At the Porla site after extraction, there was larger variation in peat thickness (0–2 m). In many places Carex/Equisetum and Equisetum/Carex peat 0.7–1 m thick was present. These layers were in some places overlain by Sphag- num peat with thickness of mainly 0.4–0.6 m. Bulk density decreased from 0.1 g cm-3 to 0.04 g cm-3 in the first years after rewetting, but returned to almost 0.1 g cm-3 after eight years. Peat physical and chemical conditions in Porla wetland changed after rewetting. The pH increased up to four years to 4.8 but decreased thereafter to 4.2 after 8 years. The concentrations of C and especially N increased to 1.6%, resulting in lower CN ratio on c. 30. Concentrations of extractable PHCl and KHCl in the upper part of the peat (0–20 cm) changed after rewetting, with P reaching higher values on 350 mg kg-1 while K decreased to 53 mg kg-1 after eight years. Amounts of both elements decreased over time and lower BD contributed to this. Prior to rewetting, the chemical composition of peat at the two sites differed, as it was more nutrient rich at site Va¨stka¨rr than at site Porla. Peat values in Va¨stka¨rr from 0 to 10 cm depth and Porla 0–20 cm gave the CN ratio, based on g g-1, on 21 and 47, respectively, with peat pH 5.0 and 4.4, respectively. However, the water chemistry showed larger differences, with pH 6–7 in water at the Va¨stka¨rr site and 5–5.5 at Porla site. Considerable differences in peat chemistry between the two sites were observed for inorganic N, P and K (Table 2). Statistical calculations All variables studied were not normally distributed, at least during some of the study period. Therefore, before studying the effects of rewetting, all data were log-transformed to meet normality and homoscedas- ticity assumptions. ANOVA tests were performed to evaluate differences in water chemistry between different periods. ANOVA tests were performed to evaluate differences in water chemistry between different periods. A threshold of 0.05 was always used for significance. SAS 9.4 was used for all statistical analyses. Water was sampled monthly at the Porla and LA¨ sites and 4–6 times per year at Va¨stka¨rr site. All samples were transported to SLU’s accredited water laboratory in Uppsala within 1–2 days. 123 Wetlands Ecol Manage (2017) 25:405–419 410 increased slightly, but this did not change the CN ratio. Exchangeable Ca and Mg concentrations increased after rewetting, while the concentrations of KHCl and PHCl decreased to 365 mg kg-1 and 565 mg kg-1, respectively. Before rewetting, the concentrations of NH4-N were negligible while NO3- N was present in higher concentrations (50 mg kg-1). After rewetting the picture was reversed and NH4-N became dominant on 115 mg kg-1 while NO3-N was almost depleted (0.7 mg kg-1). Hydrology In the humid climate of south-west Sweden with excess precipitation of 330 mm over evapotranspira- tion (Table 1), rewetting was a suitable option for restoration and the drained cut-over areas quickly received inflowing water, forming wetlands. The necessary water storage in the wetlands was estimated to make up 15% of the annual runoff. At both the Porla and Va¨stka¨rr sites, the open water reservoirs formed only a few months after rewetting, 6 and 2 months, respectively. In the Porla wetland, some initial At the nutrient-rich Va¨stka¨rr site, rewetting chan- ged the chemical characteristics in peat and lake sediment somewhat, but mainly only minor changes occurred. The pH and C and N concentrations Table 2 Chemical characteristics of the peat at the Va¨stka¨rr (depth 0–10 cm) and Porla (depth 0–20 cm) sites before rewetting CV coefficient of variation, n number of samples, dw dry weight Variable Porla CV (%) Va¨stka¨rr CV (%) n = 5 n = 10 pH 4.4 6 5.0 6 C (%) 50 4 43 12 N (%) 1.1 33 2.0 9 C/N 47 29 21 6 NO3-N, mg/kg dw 2 160 57 54 NH4-N, mg/kg dw 88 92 0 – KHCl, mg/kg dw 92 80 460 32 PHCl, mg/kg dw 320 50 650 19 12 Wetlands Ecol Manage (2017) 25:405–419 411 years 2001 and 2002 omitted because of the hydrotechnical problems at Porla site. For the period 2007–2013 the frequency of high and very high runoff values was lower than before rewetting in both catchments. However, the frequency of high flows (1–3 mm day-1) decreased more at Porla (180 days) than at the reference site (620 days), and the frequency of days (0.6% compared to 3.8%) with very high runoff ([3 mm day-1; 35 L s-1 km-2) decreased considerably more in the rewetted catchment. mishaps lowered the lake water level twice, in 2001 and 2002, to 0.1–0.2 m depth, but after that it stabilised at around 1 m depth in central parts. In the Va¨stka¨rr lakes, the water levels remained fairly stable from the beginning of rewetting, with depth between 0.5 and 1 m and occasional maximum depth up to 2 m. Filling up the water reservoirs three times at Porla resulted in short periods when the discharge was lower than in the reference LA¨ conditions, but discharge and water storage later stabilised. Hydrology Measured annual runoff from Porla wetland was lower than calculated annual runoff based on the reference LA¨ conditions. Lower annual runoff was observed for 2007–2009 and 2013, while the period 2010–2012 had runoff similar to the calculated value for non-rewetted conditions. For the period before rewetting, average runoff from the Porla catchment was 60% of the reference LA¨ value (403 mm) but after rewetting it only reached about 40% of the reference value (500 mm). This implies decreased average runoff after rewetting. Discharge (expressed in L s-1) was fairly similar for the Porla catchment and the reference before rewetting (1998/ 1999), but was lower at Porla after rewetting (Fig. 2). Before rewetting, the Porla catchment had a higher frequency (22%) of days with low daily runoff (\0.05 mm day-1) compared with the reference site (11%). In rewetting conditions (2007–2013) the frequency of days with low runoff increased to 42% at the rewetted site, while the reference catchment only had a frequency of 15%. The frequency of very low water flows (\0.01 mm day-1) or no flow at all after rewetting was also significantly higher for the rewetted area compared with the reference with 18 and 6%, respectively out of 3342 days after rewetting. Low discharge occurred in several years during summer. Effects of rewetting on high and low discharge were investigated on a daily basis. Before rewetting there was only one period (14 March–31 August 1999) with reliable daily runoff measurements for both the Porla and LA¨ catchments. During this period there were fewer days with high runoff at Porla than at the reference site. For the period after rewetting, with Va¨stka¨rr wetland Va¨stka¨rr wetland Water chemistry in the nutrient-rich Va¨stka¨rr wetland showed mainly lower ion contents after rewetting. The mainly before rewetting (water storage started in September) and 9 years after Porla rewetting 2008 (right) Fig. 2 Daily precipitation (upper bars) and discharge from the reference La¨sarmossen (LA¨ ) catchment (broken line) and the cut-over later rewetted Porla catchment (solid line) in 1999 (left) mainly before rewetting (water storage started in September) and 9 years after Porla rewetting 2008 (right) 1 3 Fig. 2 Daily precipitation (upper bars) and discharge from the reference La¨sarmossen (LA¨ ) catchment (broken line) and the cut-over later rewetted Porla catchment (solid line) in 1999 (left) mainly before rewetting (water storage started in September) and 9 years after Porla rewetting 2008 (right) 12 3 Wetlands Ecol Manage (2017) 25:405–419 412 pH increased but EC, base cations, Si and inorganic N decreased. The concentrations of Fe, Al, organic N and Ptot increased, while PO4-P decreased (Table 3). similar but decreased and increased, respectively, after rewetting, resulting in a Ninorg to Norg ratio of about 30% (Fig. 3). The Ninorg fraction of total N was c. 20%. Total N decreased somewhat after rewetting, but values were similar to those before rewetting (Table 3). In the Va¨stka¨rr cut-over peat area, mean pH before rewetting was slightly over 6. During the initial years after rewetting pH was on average 6.2, but then increased to significantly higher values than before rewetting and was on average 6.7 at 12–14 years after rewetting (Table 3). Before rewetting, mean annual DOC concentration varied between 37 and 48 mg L-1 and during the year that rewetting started (1999) it increased to 58 mg L-1. In the following years it then decreased to values that were usually significantly lower (34–38 mg L-1) than before rewetting (Table 3). Water phosphorus concentration before rewetting was 0.03 mg L-1 and almost all was present as PO4-P. In the initial 3 years after rewetting, PO4-P concen- tration remained almost unchanged, while Ptot increased about threefold (Fig. 4). In the years after rewetting, PO4-P concentration decreased to half the value before rewetting (Table 3). Total P also decreased in later years, but remained about 100% higher than before rewetting. The share of PO4-P to Ptot changed from 90% before rewetting to about 20% thereafter (Fig. 4). Va¨stka¨rr wetland Before rewetting of the Va¨stka¨rr wetland, the concentrations of inorganic and organic N were Table 3 Chemical composition (mean ± standard error) of water in the Va¨stka¨rr wetland before rewetting (1997–1998) and for four periods after rewetting Variable Units 1997–1998 1999–2001 2002–2006 2007–2010 2011–2013 n = 29 n = 35 n = 40 n = 31 n = 34 pH 6.06 ± 0.11 c 6.16 ± 0.04 c 6.66 ± 0.05 b 6.95 ± 0.06 a 6.68 ± 0.05 b EC lS cm-1 247 ± 26 a 76.5 ± 2.7 c 82.0 ± 3.2 c 89.8 ± 5.1 bc 99.2 ± 5.2 b Alkalinity mg L-1 27.2 ± 6.1 b 17.6 ± 1.7 a 19.6 ± 1.4 a 25.8 ± 3.5 a 25.6 ± 2.5 a SS mg L-1 17.4 ± 2.8 12.7 ± 2.3 9.51 ± 0.92 21.2 ± 2.9 21.5 ± 3.7 Colour mg Pt L-1 160 ± 16 c 258 ± 16 ab 175 ± 10 bc 200 ± 11 ab 187 ± 13 b DOC mg L-1 44.9 ± 3.7 ab 47.6 ± 2.2 a 33.7 ± 1.5 c 37.6 ± 1.4 bc 36.0 ± 1.3 c Ca mg L-1 41.1 ± 4.5 a 12.6 ± 0.5 cd 12.2 ± 0.6 d 15.6 ± 1.1 b 15.5 ± 1.3 bc Mg mg L-1 5.84 ± 0.65 a 1.81 ± 0.09 cd 1.70 ± 0.08 d 2.10 ± 0.12 bc 2.15 ± 0.15 b K mg L-1 2.95 ± 0.61 a 1.39 ± 0.12 b 1.41 ± 0.09 b 1.62 ± 0.10 b 1.60 ± 0.14 b Na mg L-1 5.41 ± 0.55 a 3.02 ± 0.10 c 3.35 ± 0.13 c 4.31 ± 0.17 ab 4.08 ± 0.22 b Fe mg L-1 1.67 ± 0.28 b 2.91 ± 0.26 a 2.36 ± 0.18 a 3.07 ± 0.24 a 2.77 ± 0.28 a Al mg L-1 0.39 ± 0.06 a 0.31 ± 0.03 a 0.21 ± 0.02 b 0.38 ± 0.03 a 0.32 ± 0.03 a Si mg L-1 3.48 ± 0.61 a 1.41 ± 0.17 b 1.39 ± 0.17 b 2.55 ± 0.51 b 1.45 ± 0.30 b Mn mg L-1 1.09 ± 0.24 a 0.13 ± 0.03 c 0.18 ± 0.04 b 0.28 ± 0.09 b 0.22 ± 0.06 b Cl mg L-1 4.61 ± 0.25 bc 4.00 ± 0.15 c 4.69 ± 0.20 b 5.78 ± 0.25 a 5.00 ± 0.21 b SO4-S mg L-1 3.08 ± 0.81 a 1.93 ± 0.19 b 2.62 ± 0.16 a 3.06 ± 0.17 a 2.81 ± 0.22 a NO3-N mg L-1 0.63 ± 0.19 a 0.23 ± 0.05 a 0.15 ± 0.03 b 0.14 ± 0.04 b 0.18 ± 0.04 a NH4-N mg L-1 0.64 ± 0.13 a 0.46 ± 0.07 a 0.19 ± 0.04 b 0.33 ± 0.13 ab 0.36 ± 0.10 a Norg mg L-1 1.31 ± 0.16 c 1.69 ± 0.11 ab 1.37 ± 0.07 bc 1.49 ± 0.20 c 1.70 ± 0.06 a Ntot mg L-1 2.58 ± 0.21 a 2.38 ± 0.12 a 1.70 ± 0.07 b 1.92 ± 0.25 b 2.24 ± 0.13 a PO4-P lg L-1 31 ± 10 ab 33 ± 6 a 18 ± 3 bc 10 ± 2 d 16 ± 4 cd Ptot lg L-1 34 ± 4 d 104 ± 10 a 72 ± 7 bc 58 ± 5 cd 76 ± 5 ab Qcalc. The change, DQ, was calculated in mm and as a percentage of Qcalc Qcalc. is calculated runoff based on reference LA¨ catchment data and reflects non-rewetted conditions. Qmeas. is measured runoff for the Porla catchment lculated in mm and as a percentage of Qcalc Q Q the Porla catchment Th h DQ l l d i d f Q l the Porla catchment The change DQ was calculated in mm and as a percentage of Qcalc ff based on reference LA¨ catchment data and reflects non-rewetted conditions. Qmeas. is measured runoff for Va¨stka¨rr wetland is calculated runoff based on reference LA¨ catchment data and reflects non-rewetted conditions. Qmeas. is measured runoff for the Porla catchment (mean ± standard error) of water in the Va¨stka¨rr wetland before rewetting (1997–1998) and for four Table 3 Chemical composition (mean ± standard error) of water in the Va¨stka¨rr wetland before rewetting ( periods after rewetting 12 Fig. 3 Inorganic (Ninorg) and organic (Norg) nitrogen concentrations in Va¨stka¨rr wetland, 1997–2013. Left Mean values for sub-areas and periods. Right ratio of Ninorg to Norg Fig. 4 Concentrations of total phosphorus (Ptot) and phosphate-P (PO4-P) in Va¨stka¨rr wetland, 1997–2013. Left Mean values for the sub-areas and periods. Right ratio of PO4-P to Ptot Wetlands Ecol Manage (2017) 25:405–419 413 Wetlands Ecol Manage (2017) 25:405–419 413 Fig. 3 Inorganic (Ninorg) and organic (Norg) nitrogen concentrations in Va¨stka¨rr wetland, 1997–2013. Left Mean values for sub-areas and periods. Right ratio of Ninorg to Norg Fig. 4 Concentrations of total phosphorus (Ptot) and phosphate-P (PO4-P) in Va¨stka¨rr wetland, 1997–2013. Left Mean values for the sub-areas and periods. Right ratio of PO4-P to Ptot Fig. 4 Concentrations of total phosphorus (Ptot) and phosphate-P (PO4-P) in Va¨stka¨rr wetland, 1997–2013. Left Mean values for the sub-areas and periods. Right ratio of PO4-P to Ptot Porla wetland rewetting, Norg comprised on average 52% of Ntot, but decreased to 47% of Ntot in the first 4 years of rewetting and then rose steadily to reach 79% in 12–14 years after rewetting. The concentration of NH4-N prior to rewetting was an order of magnitude higher than the NO3-N concentration, but decreased significantly in years 5–14 after rewetting. NO3-N reached lower values after rewetting than before (Fig. 5). Prior to rewetting, the pH in the outlet water was higher and displayed considerable variation compared with after rewetting, when the value stabilised at a lower level than before rewetting. In the first four-year period after rewetting changes were minor, but in the next 5–14 years the pH was on average 0.4 units lower (Table 4). Changes in chemical composition after rewetting appeared as darker water for the first 11 years, but in years 12–14 water colour decreased to the same level as before rewetting. Water DOC concentration in the first 11 years changed to a minor extent, with both increases and decreases, but in the last three-year period there was a greater decrease, i.e. in agreement with water colour (Table 4). Base cations and metals together with Si mainly showed lower values com- pared with before rewetting, with some slight devia- tions for Na, K and Al (Table 4). Bicarbonate alkalinity occurred a few times before rewetting and in the first years of rewetting, but after four years became zero in line with the low pH. Anions such as SO4, Cl and NO3 mainly decreased under rewetted conditions, with Cl as the dominant anion. Total N decreased, especially in the last six-year period, when Norg also showed lower values, agreeing with lower DOC and water colour in that period (Table 4). Before Total P increased in the first year after rewetting to over 0.02 mg L-1, but reverted to slightly lower values after 4-5 years. Median Ptot concentration was 0.015 mg L-1 before rewetting and increased to 0.019 mg L-1 after rewetting implying that slightly higher values were observed in rewetted condition (Fig. 6) The PO4-P content was 0.003 mg L-1 before rewetting and increased slightly to 0.004 mg L-1 after rewetting, making up about 20% of Ptot (Table 4; Fig. 6). Leaching of elements from the Porla wetland For the Porla catchment, where discharge measure- ments were possible, chemical element flows were calculated. Measured discharge and water chemistry concentrations were used to compute flow values and these were compared with calculated non-rewetted values estimated from the reference catchment. Leaching of elements from the Porla wetland Most 12 3 3 Wetlands Ecol Manage (2017) 25:405–419 414 Table 4 Chemical composition (mean ± SE) of water in the Porla wetland before (1998–1999) and for four periods after rewetting Variable Units 1998–1999 2000–2003 2004–2006 2007-2010 2011–2013 n = 11–17 n = 40 n = 17–18 n = 40–45 n = 34 pH 5.38 ± 0.14 a 5.08 ± 0.06 b 4.88 ± 0.03 c 4.98 ± 0.01 bc 4.99 ± 0.02 bc EC lS cm-1 43.0 ± 2.3 a 35.4 ± 1.3 b 31.3 ± 1.4 c 27.3 ± 0.5 d 29.0 ± 1.0 cd Alkalinity mg L-1 3.66 ± 1.64 a 1.36 ± 0.53 b 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 c SS mg L-1 15.5 ± 5.7 ab 11.2 ± 2.3 a 4.82 ± 0.42 ab 5.86 ± 0.63 b 4.71 ± 0.92 c Colour mg Pt L-1 223 ± 16 b 233 ± 10 ab 242 ± 10 ab 252 ± 8 a 214 ± 10 b DOC mg L-1 34.4 ± 2.1 ab 33.7 ± 1.0 a 33.8 ± 1.5 ab 33.8 ± 0.9 a 30.4 ± 1.1 b Ca mg L-1 4.43 ± 0.45 a 2.56 ± 0.31 b 1.68 ± 0.09 c 1.90 ± 0.07 bc 2.09 ± 0.10 bc Mg mg L-1 1.06 ± 0.07 a 0.56 ± 0.04 c 0.55 ± 0.03 c 0.65 ± 0.02 b 0.71 ± 0.03 b K mg L-1 0.46 ± 0.06 a 0.47 ± 0.03 a 0.50 ± 0.03 a 0.45 ± 0.02 a 0.42 ± 0.03 a Na mg L-1 2.98 ± 0.13 a 2.63 ± 0.10 b 2.70 ± 0.08 ab 2.77 ± 0.06 ab 2.83 ± 0.07 a Fe mg L-1 4.86 ± 0.57 a 3.53 ± 0.63 b 1.86 ± 0.16 c 2.36 ± 0.16 bc 2.24 ± 0.14 bc Al mg L-1 0.38 ± 0.02 ab 0.39 ± 0.02 a 0.36 ± 0.01 ab 0.35 ± 0.01 b 0.34 ± 0.01 b Si mg L-1 4.42 ± 0.61 a 2.45 ± 0.30 b 1.92 ± 0.23 b 2.00 ± 0.20 b 1.84 ± 0.24 b Mn mg L-1 0.14 ± 0.02 a 0.08 ± 0.01 bc 0.06 ± 0.01 c 0.08 ± 0.01 b 0.08 ± 0.01 b Cl mg L-1 4.41 ± 0.29 a 3.45 ± 0.09 b 3.65 ± 0.11 b 3.66 ± 0.08 b 3.67 ± 0.10 b SO4-S mg L-1 0.65 ± 0.07 a 0.54 ± 0.05 a 0.31 ± 0.06 c 0.26 ± 0.02 b 0.22 ± 0.02 bc NO3-N mg L-1 0.09 ± 0.02 a 0.07 ± 0.01 ab 0.03 ± 0.00 b 0.06 ± 0.01 ab 0.06 ± 0.01 ab NH4-N mg L-1 0.64 ± 0.06 a 0.75 ± 0.05 a 0.47 ± 0.06 b 0.30 ± 0.03 b 0.17 ± 0.02 b Norg mg L-1 0.86 ± 0.12 ab 0.70 ± 0.04 ab 0.88 ± 0.06 ab 0.73 ± 0.05 b 0.84 ± 0.03 a Ntot mg L-1 1.58 ± 0.13 a 1.51 ± 0.08 a 1.39 ± 0.08 a 1.03 ± 0.05 b 1.07 ± 0.04 b PO4-P lg L-1 3 ± 1 abc 4 ± 0 c 5 ± 0 a 4 ± 0 ab 4 ± 0 bc Ptot lg L-1 16 ± 1 a 21 ± 2 a 17 ± 2 a 19 ± 1 a 20 ± 1 a Mean values within rows with different letters are significantly different (ANOVA p \ 0.05). Leaching of elements from the Porla wetland N number of samples tion (mean ± SE) of water in the Porla wetland before (1998–1999) and for four periods after rewetting Fig. 5 Inorganic nitrogen content (NO3-N and NH4-N) in water of the Porla wetland before (1997–1999) and in four periods after rewetting. Black bars show measured concentrations and open bars the difference compared with before rewetting Fig. 5 Inorganic nitrogen content (NO3-N and NH4-N) in water of the Porla wetland before (1997–1999) and in four periods after rewetting. Black bars show measured concentrations and open bars the difference compared with before rewetting Fig. 5 Inorganic nitrogen content (NO3-N and NH4-N) in water of the Porla wetland before (1997–1999) and in four periods after rewetting. Black bars show measured concentrations and open bars the difference compared with before rewetting chemical elements and compounds showed lower export after rewetting. The main exception to lower export was for K, showing increased or unchanged export values for some periods (Table 5). Discussion K Ca Mg NO3-N NH4-N Ntot Ptot DOC SS 1999 0.02 1.3 10 2.5 0.2 1.4 3.5 0.03 74 23 2000/01 D 0.05 -0.02 0.9 -0.2 4.7 -7.3 1.2 -0.9 0.2 -0.1 1.5 -1.8 3.4 -2.2 0.04 -0.03 82 -52 17 -10 2002/04 D 0.01 -0.03 0.4 -0.5 1.5 -8.5 0.4 -1.6 0.03 -0.1 0.6 -1.5 1.3 -3.0 0.01 -0.03 30 -69 5 -16 2006/08 D 0.02 -0.02 1.0 ?0.1 3.9 -7.2 1.3 -1.0 0.1 -0.1 0.7 -0.8 2.1 -3.6 0.03 -0.01 70 -32 13 -18 2009/10 D 0.02 -0.02 0.8 -0.7 4.1 -5.1 1.3 -0.7 0.1 -0.04 0.5 -1.0 2.1 -1.7 0.04 -0 67 -24 9 -17 2011/13 D 0.02 -0.02 0.8 0 3.9 -8.9 1.3 -1.4 0.1 ?0.01 0.3 -2.1 1.9 -2.5 0.03 -0.01 55 -48 7 -18 difficulty was associated with hydrological determi- nations. The short calibration periods suffered from not having the same range of variation as the longer period after rewetting. 1995). After finished industrial peat extraction, the area must be restored to an alternative use, one being new wetland. Then, the soil surface is often prepared for incoming water, drains could be closed, bunds established and other hydrotechnical installations made. Created water reservoirs change soil organic matter content, often forming anoxic bottom sediment. Discussion Natural mires are totally altered after peat extraction, which turns the mire into a drained peatland (Wheeler 12 123 Wetlands Ecol Manage (2017) 25:405–419 415 1995). After finished industrial peat extraction, the area must be restored to an alternative use, one being new wetland. Then, the soil surface is often prepared for incoming water, drains could be closed, bunds established and other hydrotechnical installations made. Created water reservoirs change soil organic difficulty was associated with hydrological determi- nations. The short calibration periods suffered from not having the same range of variation as the longer period after rewetting. Effects on hydrology Fig. 6 Phosphorus concentration (PO4-P and Ptot, mg L-1) in water at the Porla wetland outlet before (1997–1999) and in four periods (1–14 years) after rewetting (black bars) and changes in Ptot (open bars) compared with calculated non-rewetted values (left). Share of PO4-P to Ptot (right) Table 5 Annual outflow (kg ha-1 yr-1) of chemical elements from the Porla cutover site and rewetted area in periods from 1999 to 2013, together with changes (D) compared with non- rewetting conditions Mean values within rows with different letters are significantly different (ANOVA, p \ 0.05) N number of samples H? K Ca Mg NO3-N NH4-N Ntot Ptot DOC SS 1999 0.02 1.3 10 2.5 0.2 1.4 3.5 0.03 74 23 2000/01 D 0.05 -0.02 0.9 -0.2 4.7 -7.3 1.2 -0.9 0.2 -0.1 1.5 -1.8 3.4 -2.2 0.04 -0.03 82 -52 17 -10 2002/04 D 0.01 -0.03 0.4 -0.5 1.5 -8.5 0.4 -1.6 0.03 -0.1 0.6 -1.5 1.3 -3.0 0.01 -0.03 30 -69 5 -16 2006/08 D 0.02 -0.02 1.0 ?0.1 3.9 -7.2 1.3 -1.0 0.1 -0.1 0.7 -0.8 2.1 -3.6 0.03 -0.01 70 -32 13 -18 2009/10 D 0.02 -0.02 0.8 -0.7 4.1 -5.1 1.3 -0.7 0.1 -0.04 0.5 -1.0 2.1 -1.7 0.04 -0 67 -24 9 -17 2011/13 D 0.02 -0.02 0.8 0 3.9 -8.9 1.3 -1.4 0.1 ?0.01 0.3 -2.1 1.9 -2.5 0.03 -0.01 55 -48 7 -18 Fig. 6 Phosphorus concentration (PO4-P and Ptot, mg L-1) in water at the Porla wetland outlet before (1997–1999) and in four periods (1–14 years) after rewetting (black bars) and changes in Ptot (open bars) compared with calculated non-rewetted values (left). Share of PO4-P to Ptot (right) H? Effects on hydrology Water levels in the lakes varied most in the first years of rewetting and became more stable in the longer term. The hydrological mishaps that occurred at the Porla wetland influenced discharge in two short periods, with refilling of the wetlands causing lower discharge for one period each in 2001 and 2002. In 2013 rather low runoff was measured at the Porla catchment outlet, possibly partly because of leakage at the weir for a few months. However, precipitation was low in 2013 and fairly low runoff could be expected. Restoration after peat extraction in the Porla and Va¨stka¨rr peatlands was planned within a short period of time and the commercial company involved initiated rapid restoration actions alongside ongoing peat harvesting. These conditions provided only 1–2 years for calibration measurements before rewet- ting. However, to our knowledge no other rewetting study has had long calibration period prior to rewetting (e.g. Tuittila et al. 1999). In the present study the necessary preparations were possible and the most 12 3 3 Wetlands Ecol Manage (2017) 25:405–419 416 Rewetting influenced the discharge pattern, with consequences for annual, low and high discharge. Periods with low discharge, occurring often in summer periods with low precipitation, were extended after rewetting and there was an increase in number of days with no discharge, meaning cessation of stream water flow in downstream watercourses. Despite the hydro- logical mishaps, it could be concluded that creation of reservoirs decreased annual outlet discharge in accor- dance to the hypothesis, an effect also reported in other studies (Waddington et al. 2008). Rewetting by ponding water on the peat cutover area increased the frequency of days with low or no runoff from the Porla site catchment. This could be related to the increased free water surface area leading to increased evapora- tion. When the water level in these open water areas dropped below the outflow crest, high precipitation amounts were required to create a surplus of water for outflow discharge. Consequently, this led to a higher frequency of days with low or no runoff from the rewetting area. term anoxic conditions (decades) in the sediments and peat layers, probably modified some of the chemical conditions in the sediments. The anoxic conditions in the rewetted peat at Va¨stka¨rr probably also explain the drastic change in the concentration of inorganic N fractions. Before rewetting there was no NH4-N in the peat, only NO3- N. Effects on hydrology Four years of rewetting raised NH4-N concentra- tions above 100 mg kg-1 dw, while NO3-N decreased to levels below 1 mg kg-1 dw probably dependent on anoxic conditions. NO3-N production stops at rewet- ting and instead the peat organic matter starts to release ammonium (Laine et al. 2013). Pool of NO3 would partly be denitrified and also taken up by plants increasing in coverage after 5–10 years. At the nutrient-poor Porla site no such significant changes were found and instead NH4-N levels tended to decrease. NO3-N concentrations were fairly low (1–2 mg kg-1) and decreased somewhat after rewet- ting. Inorganic nitrogen was instead dominated by NH4-N 50–200 mg kg-1. Probably, the fairly low pH and nutrient content influenced inorganic-N formation. Peatland drainage mainly decreases moderately high flows (Iritz et al. 1994). However, there have also been reports of increased peak flows and very high discharge peaks (Holden et al. 2006) but local conditions may modify this pattern (Shantz and Price 2006; Ballard et al. 2012). After rewetting at the Porla site, there was a decreased frequency of peak flows, especially lower peak flows. This effect was probably related to a higher lake percentage in the catchment after rewetting. Our observation that wetland restora- tion resulted in reduced peak flows is consistent with other studies (Wilson et al. 2010). Effects on water chemistry Changes in hydrology also affect water chemistry. Lakes and mires often act as retention areas, with chemical compounds stored in sediments and peat. Deviating from this is proton production in mires that increases, thereby giving comparatively lower pH values. The concentration of DOC may also increase, turning the water colour darker. Drainage of peatlands and wet mineral soils usually increases pH (Ramberg 1981) owing to organic material decomposition and leaching of mineral soil groundwater. Occasionally, short pulses of lower pH may occur at either very high discharges or with increasing discharge after dry periods with SO4 oxidation and H? released and washed out, (Lundin 1984). Rewetting reverses these conditions and thus in the nutrient-poor Porla site pH decreased. However, in the nutrient-rich Va¨stka¨rr wetland the pH values actually increased probably because of discharging CO2-supersaturated ground- water and CO2 evasion. Grayson et al. (2010) found that increased reveg- etation lowered peak flows compared with bare soil. However, at Porla wetland there was no trend for lower peak flows during later years after rewetting, when vegetation cover had spread to previously bare peat areas. The variation in precipitation during the years was more important for streamflow amounts and peaks. 123 Effects on the peat chemistry The concentrations of most chemical elements in the peat did not exhibit significant changes after rewetting. However, the sediment in the lakes formed due to rewetting mainly covered the superficial peat layers at the bottom of these lakes. This, together with the long- Water chemistry in the Porla wetland was charac- terised by pH values commonly found in Swedish forest streams in mires (pH *5), putting it among the 5% of lakes with the lowest pH in the Swedish national 12 123 Wetlands Ecol Manage (2017) 25:405–419 417 organic matter decomposition. However, lower phos- phate concentrations after drainage of a bog have also been reported (Lundin and Bergquist 1990). Stored P in sediments can easily be released when reducing bottom conditions occurred. At the nutrient-rich Va¨stka¨rr site, a significant increase in Ptot was observed after rewetting considered very high compared to Swedish lakes, while PO4-P decreased after a small initial increase in the first two years of rewetting, anyhow being on a high level (Fo¨lster et al. 2014). The decrease was probably related to biological uptake forming organically bound P or P release from Fe-bound P in the old, recalcitrant peat at the bottom of the newly established shallow lakes (Jordan et al. 2007; Zak et al. 2008). As the Va¨stka¨rr area had been used for agriculture, it would be possible fertilization had been used but since about two metres of peat was removed, influence would be limited. After the initial pulse of P release, the P concentration decreased somewhat in the next 5–11 years after rewetting, but was still higher 12–15 years after rewetting than before rewetting. However, at the nutrient-poor Porla site there were no significant effects on Ptot or PO4-P after rewetting both being on 75% percentile values compared to Swedish lakes (Fo¨lster et al. 2014), although there was a tendency for increased Ptot values. This could be related to the vegetation development as in Va¨stka¨rr site rather fast vegetation development occurred and for Porla area after about 5 years vegetation increased and with sphagnum cov- erage increasing after seven years (Kozlov et al. 2016). inventory (Fo¨lster et al. 2014). The water was dark, with a colour rating of *240 mg Pt L-1, whereas the median value for Swedish lakes is 60 mg Pt L-1. Effects on the peat chemistry Total organic carbon (TOC) in Porla wetland was 33 mg L-1, compared with a median of 9 mg L-1 for Swedish lakes (Fo¨lster et al. 2014). Moreover, the content of base cations was fairly high (0.3 mEq L-1) compared with the Swedish median (0.2 mEq L-1). The mean value Ntot value was 1.2 mg L-1 (Swedish median 0.31 mg L-1) and the mean Ptot value was 0.02 mg L-1 (Swedish median 0.01 mg L-1) (ibid). Studies of peatland drainage often report lower water colour and DOC concentrations after drainage (Lundin 1988). The opposite could be expected on rewetting, but the two wetlands studied here showed mainly similar effects in drained conditions, perhaps caused by dilution of easily soluble organic matter in remaining peat by the additional water inflow. In the long run, new organic matter will be formed and this might change the water quality. The time factor is of course important. Before stable conditions are reached in the rewetted area, easily soluble substances are leached out from the superficial peat layers and sediments. Ditch blocking in blanket peatlands usually decreases DOC or water colour (Wilson et al. 2011; Armstrong et al. 2010; Strack and Zuback 2013), but some studies have found the opposite or no change (Worrall et al. 2007; Ramchunder et al. 2012). Some of the studies reporting increased levels of DOC were carried out during the first 1–3 years after restoration (Worrall et al. 2007). Outflow of elements depended on discharge and the concentrations in water. In low water flow conditions, the concentrations of elements and compounds were often high, but with low water flow the contribution to fluxes was limited. In high discharge situations even low concentrations resulted in high fluxes, with consecutive consequences for annual outflows. How- ever, the changes in leaching, with decreased flux of many elements, coincide fairly well in contrast with findings after peatland drainage, where only proton concentrations decreased (Lundin 1988). The nutrient load on downstream watercourses could be considered to decrease but Ptot outflow possibly being higher. The concentrations of base cations and metals often increase after peatland drainage (Sallantaus 1989) and thus rewetting could be expected to result in lower concentrations. This was in fact mainly the case in the two rewetted areas studied. The influence of the new water bodies in sedimentation storage was probably the reason. Effects on the peat chemistry Following peatland drainage, N outflows are often reported to increase, especially inorganic N flows, as the peat starts to decompose and oxidising conditions occur (Lundin 1988). Accordingly, rewetting lowered the N concentrations, but although the share of organic N increased, the Ntot content did not. The values were in the common range for Swedish mire and forest stream with c. 20% inorganic N (Fo¨lster et al. 2014). Phosphorus availability to plants is restricted in peatlands. Drainage tends to give small increases in concentration (Hooper and Morris 1982) mainly due to References Armstrong A, Holden J, Kay P, Francis B, Foulger M, Gledhill S, McDonald AT, Walker A (2010) The impact of peatland drain-blocking on dissolved organic carbon loss and dis- colouration of water; results from a national survey. J Hy- drol 381:112–120 Ballard CE, McIntyre N, Wheater HS (2012) Effects of peatland drainage management on peak flows. Hydrol Earth Syst Sci 16:2299–2310 Blankenburg J, Tonnis W (eds) (2004) Guidelines for wetland restoration of peat cutting areas—the BRIDGE project. EC D-RTD, EN V4-CT98-0766. Geological Survey of Lower Saxony, Bremen, p 56 Wetland restoration at the Porla and Va¨stka¨rr sites was successful, as two functioning wetland ecosys- tems with stable hydrology have established after 15 years since rewetting and characteristic peatland vegetation, both fen vegetation and Sphagnum mosses have developed (Kozlov et al. 2016). This is an important prerequisite for new peat growth and a future C sink. However, the observed reduced peak flows, lower runoff and increased frequency of days with low or no runoff may have severe consequences for aquatic organisms in downstream watercourses. Braekke FH (1970) Myrgro¨fting for skogsproduktion. Inflytelse pa˚ vannhusholding og flomfare. Tidskrift Skogsbruk 78:227–238 (in Norwegian) Eggelsmann R, Heathwaite AL, Grosse-Brauckmann G, Ku¨ster E, Naucke W, Schuch M, Schweickle (1993) Physical processes and propertries of mires. In: Heathwaite AL, Go¨rrlich Kh (eds) Mires-Process. Exploitation and Con- servation, Wiley & Sons.Chichester Fo¨lster J, Johnson RK, Futter MN, Wilander A (2014) The Swedish monitoring of surface waters: 50 years of adaptive monitoring. In: Lydersen, E., Lo¨fgren, S., Fo¨lster, J., Fut- ter, M. and Johnson, R.K. (eds.) Fifty years of freshwater monitoring in Sweden—Special Issue of Ambio, Vol. 43. (Suplement 1); 3-18. doi 10.1007/s13280-014-0558-z The concentrations of DOC, base cations and NO3- N, NH4-N and Ntot were still lower 15 years after rewetting, but varied somewhat over time. The Ptot concentration increased, especially at the nutrient-rich Va¨stka¨rr site. Grayson R, Holden J, Rose R (2010) Long-term change in storm hydrographs in response to peatland vegetation change. J Hydrol 389:336–343 Grip H (1982) Water chemistry and runoff in forest streams at Kloten. UNGI Report 58. Uppsala University While the two former cutover peatlands have reverted to natural or semi-natural lake conditions after 15 years of rewetting, there will still be changes in peat and water chemistry influencing the future wetland environment. Holden J, Evans MG, Burt TP, Horton MM (2006) Impact of land drainage on peatland hydrology. J Environ Qual. Conclusions Studies at the rewetted Porla and Va¨stka¨rr sites revealed storage of material, base cations and metals 12 3 3 Wetlands Ecol Manage (2017) 25:405–419 418 in the newly established wetlands. In particular, protons, organic nitrogen and phosphorus showed high contents in lake sediments and water. This lowered the eutrophication load, through lower flows of base cations and inorganic nitrogen being released to downstream water courses. However, in dry periods lower water runoff could have lethal consequences for surface water organisms at ceasing flow. In the long run though, as wetland vegetation develops in the area, the wetlands will revert to natural conditions, with consequences for the environment such as decreased water flow, mainly lower nutrient load but perhaps lower pH and higher water colour. In this study, significant effects on water chemistry appeared more clearly 8–11 years after rewetting, demonstrat- ing the importance of monitoring for long periods after restoration to determine the full effects of rewetting. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unre- stricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons license, and indicate if changes were made. Funding sources Swedish Energy Agency and the Swedish Peat Research Foundation. References 35:1764–1778 Hooper FF, Morris LS (1982) Mat-water phosphorus exchange in an acid bog lake. Ecology 63:1411–1421 Iritz L, Johansson B, Lundin L (1994) Impacts of forest drainage on flodds. Hydrol Sci J 39:637–661 Acknowledgements This project was carried out at the Swedish University of Agricultural Sciences with financial support from the Swedish Energy Agency and the Swedish Peat Research Foundation. Sites were provided by Neova Company and the private landowner Per-Olof Sta˚lhammar, who both provided valuable contributions to fieldwork. Local field observations were carried out by Sten-Ove Pettersson and Lotta Pettersson. Schumann M, Joosten, H (2008) Global peatland restoration manual. http://www.imcg.net/media/download_gallery/ books/gprm_01.pdf. Accessed 20 April 2016 Joosten H, Clarke D (2002) Wise use of mires and peatlands— background and principles including a framework for decision-making. International Mire Conservation Group and International Peat Society. Saarija¨rvi. pp. 304 ISBN 951-97744-8-3 Funding sources Swedish Energy Agency and the Swedish Peat Research Foundation. Jordan S, Velty S, Zeitz J (2007) The influence of degree of peat decomposition on phosphorus binding forms in fens. Mires 12 3 Wetlands Ecol Manage (2017) 25:405–419 419 and Peat 2: Art. 7. (http://www.mires-and-peat.net/pages/ volumes/map02/map0207.php). Accessed 02 Nov 2016 and Peat 2: Art. 7. (http://www.mires-and-peat.net/pages/ volumes/map02/map0207.php). Accessed 02 Nov 2016 Sphagnum dominated peatlands. Wetlands Ecol Manage 11:3–20 Jordan S, Stro¨mgren M, Fiedler J, Lundin L, Lode E, Nilsson T (2016) Ecosystem respiration, methane and nitrous oxide emission fluxes from ecotopes in a rewetted extracted peatland in Sweden. Mires Peat 17:1–23 Sallantaus T (1989) Loading of watercourses due to peat mining. Peatland ecosystem and man—an impact assessment, International Peat Society/Mires Research Group, Dundee. pp 8 Kozlov SA, Lundin L, Avetov NA (2016) Revegetation dynamics after 15 years of rewetting in two extracted peatlands in Sweden. Mires Peat 18:1–17. doi:10.19189/ MaP.2015.OMB.204 Shantz MA, Price JS (2006) Characterization of surface storage and runoff patterns following peatland restoration, Quebec, Canada. Hydrol Process 20:3799–3814 SLU (2016) http://www.slu.se/institutioner/vatten-miljo/ laboratorier/vattenkemiska-laboratoriet/vattenkemiska- analysmetoder/. 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J Hydrol 337:315–325 Raab B, Vedin, H (1995) Klimat, sjo¨ar och vattendrag. Sveriges Nationalatlas. pp 176. Stockholm. (in Swedish) Ramberg L (1981) Increase in stream pH after a forest drainage. Ambio Vol. X. No 1 Zak D, Gelbrecht J, Wagner C, Steinberg EW (2008) Evaluation of phosphorus mobilization potential in rewetted fens by an improved sequential chemical extraction procedure. References Eur J Soil Sci 59:1191–1201. doi:10.1111/j.1365-2389.2008. 01081.x Ramchunder SJ, Brown LE, Holden J (2012) Catchment-scale peatland restoration benefits stream ecosystem biodiver- sity. J Appl Ecol 49:182–191 Rochefort L, Quinty F, Campeau S, Johnson K, Malterer T (2003) North American approach to the restoration of 123

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Sustainability in the restoration and management of the historic architecture in Palermo

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Energy performance and efficiency, Historic Architecture, Thermophysical properties, Categorization method Energy performance and efficiency, Historic Architecture, Thermophysical properties, Categorization metho * Corresponding author. Tel.: +39-3407940974; e-mail: [email protected], [email protected] Highlights The energy and environmental improvement of the historic architecture has to be referred to its local peculiarities. Hence, for a specific context, the geometric and distribution features of historic constructions, together with the thermophysical properties of traditional materials and building components, have to be studied. For this purpose, this research analyses the historic architecture of Palermo on the base of representative buildings, selected through a categorization method. For some of these constructions, the research is investigating the current energy performance and the potential to combine its improvement with the conservation of their material and aesthetic features. Sustainability in the restoration and management of the historic architecture in Palermo Enrico Genovaa* Enrico Genovaa* a Università degli Studi di Palermo, Dipartimento di Architettura, Viale delle Scienze, edificio 8, Palermo, 90128, Italia a Università degli Studi di Palermo, Dipartimento di Architettura, Viale delle Scienze, edificio 8, Palermo, 90128, Italia Abstract This research investigates the energy and environmental sustainability in the restoration and conservation of historic buildings. It focuses on the architectural heritage of Palermo, which can be a significant case study for the Mediterranean area. Its objective is to analyse the current energy performance of this historic architecture and its potential for energy improvement. Therefore, it aims at proposing a methodology to combine the enhancement of energy and environmental performances of the historic architecture of Palermo with the need of its material and aesthetic conservation, in the frame of the current regulations. 2. STATO DELL’ARTE Anche per l’architettura storica, come per le recenti e le nuove costruzioni, la gran parte delle ricerche sul miglioramento delle prestazioni energetiche e ambientali interessa aree in cui l’esigenza prevalente è quella del riscaldamento (sebbene recentemente un numero crescente di studi si concentri sul contesto mediterraneo). Tra le prime ricerche sul tema, rilevanti sono gli studi promossi, in Gran Bretagna, dagli enti di tutela English Heritage [2] e Historic Scotland [3, 4]: con particolare attenzione all’edilizia abitativa, essi pongono l’accento sul differente approccio al miglioramento energetico che il comportamento termoigrometrico del costruito storico richiede; significativo, in proposito, è anche il repertorio interattivo di tecniche d’intervento proposto da STBA (Sustainable Traditional Buildings Alliance) [5]. Altri studi si focalizzano invece sul riscaldamento dei grandi ambienti, in particolare delle chiese [6]; questo aspetto è affrontato in varie ricerche anche in Svezia (in particolare nel contesto del progetto Spara och Bevara) e si è tradotto in applicazioni pratiche su edifici museali e di culto. In Italia, alle ricerche che si concentrano su singoli casi di studio si accompagna la redazione di linee generali d’indirizzo e sistemi di valutazione: si citano a tal proposito le linee guida per la sostenibilità dell’architettura storica elaborate nel progetto A.T.T.E.S.S. [7], quelle recentemente pubblicate dall’AICARR [8] e il protocollo GBC Historic Buildings [9], basato sul sistema internazionale LEED. Come anche nelle ricerche condotte in Francia (BATAN, TUFFEAU), in Germania, in Svizzera (SuRHiB), il tema è affrontato sotto vari punti di vista: alcuni studi analizzano le prestazioni energetiche e le potenzialità di miglioramento per interi centri storici; altre si concentrano su singoli edifici, per sviluppare strumenti di simulazione e tecniche d’intervento. Questi approcci, che spaziano dalla scala urbana a quella degli elementi tecnici, sono compresenti in alcuni progetti europei, fra i quali 3ENCULT ed EFFESUS: in essi, lo sviluppo di procedure accurate per la diagnosi e la simulazione, e di tecnologie per incrementare le prestazioni dell’involucro, si accompagna all’elaborazione di metodologie volte a definire strategie di miglioramento energetico e ambientale sulla base delle caratteristiche costruttive, del regime di tutela, delle condizioni d’uso e del degrado degli edifici. 1. INTRODUZIONE All’architettura storica si attribuisce oggi un ruolo potenzialmente rilevante nel perseguimento degli obiettivi europei di sostenibilità per il settore delle costruzioni, in quanto costituisce una porzione significativa del patrimonio edilizio comunitario. Al contempo, permane l’esigenza di conservarne i caratteri formali e materiali: da ciò discende, ad esempio, l’esenzione dal rispetto di requisiti minimi di efficienza energetica che la normativa europea, e quasi tutte le disposizioni nazionali di recepimento, ammettono per gli edifici ufficialmente tutelati, ma non per buona parte dell’architettura storica di base. Molti recenti studi propongono quindi di adottare per il costruito storico, nell’ambito dell’efficienza energetica e degli altri aspetti che con questa concorrono a definirne un recupero e una gestione sostenibili, lo stesso approccio di miglioramento già messo in pratica in materia antisismica e di accessibilità: ovvero accrescere le prestazioni dell’edificio storico nei limiti consentiti dalla conservazione dei suoi caratteri [1]. Questa ricerca indaga il concetto di sostenibilità nell’architettura storica concentrandosi sull’aspetto delle prestazioni energetiche e ambientali. Del miglioramento di queste, inoltre, analizza la possibile integrazione nelle pratiche attuali del recupero e della conservazione, facendo specifico riferimento all’architettura storica di Palermo, caso di studio significativo per l’area mediterranea. 3. OBIETTIVI E METODO DELLA RICERCA Questa ricerca indaga l’applicazione del principio di sostenibilità ambientale agli edifici storici, realizzati con tecniche costruttive tradizionali, e si concentra sull’integrazione fra il recupero di tali architetture e l’accrescimento delle loro prestazioni energetiche e ambientali. Affinché tale istanza di miglioramento sia perseguita nel rispetto dei caratteri formali, materici e costruttivi del patrimonio storico, è necessario tener conto delle specificità di questo e riferirsi a contesti locali in cui esse siano sufficientemente omogenee. Lo studio si focalizza, quindi, sull’architettura storica di Palermo: obiettivo della ricerca è analizzarne le prestazioni attuali, indagarne le potenzialità di miglioramento energetico e proporre delle linee generali d’indirizzo volte a inquadrare quest’ultimo nella cornice delle disposizioni normative vigenti. Attraverso lo studio delle sue caratteristiche geometriche e distributive, il patrimonio del centro storico cittadino è suddiviso in categorie, in modo da individuare un numero contenuto di gruppi, e quindi di architetture rappresentative; per alcuni edifici così scelti, dopo aver indagato le caratteristiche termofisiche di materiali ed elementi tecnici tipici del costruito storico locale, si analizzano le prestazioni energetiche e si simulano possibili strategie di miglioramento. 3.1 Descrizione dell’architettura storica palermitana per categorie Il Piano Particolareggiato Esecutivo (1993) che regola le attività edilizie sul centro storico di Palermo adotta per esso un approccio tipologico (poi esteso anche al costruito storico esterno al perimetro dell’antica città murata): le tipologie introdotte dal piano rendono conto della funzione storica dell’edificio ma anche, in una certa misura, delle sue caratteristiche dimensionali, distributive e di aggregazione nel tessuto urbano; soprattutto, per ciascuna tipologia sono indicati gli elementi caratteristici da salvaguardare e le modalità d’intervento, più o meno restrittive, consentite. A supporto della presente ricerca, si è applicato al caso palermitano un metodo di categorizzazione, proposto dal progetto europeo EFFESUS [10] e volto all’analisi del patrimonio costruito dei centri storici (historic districts). L’applicazione ha riguardato il “mandamento” Castellammare, una delle quattro parti in cui il centro storico di Palermo è tradizionalmente suddiviso, e ha coinvolto più di cinquecento edifici (dallo studio sono escluse le chiese). Per ciascuno di questi, sono stati raccolti in Quantum GIS (QGIS), ed elaborati in Microsoft Office Excel, i dati concernenti la tipologia assegnata dal P.P.E., il numero di piani, l’area di sedime, il volume, la frazione del perimetro di base condivisa con le unità edilizie adiacenti. Quest’ultima caratteristica, impiegata per esprimere le modalità di aggregazione nel tessuto urbano, rende anche conto dell’eventuale presenza di cortili, che lo strumento urbanistico identifica come caratteristica di alcune tipologie. L’analisi è volta a correlare più direttamente le tipologie del P.P.E. con le caratteristiche che influenzano il comportamento energetico e ambientale delle costruzioni. Infatti la descrizione per categorie consente di studiare le prestazioni dell’architettura storica palermitana attraverso un numero contenuto di edifici rappresentativi, ma anche di analizzare l’applicabilità e l’efficacia di possibili misure di miglioramento [11]; al contempo, permette di riferire direttamente queste ultime alle modalità d’intervento previste dallo strumento urbanistico vigente. 3.3 Prestazioni degli edifici e possibili strategie di miglioramento Una parte del convento di Sant’Anna alla Misericordia, che ospita gli uffici della Galleria, è analizzata per confrontarne il comportamento energetico attuale con quello conseguibile se si applicassero tecniche d’incremento prestazionale, desunte dai repertori disponibili in letteratura per le architetture storiche. Le simulazioni, in corso di svolgimento, sono condotte con il programma WUFI Plus, che consente il calcolo termoigrometrico in regime dinamico. Lo studio è condotto anche per un edificio elencale (“catoio”), rappresentativo di una delle categorie in cui l’analisi condotta ha articolato le tipologie del P.P.E. Ciò consente di comparare l’efficacia che le medesime tecniche possono avere su edifici diversi, ma anche il miglioramento prestazionale conseguibile in architetture sulle quali differenti sono le modalità d’intervento ammesse dalla normativa vigente. 3.2 Studio dei materiali e degli elementi tecnici dell’architettura storica di Palermo Lo studio del fabbisogno energetico di un edificio richiede la conoscenza di numerosi parametri, molti dei quali inerenti alle proprietà termofisiche dei materiali e alla costituzione dei componenti, soprattutto d’involucro. Anche per l’architettura storica di Palermo, si è riscontrata in quest’ambito la disponibilità di pochi dati: da ciò l’attenzione di questa ricerca anche alla scala degli elementi tecnici. Vengono dunque analizzate le prestazioni delle soluzioni tipiche della tradizione costruttiva palermitana [12, 13, 14] e le caratteristiche termofisiche dei materiali impiegati [15, 16], per le quali preliminarmente si fa riferimento alle raccolte di dati esistenti nella normativa tecnica (UNI 10351:1994, UNI EN ISO 10456:2008 e UNI EN 1745:2012). La letteratura sul tema, tuttavia, mette in luce scostamenti anche considerevoli tra valori calcolati e misurati [5, 17, 18]. Pertanto, nel caso delle murature, le determinazioni di calcolo sono confrontate con i risultati di una campagna di misure in situ di conduttanza termica (ISO 9869:1994), che si sta svolgendo presso la Galleria d’Arte Moderna della città, ospitata nell’ex convento di Sant’Anna alla Misericordia. Infatti il complesso, per via della sua articolata storia costruttiva, è caratterizzato da una ricca casistica di murature in conci di calcarenite, che si differenziano per provenienza dei materiali, per epoca, per dimensioni e modalità costruttive; per esso, inoltre, si dispone dei documenti progettuali relativi al restauro svolto nel 1996-2006. Le misure sono condotte raccogliendo in un data-logger Ahlborn ALMEMO 2690-8 i valori rilevati da cinque sensori, ad esso collegati con fili: una piastra per la misura del flusso termico posta sulla superficie interna della muratura; quattro termocoppie per la misura della temperatura superficiale, due sul paramento esterno e due su quello interno. Con il metodo delle medie progressive indicato nella ISO 9869:1994, dai valori misurati si determina la conduttanza termica della parete; la trasmittanza è desunta da questa ricorrendo alle resistenze superficiali indicate nella norma UNI EN ISO 6946:2008. Una prima serie di nove misure [19, 20] è stata condotta (inverno 2013-2014) sulle tre elevazioni di una delle pareti del chiostro secentesco, esposta a nord. Una seconda serie riguarda invece le murature della parte quattro-cinquecentesca del complesso, e consta di determinazioni estive (giugno-settembre 2014) e invernali (iniziate a dicembre 2014 e attualmente in corso) su sette punti di misura. Galleria d’Arte Moderna della città, ospitata nell’ex convento di Sant’Anna alla Misericordia. 3.2 Studio dei materiali e degli elementi tecnici dell’architettura storica di Palermo Infatti il complesso, per via della sua articolata storia costruttiva, è caratterizzato da una ricca casistica di murature in conci di calcarenite, che si differenziano per provenienza dei materiali, per epoca, per dimensioni e modalità costruttive; per esso, inoltre, si dispone dei documenti progettuali relativi al restauro svolto nel 1996-2006. Le misure sono condotte raccogliendo in un data-logger Ahlborn ALMEMO 2690-8 i valori rilevati da cinque sensori, ad esso collegati con fili: una piastra per la misura del flusso termico posta sulla superficie interna della muratura; quattro termocoppie per la misura della temperatura superficiale, due sul paramento esterno e due su quello interno. Con il metodo delle medie progressive indicato nella ISO 9869:1994, dai valori misurati si determina la conduttanza termica della parete; la trasmittanza è desunta da questa ricorrendo alle resistenze superficiali indicate nella norma UNI EN ISO 6946:2008. Una prima serie di nove misure [19, 20] è stata condotta (inverno 2013-2014) sulle tre elevazioni di una delle pareti del chiostro secentesco, esposta a nord. Una seconda serie riguarda invece le murature della parte quattro-cinquecentesca del complesso, e consta di determinazioni estive (giugno-settembre 2014) e invernali (iniziate a dicembre 2014 e attualmente in corso) su sette punti di misura. Le misure sono condotte raccogliendo in un data-logger Ahlborn ALMEMO 2690-8 i valori rilevati da cinque sensori, ad esso collegati con fili: una piastra per la misura del flusso termico posta sulla superficie interna della muratura; quattro termocoppie per la misura della temperatura superficiale, due sul paramento esterno e due su quello interno. Con il metodo delle medie progressive indicato nella ISO 9869:1994, dai valori misurati si determina la conduttanza termica della parete; la trasmittanza è desunta da questa ricorrendo alle resistenze superficiali indicate nella norma UNI EN ISO 6946:2008. Una prima serie di nove misure [19, 20] è stata condotta (inverno 2013-2014) sulle tre elevazioni di una delle pareti del chiostro secentesco, esposta a nord. Una seconda serie riguarda invece le murature della parte quattro-cinquecentesca del complesso, e consta di determinazioni estive (giugno-settembre 2014) e invernali (iniziate a dicembre 2014 e attualmente in corso) su sette punti di misura. 4. RISULTATI PRELIMINARI Applicando il metodo di categorizzazione del progetto EFFESUS al mandamento Castellammare di Palermo, sono state definite dodici categorie, significative rispetto al campione esaminato sia per percentuale di superficie e di volume sia per numero di edifici. La classificazione proposta è basata su tre caratteristiche: la modalità di aggregazione dell’unità edilizia nel tessuto urbano (espressa attraverso la frazione di perimetro condivisa con le unità adiacenti), le dimensioni (in termini di volume), i vincoli di tutela (attraverso la definizione di tre “livelli”). Quest’ultimo aspetto, in particolare, costituisce il legame fra le categorie proposte e le tipologie del citato P.P.E. I risultati della descrizione per categorie sono stati adoperati per identificare un edificio rappresentativo di una parte dell’architettura elencale cittadina, e per suffragare la scelta del convento di Sant’Anna per l’analisi del costruito monumentale. Figura 1. Descrizione per categorie di una porzione del mandamento Castellammare. Figura 1. Descrizione per categorie di una porzione del mandamento Castellammare. Nella definizione dei dati d’ingresso, i risultati preliminari della prima serie di misure di conduttanza termica (chiostro) hanno evidenziato, per le murature indagate, una buona sovrapposizione con i valori calcolati (UNI EN ISO 6946:2008) adoperando la conducibilità termica del “tufo” (ρ=1.500 kg/m3, λ=0,63 W/mK, UNI 10351:1994); invece se ci si riferisce, in modo teoricamente più appropriato, alle “rocce naturali sedimentarie leggere” (ρ=1.500 kg/m3, λ=0,85 W/mK, UNI EN ISO 10456:2008), si tende a sovrastimare la trasmittanza termica delle murature in misura compresa fra il 10% e il 39%. Ciò conferma l’opportunità di definire, attraverso campagne estese di misura, abachi locali sulle caratteristiche termofisiche dei materiali e degli elementi tecnici tipici dell’architettura storica locale. Figura 2. Confronto, per la prima serie di misure (chiostro), fra i valori di trasmittanza calcolati (“tufo”, ρ=1.500 kg/m3, UNI 10351:1994, linea rossa; “rocce naturali sedimentarie leggere”, UNI EN ISO 10456:2008, linea azzurra) e quelli desunti dalle misure (banda grigia). Figura 2. Confronto, per la prima serie di misure (chiostro), fra i valori di trasmittanza calcolati (“tufo”, ρ=1.500 kg/m3, UNI 10351:1994, linea rossa; “rocce naturali sedimentarie leggere”, UNI EN ISO 10456:2008, linea azzurra) e quelli desunti dalle misure (banda grigia). L’analisi degli edifici selezionati, in corso di svolgimento, costituirà infine un’applicazione, limitata ad alcune delle categorie individuate, della metodologia proposta per l’elaborazione di strategie di miglioramento energetico per l’architettura storica palermitana. 5. CONCLUSIONI Le linee guida e i sistemi di certificazione per l’efficienza energetica e la qualità ambientale dell’architettura storica, per non risolversi nella semplice omologazione di procedure e tecniche d’intervento, devono riferirsi ai suoi caratteri dimensionali, distributivi, materico-costruttivi. In quest’ottica s’inserisce la metodologia proposta per l’analisi dell’architettura storica palermitana, mirata ad elaborare per essa delle strategie d’incremento delle prestazioni energetiche e ambientali che possano coniugarsi con le pratiche consolidate del recupero e della conservazione. L’analisi delle caratteristiche generali del patrimonio storico di un ambito locale è certamente necessaria per la pianificazione d’interventi di miglioramento energetico alla scala urbana; al contempo, un approccio tipologico, così come la definizione di valori di riferimento per le proprietà termofisiche dei materiali costruttivi tradizionali, delineano un quadro preliminare di conoscenze utile nello studio del singolo edificio, delle cui peculiarità è comunque necessario tener conto: anche l’intervento di miglioramento energetico, infatti, deve risultare da un’approfondita conoscenza della fabbrica storica, che è il fondamento del suo recupero. 6. RIFERIMENTI BIBLIOGRAFICI [1] Lucchi E, Pracchi V, editors. Efficienza energetica e patrimonio costruito. La sfida del miglioramento delle prestazioni nell’edilizia storica. Santarcangelo di Romagna (RM): Maggioli Editore; 2013. g g ( ) gg [2] English Heritage. Energy efficiency in historic buildings. English Heritage; 2010-2014. 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An internet-based intervention with brief nurse support to manage obesity in primary care (POWeR+): a pragmatic, parallel-group, randomised controlled trial

˜The œLancet. Diabetes & endocrinology

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Lancet Diabetes Endocrinol 2016; 4: 821–28 Methods We did this pragmatic, parallel-group, randomised controlled trial at 56 primary care practices in central and south England. Eligible adults aged 18 years or older with a BMI of 30 kg/m² or more (or ≥28 kg/m² with hypertension, hypercholesterolaemia, or diabetes) registered online with POWeR+—a 24 session, web-based, weight management intervention lasting 6 months. After registration, the website automatically randomly assigned patients (1:1:1), via computer-generated random numbers, to receive evidence-based dietetic advice to swap foods for similar, but healthier, choices and increase fruit and vegetable intake, in addition to 6 monthly nurse follow-up (control group); web-based intervention and face-to-face nurse support (POWeR+Face-to-face [POWeR+F]; up to seven nurse contacts over 6 months); or web-based intervention and remote nurse support (POWeR+Remote [POWeR+R]; up to fi ve emails or brief phone calls over 6 months). Participants and investigators were masked to group allocation at the point of randomisation; masking of participants was not possible after randomisation. The primary outcome was weight loss averaged over 12 months. We did a secondary analysis of weight to measure maintenance of 5% weight loss at months 6 and 12. We modelled the cost-eff ectiveness of each intervention. We did analysis by intention to treat, with multiple imputation for missing data. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN21244703. Findings Between Jan 30, 2013, and March 20, 2014, 818 participants were randomly assigned to the control group (n=279), the POWeR+F group (n=269), or the POWeR+R group (n=270). Weight loss averaged over 12 months was recorded in 666 (81%) participants. The control group lost almost 3 kg over 12 months (crude mean weight: baseline 104·38 kg [SD 21·11; n=279], 6 months 101·91 kg [19·35; n=136], 12 months 101·74 kg [19·57; n=227]). The primary imputed analysis showed that compared with the control group, patients in the POWeR+F group achieved an additional weight reduction of 1·5 kg (95% CI 0·6–2·4; p=0·001) averaged over 12 months, and patients in the POWeR+R group achieved an additional 1·3 kg (0·34–2·2; p=0·007). 21% of patients in the control group had maintained a clinically important 5% weight reduction at month 12, compared with 29% of patients in the POWeR+F group (risk ratio 1·56, 0·96–2·51; p=0·070) and 32% of patients in the POWeR+R group (1·82, 1·31–2·74; p=0·004). Lancet Diabetes Endocrinol 2016; 4: 821–28 The incremental overall cost to the health service per kg weight lost with the POWeR+ interventions versus the control strategy was £18 (95% CI –129 to 195) for POWeR+F and –£25 (–268 to 157) for POWeR+R; the probability of being cost-eff ective at a threshold of £100 per kg lost was 88% and 98%, respectively. No adverse events were reported. Interpretation Weight loss can be maintained in some individuals by use of novel written material with occasional brief nurse follow-up. However, more people can maintain clinically important weight reductions with a web-based behavioural program and brief remote follow-up, with no increase in health service costs. Future research should assess the extent to which clinically important weight loss can be maintained beyond 1 year. Funding Health Technology Assessment Programme of the National Institute for Health Research. ding Health Technology Assessment Programme of the National Institute for Health Research. An internet-based intervention with brief nurse support to manage obesity in primary care (POWeR+): a pragmatic, parallel-group, randomised controlled trial Paul Little, Beth Stuart, FD Richard Hobbs, Jo Kelly, Emily R Smith, Katherine J Bradbury, Stephanie Hughes, Peter W F Smith, Michael V Moore, Mike E J Lean, Barrie M Margetts, Chris D Byrne, Simon Griffi n, Mina Davoudianfar, Julie Hooper, Guiqing Yao, Shihua Zhu, James Raftery, Lucy Yardley Cambridge, UK (Prof S Griffin MD) Articles Articles Articles Articles www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 Summary y Background The obesity epidemic has major public health consequences. Expert dietetic and behavioural counselling with intensive follow-up is eff ective, but resource requirements severely restrict widespread implementation in primary care, where most patients are managed. We aimed to estimate the eff ectiveness and cost-eff ectiveness of an internet-based behavioural intervention (POWeR+) combined with brief practice nurse support in primary care. Correspondence to: Prof Paul Little, Primary Care and Population Sciences Division, Aldermoor Health Centre, University of Southampton, Southampton SO16 5ST, UK [email protected] Implications of all the available evidence The weight loss achieved with POWeR+ was similar to that achieved with the best performing interventions evaluated in a primary care setting over a 12 month period, including those produced by face-to-face commercial programmes. When combined with very brief staff support, the POWeR+ program could be feasibly used in most practices and could make a clinically important contribution to the management of obesity. Future research should assess the extent to which clinically important weight loss can be maintained beyond 1 year. behaviour-change expertise, and the little time available for counselling and follow-up, makes this approach challenging in the progressively resource-constrained primary care environment. A review6 of randomised controlled trials in primary care (excluding trials with more than 30% attrition at 12 months, which is common in studies of obesity) showed little evidence of appropriately intensive behavioural counselling and suggested trained interventionists. cost- eff ective if a 1 kg diff erence in weight was maintained for life. Automated interventions could be enhanced by human support, but although intensive support improves web-based weight management,9 we are aware of no studies examining the eff ectiveness of brief support by primary care staff that is likely to be both more feasible and cost-eff ective. A 2015 review10 identifi ed nine studies comparing internet support with minimal intervention, but two studies had unusual populations (university staff ; lactating women), and the only study to report fewer than ten contacts by behavioural counsellors documented less than 1·5 kg of weight loss and high attrition at 12 months, with only 49% of individuals followed up. One alternative to a cadre of highly trained inter- ventionists is to harness the capacity of the internet to help support behaviour change. Evidence before this study p<0·001). However, two studies had unusual populations (university staff ; lactating women), and the only study to report fewer than ten contacts by behavioural counsellors documented less than 1·5 kg weight loss and high attrition at 12 months, with only 49% of individuals followed up. Thus, evidence for the eff ectiveness of internet interventions using brief behavioural support in a primary care setting is poor. We did not repeat the most recent systematic review, done by Hutchesson and colleagues, which included studies of adults aged 18 years or older that assessed weight loss or weight maintenance interventions with an e-Health component. Between 1995, and Sept 17, 2014, Hutchesson and colleagues searched the Cochrane Library, MEDLINE and PreMEDLINE, Embase, CINAHL, Web of Science, Scopus, PubMed, and PsycINFO. Search terms were obesity/ or obesity, abdominal/ or obesity, orbid/Overweight/Adiposity/ obese.mp.ehealth.mp. or telemedicine/ telehealth.mp.Text Messaging/mhealth.mp. Computers, Handheld/(tablet* and (mac or ipad or android* or microsoft or windows)).mp. [mp=title, abstract, original title, name of substance word, subject heading word, keyword heading word, protocol supplementary concept, rare disease supplementary concept, unique identifi er]exp Internet/world wide web.mp.web based.mp.((web* or remote or online) adj3 deliver*).mp. [mp=title, abstract, original title, name of substance word, subject heading word, keyword heading word, protocol supplementary concept, rare disease supplementary concept, unique identifi er]website*.mp.online.ab,ti.smart phone*.mp.digital game*.mp.smartphone*.mp.Computer Simulation/ or virtual reality.mp. exp diet/healthy eating.tw. nutrition.tw.physical activity.tw.exp exercise/*motor activity/*Physical Fitness. 23 (27·4%) studies were classifi ed as higher quality (quality and risk of bias), 57 (67·9%) studies were classifi ed as moderate quality, and nine (10·7%) studies were lower quality. Meta-analysis showed signifi cantly greater weight loss (kg) with e-Health interventions than with minimal interventions (mean diff erence −1·40, 95% CI −1·98 to −0·82; Implications of all the available evidence Reviews suggest that automated interactive web-based interventions without human input can achieve greater weight loss than does no treatment or minimal interventions, but the evidence base included too many small, short-term trials in volunteer samples, arguing the need for large, pragmatic trials with at least 1 year of follow-up and including assessments of cost-eff ectiveness.7,8 The trials identifi ed by NICE mostly had expert lifestyle and behavioural input, and followed up patients intensively—on average 13 times per year during the fi rst 12 months.4 NICE also estimated that any intervention costing £100 per kg lost was likely to be We did the POWeR+ trial to estimate the eff ectiveness and cost-eff ectiveness of a web-based behavioural weight management intervention (POWeR+) with either brief face-to-face nurse support or brief remote nurse support for obese patients managed in primary care. Added value of this study Our results show that clinically important weight reduction is possible with a web-based behavioural program lasting 6 months combined with brief remote follow-up. Patients assigned to the web-based intervention and either face-to-face nurse support (POWeR+Face-to-face) or remote nurse support (POWeR+Remote) achieved roughly 1·5 kg more weight loss than those assigned to the control group (evidence-based dietetic advice and 6 monthly nurse follow-up). Furthermore, 30% of patients in the POWeR+ groups maintained 5% weight loss by 12 months, with less recourse to other weight loss activities and with no increase in health service costs. www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 Introduction including that from the National Institute for Health and Care Excellence (NICE),4 advocate dietary and physical activity intervention supported by intensive behavioural techniques. The low availability of high-level dietetic and including that from the National Institute for Health and Care Excellence (NICE),4 advocate dietary and physical activity intervention supported by intensive behavioural techniques. The low availability of high-level dietetic and Obesity is a major threat to public health,1,2 the prevalence has risen sharply since the early 1990s,3 and most patients are managed in primary care.4 Systematic reviews,5 821 www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 Articles Study design and patients We did this pragmatic, parallel-group, randomised controlled trial at 56 primary care practices in central and 822 Articles south England (around Southampton and Oxford). Up to 100 patients from each practice were randomly chosen, by use of computer-generated numbers, and invited to a screening appointment. Patients could also be referred opportunistically from routine practice appointments. choices (healthy foods swap sheet), or to increase fruit and vegetable intake (we used the National Health Service [NHS] fi ve-a-day sheet). Our previous trial11 documented 1·2 kg weight loss at 6 months when patients with hypertension received prompt sheets promoting healthy eating compared with a generic advice booklet. To enhance retention, participants were informed that this intervention had been shown to support weight loss. Nurses arranged brief follow-up (5–10 min appointments) with suffi cient time to measure weight at 6 months and 12 months, but not to provide explicit counselling. y We enrolled individuals aged 18 years or older with a BMI of 30 kg/m² or more (or ≥28 kg/m² with hypertension, hypercholesterolaemia, or diabetes) as identifi ed from general practitioner (GP) routine electronic health records. We excluded patients with severe mental health problems (eg, psychosis; diffi culty completing outcomes), patients who were too ill to take part in a study such as this one or who were unable to change their diet (eg, individuals with severe heart, lung, kidney, bowel, or liver diseases), patients who were pregnant or breastfeeding, patients with a perceived inability to walk 100 m (physical activity diffi cult), and patients with another member of the household taking part or no regular access to the internet. The study was approved by the National Research Ethics Service Committee South Central—Southampton B (reference 11/SC/0455). All participants provided written informed consent. The aim of the POWeR+F intervention was to provide automated behavioural counselling, with just three scheduled (and four optional) face-to-face nurse support sessions, thus requiring substantially less health professional skill and time than the evidence-based lifestyle interventions documented in the NICE review,4 and hence being much easier to implement in the NHS. In addition to weight recording every 6 months, as in the control group, participants had three scheduled face-to- face appointments in the fi rst 3 months, and then up to four more appointments during a further 3 months if needed (ie, 6 months in total). Study design and patients Weight gain on two consecutive logins triggered an automated email to the nurse advising that the patient needed further support, or patients could request additional support. Outcomes h The primary outcome was weight loss averaged over 12 months. Weight loss was measured with participants lightly clothed without shoes, at the same time every day when possible, with automated digital scales (Tanita Europe BV, Amsterdam, the Netherlands). Piloting suggested that intensive follow-up for anything but the primary outcome would increase dropout, we therefore focused on weight. As such, the fi nal analysis plan (July 1, 2015), matching the clinical rationale for our original sample size, included a secondary analysis of weight—maintenance of 5% weight loss. This outcome is very important clinically,12,13 and facilitated direct comparison with a previous UK primary care trial14 published after our study commenced. Although there was no explicit measurement of the work of participants (ie, how much time was spent doing the intervention activities), other secondary outcomes included health service resource use, use of the website (pages accessed and time taken, recorded automatically by the website), how enabled patients felt (measured with the modifi ed Patient Enablement Instrument15), and any Randomisation and masking Patients were given details of how to log in and register to the POWeR+ intervention, whereupon individuals were presented with baseline questionnaires. Upon completion of the questionnaires, the website automatically randomly assigned patients (1:1:1), via computer-generated random numbers, to one of three intervention groups: evidence- based dietetic advice and 6 monthly nurse follow-up (control group); web-based intervention and face-to-face nurse support (POWeR+Face-to-face [POWeR+F] group); or web-based intervention and remote support (POWeR+Remote [POWeR+R group]). Participants and investigators were masked to group allocation at the point of randomisation; masking of participants was not possible after randomisation. The 12 month weight measurement was masked whenever possible. The aim of the POWeR+R intervention was to assess whether even briefer professional support for the web intervention could be eff ective. Patients could access the same web-based intervention as in the POWeR+F group. In addition to weight recording every 6 months, as in the control group, participants had three scheduled phone or email contacts and up to two optional phone or email contacts in the fi rst 6 months (triggered by weight gain or patient request, as in the POWeR+F group). www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 See Online for appendix Articles eff ect size of 0·33 (equivalent to 2–3 kg diff erence assuming a standard deviation of change of 6·5–7·5 kg16,17), and 80% power, required 174 patients per group with complete data or 654 patients in total, allowing for a 20% loss to follow-up. After liaison with both the funder of the study and the Trial Steering Committee, the power calculation was revised on Sept 25, 2013, to allow for modest clustering at practice level if signifi cant clustering was recorded. We assumed recruitment of 18 patients per practice to achieve 15 patients at follow-up, of whom roughly fi ve to six patients would be in each of the two intervention groups at follow-up. Assuming fi ve patients per group in each practice for an intracluster correlation of 0·05—ie, a design eff ect of 1·2 (1 + [(5–1) × 0·05])—resulted in a minimum of 654 × 1·2=785 patients. additional activities participants used to help lose weight. We also documented various other secondary outcomes (waist measurement, blood pressure, HbA1c, liver function tests, self-reported measures of physical activity, and diet; appendix p 11). We planned to use Actiheart monitors in a randomised subset of individuals, but organising this method at a time of intensive fi nal follow-up became too diffi cult. Participants had appointments for weight measurement with the practice nurse at baseline and 6 months, and an appointment or home visit by a nurse researcher masked to group allocation at 12 months. When a blinded weight measurement could not be obtained, we used practice nurses’ recorded weights, and when that was not possible, we used participants’ reported weights. Participants received a £10 gift voucher with the 12 month appointment notifi cation letter as a thank you for participation, irrespective of whether an appointment was made. For self-reported measures, participants received three emails prompting online completion followed by a postal version of the questionnaire. To avoid loss of data, on July 1, 2015, repeated measures ANOVA for the principal continuous outcome (ie, weight) was changed to mixed multivariate regression models to enable data to be used from any participant who had 6 months’ or 12 months’ data. We modelled the risk ratios compared with the control group for the number of patients achieving 5% weight loss. Articles All regression models controlled for weight at baseline, sex, age, smoking, diabetes, medications (including orlistat used at baseline), any comorbidities, deprivation (Index of Material Deprivation 2010), and any clustering by practice. No interim analyses were undertaken. Procedures POWeR+ is a theory and evidence-based intervention to teach patients self-regulation and cognitive-behavioural techniques to form sustainable eating and physical activity habits for long-term weight management in a series of 24 web-based sessions designed to be used over 6 months, with novel content, links to external content, and email reminders. Patients initially chose either a low-calorie or a low-carbohydrate eating plan, but could change plans at any stage if they wished (appendix pp 1–4). Participants assigned to the control group were directed to a set of two printable web-based pages with brief structured advice. This intervention was active, since it was intended to aid weight loss. The materials were developed by the Institute of Food Research to provide appealing strategies to minimise the pressure to cut down favourite foods, and to instead swap less healthy foods for healthier 823 Articles Articles Statistical analysis On Sept 25, 2013, the Trial Steering Committee advised an increase to the sample size to allow for clustering; this amendment required an extension to the planned recruitment period. We allowed all groups to be compared with each other (α 0·05/3=0·017), but for the primary analysis compared each intervention group with the control group. For the primary outcome, we estimated that a standardised p y Intention-to-treat analysis used both measured and reported weights in a multiply imputed dataset (based on 830 patients screened for eligibility 3059 patients invited from 25 practices 31 practices did not provide information about the number of patients involved 826 patients from 56 practices randomly assigned and had weight recorded at baseline 4 ineligible 2 had low BMI 2 unable to walk 100 m 269 assigned to the POWeR+Face-to-face group 3 ineligible post-randomisation (low BMI) and excluded 279 assigned to the control group 4 ineligible post-randomisation (low BMI) and excluded 270 assigned to the POWeR+Remote group 1 ineligible post-randomisation (low BMI) and excluded 148 had weight recorded at 6 months 221 had weight recorded at 12 months 15 withdrew* 33 lost to follow-up 136 had weight recorded at 6 months 227 had weight recorded at 12 months 18 withdrew* 34 lost to follow-up 155 had weight recorded at 6 months 218 had weight recorded at 12 months 18 withdrew* 34 lost to follow-up Figure: Trial profi le *Reasons for withdrawal: the patient’s situation changed (eg, pregnancy, moved house; n=18), the patient disliked their group (n=8), the patient had no time to participate or changed their mind (n=4), or no reasons were given (n=21). 3059 patients invited from 25 practices 31 practices did not provide information about the number of patients involved 826 patients from 56 practices randomly assigned and had weight recorded at baseline 270 assigned to the POWeR+Remote group 1 ineligible post-randomisation (low BMI) and excluded 269 assigned to the POWeR+Face-to-face group 3 ineligible post-randomisation (low BMI) and excluded Figure: Trial profi le *Reasons for withdrawal: the patient’s situation changed (eg, pregnancy, moved house; n=18), the patient disliked their group (n=8), the patient had no time to participate or changed their mind (n=4), or no reasons were given (n=21). www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 824 Articles 50 replications). Secondary analyses used both complete cases and measured weights. Statistical analysis session and 404 (75%) participants completed all three core sessions (n=208 in the POWeR+F group and n=196 in the POWeR+R group). Participants completed a mean of 10·97 (SD 12·65) weight and goal reviews (range zero to 52), with a mean of 10·16 (11·92) completed reviews in the POWeR+F group and 11·85 (13·38) in the POWeR+R group. The median number of nurse contacts was four (range zero to seven) in both intervention groups, with a median of two (IQR one to three) face-to-face contacts, one (zero to two) phone contact, and one (zero to two) email contact in the POWeR+F group, and a median of one (zero to two) phone contact and three (two to four) email contacts in the POWeR+R group. We recorded a 2–2·5 kg diff erence in weight reduction for participants who completed more than the fi rst basic stage of the program. We explored whether there was signifi cant eff ect modifi cation in subgroups using interaction terms in the models. Key pre-identifi ed subgroups were baseline waist measurement (high or very high waist vs low waist; men: high 94 cm, very high 102 cm; women: high 80 cm, very high 88 cm), and presence of metabolic syndrome (syndrome vs no syndrome). Metabolic syndrome18 was defi ned as the presence of three out of fi ve conditions: elevated waist circumference (>94 cm for men, 80 cm for women), triglyceride concentrations of 1·7 mmol/L or more, reduced HDL-cholesterol (<1·00 mmol/L in men, <1·3 mmol/L in women), raised blood pressure (systolic >130 mm Hg or diastolic >85 mm Hg, or treatment of high blood pressure), and elevated fasting glucose (≥5·6 mmol/L).18 We also explored outcomes according to the type of diet chosen by patients. Patients in the control group maintained a weight loss of almost 3 kg in total over 12 months (table 2). The primary imputed analysis showed that compared with the control group, patients in the POWeR+F group achieved an estimated additional 1·5 kg reduction averaged over 12 months and patients in the POWeR+R group achieved an additional 1·3 kg reduction (table 3). A secondary analysis of only the complete cases showed a greater weight reduction at 12 months in the intervention groups than in the control group (POWeR+F: –1·78 kg, 95% CI –2·81 to –0·76; p=0·001; POWeR+R: –1·6 kg, –2·6 to –0·6; p=0·002; appendix pp 9, 10). Role of the funding source g The funder of the study had no role in study design, data collection, data interpretation, data analysis, or writing of the report. The corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. Statistical analysis We obtained data for health service resource use from case-note review of medication, primary care visits, outpatient consultant, obesity-related admissions to accident and emergency and hospital (eg, for management of obesity and disorders aff ected by obesity, such as cardiovascular disease, control of asthma, and musculoskeletal problems [hip, back, and knee]; appendix pp 5–8). For the cost-eff ectiveness analysis, we measured outcomes as incremental costs per kg weight loss; we imputed missing data and used bootstrapping methods to produce confi dence intervals. We did analysis with Stata (version 14). By month 12, with data for complete cases, the proportion of patients maintaining a 5% reduction in weight or more By month 12, with data for complete cases, the proportion of patients maintaining a 5% reduction in weight or more Control group (n=279) POWeR + Face-to- face group (n=269) POWeR + Remote group (n=270) Age (years) 52·69 (13·25) 53·70 (13·21) 54·74 (12·95) Sex Female 185 (66%) 175 (65%) 160/269 (60%) Male 94 (34%) 94 (36%) 109/269 (40%) Smoker 24 (9%) 21 (8%) 25/269 (9%) Diabetes 48 (17%) 46/268 (17%) 42 (16%) Orlistat use 3/270 (1%) 5/262 (2%) 5/266 (2%) Comorbidity 48 (17%) 55 (20%) 55 (20%) Deprivation score (IMD 2010) 14·32 (10·45) 13·73 (10·28) 13·29 (10·17) Weight (kg) 104·38 (21·11) 102·40 (16·87) 102·93 (18·26) BMI (kg/m²) 37·10 (5·97) 36·66 (5·36) 36·28 (5·65) Data are mean (SD), n (%), or n/N (%), unless otherwise specifi ed. Data were missing for some individuals. IMD=Index of Material Deprivation. Table 1: Baseline characteristics Baseline 6 months 12 months Control group 104·38 (21·11); n=279 101·91 (19·35); n=136 101·73 (19·57); n=227 POWeR+Face-to-face group 102·40 (16·87); n=269 97·55 (15·99); n=148 98·56 (15·95); n=221 POWeR+Remote group 102·93 (18·26); n=270 98·30 (18·34); n=155 99·72 (18·88); n=218 Data in parentheses are SDs. Table 2: Crude mean weights for complete cases at baseline, 6 and 12 months Control group (n=279) POWeR + Face-to- face group (n=269) POWeR + Remote group (n=270) Age (years) 52·69 (13·25) 53·70 (13·21) 54·74 (12·95) Sex Female 185 (66%) 175 (65%) 160/269 (60%) Male 94 (34%) 94 (36%) 109/269 (40%) Smoker 24 (9%) 21 (8%) 25/269 (9%) Diabetes 48 (17%) 46/268 (17%) 42 (16%) Orlistat use 3/270 (1%) 5/262 (2%) 5/266 (2%) Comorbidity 48 (17%) 55 (20%) 55 (20%) Deprivation score (IMD 2010) 14·32 (10·45) 13·73 (10·28) 13·29 (10·17) Weight (kg) 104·38 (21·11) 102·40 (16·87) 102·93 (18·26) BMI (kg/m²) 37·10 (5·97) 36·66 (5·36) 36·28 (5·65) www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 Articles was 19% in the control group, 28% in the POWeR+F group, and 32% in the POWeR+R group; the imputed estimates were 21%, 29%, and 32%, respectively (table 4). pressurising participants to have blood taken was off - putting, achievement of high follow-up for blood samples was necessarily a secondary priority; therefore, only a small number of participants had follow-up blood measurements. The available results suggest generally positive directions of outcomes in the POWeR+ groups versus the control group, although changes were mostly non-signifi cant (raised HDL cholesterol, lower aspartate transaminase and alanine transaminase, lower HbA1C; appendix pp 12–14). The low completion rate of the EuroQol-5D (EQ-5D) questionnaire reduces the reliability of these data; as such, the direction of change in EQ-5D scores is diffi cult to interpret, particularly because the estimate of direction of change varies dependent on whether we controlled for baseline values (appendix p 18). Body fat percentage and blood pressure were recorded in most participants at follow-up (n=454 and n=494, respectively), and although there were no consistent changes in blood pressure in any of the three groups, fat mass was slightly reduced at 12 months in both POWeR+ groups (appendix p 15). pressurising participants to have blood taken was off - putting, achievement of high follow-up for blood samples was necessarily a secondary priority; therefore, only a small number of participants had follow-up blood measurements. The available results suggest generally positive directions of outcomes in the POWeR+ groups versus the control group, although changes were mostly non-signifi cant (raised HDL cholesterol, lower aspartate transaminase and alanine transaminase, lower HbA1C; appendix pp 12–14). The low completion rate of the EuroQol-5D (EQ-5D) questionnaire reduces the reliability of these data; as such, the direction of change in EQ-5D scores is diffi cult to interpret, particularly because the estimate of direction of change varies dependent on whether we controlled for baseline values (appendix p 18). estimates were 21%, 29%, and 32%, respectively (table 4). We reviewed 753 case notes from the GP electronic health records for 12 months after recruitment. Mean intervention costs were low both per person using the services (£22 [95% CI 21–23] for POWeR+F and £12 [11–13] for POWeR+R) and for all participants in each group (£17 [15–18] and £9 [8–10], respectively). Discussion This study is one of few to compare simple weight management interventions using primary care staff for support to manage obesity. Clinically important weight loss (5% reduction) was achieved by some participants in the control group, and the crude mean weight reduction by 12 months was not signifi cantly lower in the POWeR+ behavioural web-based intervention groups. However, in the POWeR+ groups, signifi cantly more weight loss occurred over 12 months, and more participants maintained clinically important weight reduction and felt enabled to manage their weight. This outcome was achieved with no increase in health service costs despite brief nurse remote support. Due to the priority to obtain follow-up data for weight, and the clear feedback from piloting suggesting that the POWeR+ groups, signifi cantly more weight loss occurred over 12 months, and more participants maintained clinically important weight reduction and felt enabled to manage their weight. This outcome was achieved with no increase in health service costs despite brief nurse remote support. Individuals who take part in trials are likely to be fairly well motivated, but participants are also the intended target group in whom intervention is likely to be most helpful. The present study was large and pragmatic, Complete cases Imputed data ≥5% reduction in weight Risk ratio versus control ≥5% reduction in weight Risk ratio versus control 6 months 12 months 6 months 12 months 6 months 12 months 6 months 12 months Control 16/136 (12%) 42/227 (19%) 1·00 1·00 15·9% 20·8% 1·00 1·00 POWeR+ Face-to-face 59/148 (40%) 62/221 (28%) 3·42 (2·10–5·56; p<0·001) 1·46 (1·02–2·08; p=0·036) 36·8% 29·2% 3·10 (1·85–5·18; p<0·001) 1·56 (0·96–2·51; p=0·070) POWeR+Remote 55/155 (35%) 69/218 (32%) 3·02 (1·89–4·83; p<0·001) 1·67 (1·17–2·37; p=0·004) 33·7% 32·4% 2·64 (1·60–4·36; p<0·001) 1·82 (1·21–2·74; p=0·004) Data are n/N (%) or risk ratio (95% CI; p value), unless otherwise specifi ed. Table 4: Patients maintaining at least 5% weight loss from baseline to months 6 and 12 6 months 12 months Overall* POWeR+Face-to-face group –2·54 (–3·66 to –1·42; p<0·001) –0·37 (–1·66 to 0·92; p=0·566) –1·49 (–2·41 to –0·58; p=0·001) POWeR+Remote group –1·97 (–3·18 to –0·76; p=0·002) –0·58 (–1·88 to 0·72; p=0·375) –1·27 (–2·19 to –0·34; p=0·007) Data in parentheses show 95% CI; p value. Analysis based on 50 imputations. *Repeated measures. Articles Addition of the estimated cost of the website raised the overall costs for all participants to £18 [95% CI 16–19] in the POWeR+F group and £10 [9–11] in the POWeR+R group. We recorded greater diff erences in overall costs for estimated overall NHS resource use related to obesity in the intervention versus the control groups (appendix pp 7, 8). Bootstrapping analysis showed that diff erences in costs compared with the control group were £23 for POWeR+F and –£36 for POWeR+R; neither of these diff erences was statistically signifi cant (table 5). The estimated incremental overall NHS cost per kg weight loss was £18 for POWeR+F and –£25 for POWeR+R (table 5)—ie, POWeR+R was more eff ective and cost less than the control strategy. The probability of cost-eff ectiveness was more than 80% in cost-eff ectiveness acceptability curves for both POWeR+F and POWeR+R versus control (88% and 98%, respectively; appendix pp 8, 9). Body fat percentage and blood pressure were recorded in most participants at follow-up (n=454 and n=494, respectively), and although there were no consistent changes in blood pressure in any of the three groups, fat mass was slightly reduced at 12 months in both POWeR+ groups (appendix p 15). In subgroup analysis, we recorded some evidence of a reduced eff ect of the POWeR+F group at 6 months in patients with metabolic syndrome, but no signifi cant eff ect of type of diet chosen (appendix p 9). No harms were reported during the study For patients returning the fi nal questionnaire, almost half the people in the control group were doing additional activities, compared with almost two-fi fths in the POWeR+F group and only a quarter in the POWeR+R group (table 6). Participants in the intervention groups also felt more enabled to manage their weight problem than did those in the control group (table 6). Results Figure 1 shows the trial profi le. Between Jan 30, 2013, and March 20, 2014, 826 patients underwent randomisation. After exclusion of eight participants identifi ed as ineligible post-randomisation, 818 individuals were randomly assigned to the control group (n=279), the POWeR+F group (n=269), or the POWeR+R (n=270). 439 (54%) patients had a weight recorded at 6 months’ follow-up and 666 (81%) patients had a weight recorded at 12 months. 510 (77%) of the 666 recordings were blinded, 28 (4%) were unblinded, and 128 (19%) were self-reported. The number of self-reported measurements was similar between groups (control n=40, POWeR+F n=48, POWeR+R n=40). Baseline characteristics were generally well balanced (table 1). The practice intracluster correlation for weight in the repeated measures analysis was 0·01 (95% CI 0·003–0·09) when controlling for baseline weight. Data are mean (SD), n (%), or n/N (%), unless otherwise specifi ed. Data were missing for some individuals. IMD=Index of Material Deprivation. Table 1: Baseline characteristics Baseline 6 months 12 months Control group 104·38 (21·11); n=279 101·91 (19·35); n=136 101·73 (19·57); n=227 POWeR+Face-to-face group 102·40 (16·87); n=269 97·55 (15·99); n=148 98·56 (15·95); n=221 POWeR+Remote group 102·93 (18·26); n=270 98·30 (18·34); n=155 99·72 (18·88); n=218 Data in parentheses are SDs. Table 2: Crude mean weights for complete cases at baseline, 6 and 12 months ) g g Of the 539 participants randomised to the POWeR+ intervention groups, 524 (97%) participants started the fi rst www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 825 Articles Articles Discussion As a result, even though estimates used multiple imputation, the results for blood samples should be interpreted cautiously. Nevertheless, we recorded signifi cant weight loss irrespective of the method of specifying the weight outcome (average reduction over 12 months as specifi ed in the funding application, or clinically important [5%] weight loss at 6 months or 12 months), and most secondary outcomes also changed in positive directions, albeit mostly not signifi cantly, suggesting that selective reporting and type I error are less likely. Mean diff erence in overall costs (£) Mean diff erence in weight lost (kg) Incremental cost (£) per kg of weight lost POWeR+ Face-to-face versus control 23 (–105 to 152) 1·49 (0·58 to 2·41) 18 (–129 to 195) POWeR+Remote versus control –36 (–154 to 81) 1·27 (0·34 to 2·19) –25 (–268 to 157) Based on imputed data with repeated measures. Data in parentheses are 95% CIs. Table 5: Incremental overall National Health Service cost per kg of weight lost in the POWeR+ groups versus the control group In the control group, we observed 3% loss in weight, and 12% and 19% of participants in this group maintained a clinically important weight loss (5%) at 6 and 12 months, respectively. These results are consistent with our previous fi ndings,11 showing that a brief intervention promoting the use of simple sheets for food swaps and fi ve-a-day fruit and vegetable consumption aids weight control. Some of the eff ective weight management in this group might also be due to the motivational eff ects of regular follow-up weighing by the nurse, and to the undertaking of additional activities to help themselves (which about half of participants did). By contrast, fewer individuals off ered the POWeR+ interventions undertook other activities, and they felt signifi cantly more enabled to manage their weight. based on a review of predominantly motivated volunteer samples.20 Improved weight loss (around 5 kg) with an internet-based program has only been achieved with much more intensive human support (eg, weekly 20 min contact for 12 weeks, and then monthly up to 24 month follow-up9). Encouragingly, the weight loss achieved with POWeR+ was similar to that achieved with the best- performing interventions21 evaluated over 12 months in a primary care setting, including those produced by face-to- face commercial programmes. Discussion Table 3: Diff erence in weight loss (kg) for POWeR+ versus control, with imputed data 6 months 12 months Overall* POWeR+Face-to-face group –2·54 (–3·66 to –1·42; p<0·001) –0·37 (–1·66 to 0·92; p=0·566) –1·49 (–2·41 to –0·58; p=0·001) POWeR+Remote group –1·97 (–3·18 to –0·76; p=0·002) –0·58 (–1·88 to 0·72; p=0·375) –1·27 (–2·19 to –0·34; p=0·007) Data in parentheses show 95% CI; p value. Analysis based on 50 imputations. *Repeated measures. Table 3: Diff erence in weight loss (kg) for POWeR+ versus control, with imputed data Individuals who take part in trials are likely to be fairly well motivated, but participants are also the intended target group in whom intervention is likely to be most helpful. The present study was large and pragmatic, Complete cases Imputed data ≥5% reduction in weight Risk ratio versus control ≥5% reduction in weight Risk ratio versus control 6 months 12 months 6 months 12 months 6 months 12 months 6 months 12 months Control 16/136 (12%) 42/227 (19%) 1·00 1·00 15·9% 20·8% 1·00 1·00 POWeR+ Face-to-face 59/148 (40%) 62/221 (28%) 3·42 (2·10–5·56; p<0·001) 1·46 (1·02–2·08; p=0·036) 36·8% 29·2% 3·10 (1·85–5·18; p<0·001) 1·56 (0·96–2·51; p=0·070) POWeR+Remote 55/155 (35%) 69/218 (32%) 3·02 (1·89–4·83; p<0·001) 1·67 (1·17–2·37; p=0·004) 33·7% 32·4% 2·64 (1·60–4·36; p<0·001) 1·82 (1·21–2·74; p=0·004) Data are n/N (%) or risk ratio (95% CI; p value), unless otherwise specifi ed. Table 4: Patients maintaining at least 5% weight loss from baseline to months 6 and 12 www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 826 Articles Mean diff erence in overall costs (£) Mean diff erence in weight lost (kg) Incremental cost (£) per kg of weight lost POWeR+ Face-to-face versus control 23 (–105 to 152) 1·49 (0·58 to 2·41) 18 (–129 to 195) POWeR+Remote versus control –36 (–154 to 81) 1·27 (0·34 to 2·19) –25 (–268 to 157) Based on imputed data with repeated measures. Data in parentheses are 95% CIs. Discussion Table 5: Incremental overall National Health Service cost per kg of weight lost in the POWeR+ groups versus the control group Control group POWeR+Face-to- face group POWeR+Remote group Additional activities Took part in regular activity for long enough to produce sweat Rarely 50/135 (37%) 38/134 (28%) 39/146 (27%) Sometimes 57/135 (42%) 69/134 (51%) 77/146 (53%) Often 28/135 (21%) 27/134 (20%) 30/146 (21%) Took part in another weight loss 64/136 (47%) 51/137 (37%) 40/150 (27%) WeightWatchers or Slimming World (or similar) meetings 23/136 (17%) 22/137 (16%) 14/150 (9%) Another weight management website 0/135 4/136 (3%) 4/150 (3%) Mobile phone application 13/136 (10%) 8/136 (6%) 10/149 (7%) Weight loss pills 5/136 (4%) 4/137 (3%) 2/150 (1%) Health trainer programme 4/136 (3%) 2/136 (1%) 3/148 (2%) Exercise referral scheme 4/136 (3%) 7/137 (5%) 4/148 (3%) Another weight loss scheme 8/136 (6%) 13/136 (10%) 8/149 (5%) Any other weight management method 22/97 (23%) 13/87 (15%) 8/103 (8%) Patient enablement Item score at baseline 3·19 (1·27) 3·42 (1·19) 3·31 (1·26) Item score at 12 months 3·23 (1·57) 4·10 (1·28) 3·85 (1·35) Diff erence in enablement score versus control group ·· 0·70 (0·39–1·01; p<0·0001) 0·54 (0·24–0·85; p<0·0001) Data are n/N (%), mean (SD), or diff erence (95% CI; p value). Table 6: Additional activities undertaken over 12 months, and patients’ enablement in managing their weight mimicking the everyday conditions in primary care settings; therefore, patients in the control group were not closely regulated and so undertook other activities to lose weight. However, this factor is also a strength because resulting estimates are more refl ective of real-world practice. Participants with obesity in primary care settings are notoriously diffi cult to follow up, but we achieved follow-up of more than 80% of individuals at 12 months. Loss to follow-up was similar between groups, which reduces the eff ect of missing data. Furthermore, we imputed data for the primary analysis, thus providing more conservative estimates of eff ectiveness than complete cases. However, in practice, multiple imputation modifi ed the estimates only slightly, which suggests that attrition bias was not a major issue. On the basis of the experience of piloting, whereby pressure to achieve follow-up of both primary and secondary outcomes resulted in participants dropping out, our eff orts were concentrated on maximising the primary outcome, which resulted in fewer secondary outcomes being available. www.thelancet.com/diabetes-endocrinology Vol 4 October 2016 Contributors PL devised the protocol, led protocol development and the funding application, supervised the running of the lead study centre and coordination of centres, contributed to the analysis, and led the drafting of the report. FDRH developed the protocol for funding, contributed to management of the study, supervised the Oxford study centre, and contributed to drafting of the report. JK and ERS (senior trial managers) developed the protocol, provided day-to-day overall management of the study, coordinated recruitment in the lead study centre and coordination of other centres, and commented on drafts of the report. MD coordinated the Oxford study centre. JH provided coordination and data management for the Southampton study centre. GY, SZ, and JR (health economists) developed the protocol for analysis of data from the medical notes reviews and contributed to the drafting of the paper. JR contributed to the management of the study and supervised the analysis of data for resource use. KJB and SH (health psychologists) contributed to protocol development, were responsible for the day-to-day development and piloting of the intervention, and commented on drafts of the report. MVM developed the protocol for funding, contributed to management of the study, and contributed to the analysis and drafting of the report. BS (study statistician) and PWFS developed the analysis protocol, did the quantitative analysis, and contributed to drafting of the report. LY developed the protocol and funding application with PL, led the development of the intervention, contributed to daily supervision of website issues, contributed to broader study management, and contributed to drafting of the report. MEJL, BMM, and CDB developed the protocol for funding, contributed to management of the study, and contributed to drafting of the report. 9 Appel LJ, Clark JM, Yeh H-C, et al. Comparative eff ectiveness of weight-loss interventions in clinical practice. N Engl J Med 2011; 365: 1959–68. 9 Appel LJ, Clark JM, Yeh H-C, et al. Comparative eff ectiveness of weight-loss interventions in clinical practice. N Engl J Med 2011; 365: 1959–68. 10 Hutchesson MJ, Rollo ME, Krukowski R, et al. eHealth interventions for the prevention and treatment of overweight and obesity in adults: a systematic review with meta-analysis. Obes Rev 2015; 16: 376–92. 11 Little P, Kelly J, Barnett J, Dorward M, Warm D, Margetts B. Randomised controlled factorial trial of dietary advice for patients with a single high blood pressure reading in primary care. BMJ 2004; 328: 1054–60. 1 Troiano RP, Frongillo EA Jr, Dobal J, Levitsky DA. The relationship between body weight and mortality: a quantitative analysis of combined information from existing studies. Int J Obes Rel Metabol Dis 1996; 20: 63–75. Declaration of interests We declare no competing interests. The views expressed in this publication are those of the authors and not necessarily those of the Health Technology Assessment Programme (HTA), the National Health Service, the National Institute for Health Research (NIHR), or the Department of Health. 17 17 Rothert K, Strecher V, Doyle L, et al. Web-based weight management programs in an integrated health care setting: a randomized, controlled trial. Obesity 2006; 14: 266–72. 18 18 Alberti K, Eckel R, Grundy S, et al. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 2009; 120: 1640–45. Contributors 12 Tuomilehto J, Lindstrom J, Eriksson J, et al. Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. N Engl J Med 2001; 344: 1343–50. 13 Knowler W, Barrett-Connor E, Fowler S, et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002; 346: 393–403. 14 Jolly K, Lewis A, Beach J, et al. Comparison of range of commercial or primarycare led weight reduction programmes with minimal intervention control for weight loss in obesity: Lighten Up randomised controlled trial. BMJ 2011; 343: d6500. 15 Little P, Lewith G, Webley F, et al. Randomised controlled trial of Alexander technique lessons, exercise, and massage (ATEAM) for chronic and recurrent back pain. BMJ 2008; 337: a884. 16 16 Sjostrom L, Rissanen A, Andersen T, et al. Randomised placebo-controlled trial of orlistat for weight loss and prevention of weight regain in obese patients. European Multicentre Orlistat Study Group. Lancet 1998; 352: 167–72. Articles Behavioral treatment of obesity in patients encountered in primary care settings: a systematic review. JAMA 2014; 312: 1779–91. 7 Wieland LS, Falzon L, Sciamanna CN, et al. Interactive computer-based interventions for weight loss or weight maintenance in overweight or obese people. Cochrane Database Syst Rev 2012; 8: CD007675. 8 Tang J, Abraham C, Greaves C, Yates T. Self-directed interventions to promote weight loss: a systematic review of reviews. J Med Internet Res 2014; 16: e58. Articles their own condition without needing signifi cant face-to- face contact, this could empower self-management more generally. Previous modelling by NICE showed that at least a 1 kg per head weight loss among overweight or obese adults is likely to be cost- eff ective, provided that the cost per person of intervening is less than £100 and the weight diff erence is maintained for life. The present results suggests that irrespective of whether intervention costs or overall costs are used, both POWeR+ interventions achieve weight losses at a cost per kg that is less that required by NICE.4 The POWeR+ intervention was for 6 months, but the number of patients maintaining clinically important weight loss remained steady up to 1 year, which suggests that long-term benefi t could be achieved. However, future research should assess the extent to which clinically important weight loss can be maintained beyond 1 year. 2 National Institutes of Health (NIH), National Heart Lung and Blood Institute. Clinical guidelines on the identifi cation and treatment of overweight and obesity in adults. Bethesda, MD: NIH, 1998. 2 National Institutes of Health (NIH), National Heart Lung and Blood Institute. Clinical guidelines on the identifi cation and treatment of overweight and obesity in adults. Bethesda, MD: NIH, 1998. 3 Department of Health; Joint Surveys Unit of Social and Community Planning Research; Department of Epidemiology and Public Health, University College London. Health Survey for England 1996. London: HMSO, 1998. 4 Obesity guideline development group, National Institute for Health and Care Excellence. Obesity full guideline. 2006. http://guidance. nice.org.uk/CG43/guidance (May 15, 2016). 4 Obesity guideline development group, National Institute for Health and Care Excellence. Obesity full guideline. 2006. http://guidance. nice.org.uk/CG43/guidance (May 15, 2016). 5 Avenell A, Broom J, Brown T, et al. Systematic review of the long term eff ects and economic consequences of treatment for obesity and the implications for health improvement. Health Technol Assess 2004; 8: iii–iv, 1–182. 5 Avenell A, Broom J, Brown T, et al. Systematic review of the long term eff ects and economic consequences of treatment for obesity and the implications for health improvement. Health Technol Assess 2004; 8: iii–iv, 1–182. 6 Wadden T, Butryn M, Hong P, Tsai A. Behavioral treatment of obesity in patients encountered in primary care settings: a systematic review. JAMA 2014; 312: 1779–91. 6 Wadden T, Butryn M, Hong P, Tsai A. Discussion Systematic reviews4,6,19 of interventions in other settings and primary care suggest that both intensive dietetic behavioural counselling and intensive follow-up are usually necessary to achieve eff ective weight reduction. However, we found that a behavioural web-based intervention such as POWeR+ was eff ective with just a few brief phone calls and emails, plus weight monitoring every 6 months. The weight loss achieved with POWeR+ compares favourably with other internet-based interventions, which, on average (albeit with high heterogeneity), have led to only short-term weight loss, of less than 1 kg weight loss compared with no treatment, or less than 2 kg when combined with face-to-face support, The estimates of resource use suggest that neither intervention was resource intensive. Considering overall NHS costs, fewer NHS resources were used in the POWeR+R group, mainly due to reduced primary care costs. In view of the variability of cost estimates, these results should be treated with some caution, but health service costs are unlikely to be signifi cantly increased with the remote POWeR+ intervention. We also recorded that POWeR+ users felt signifi cantly more enabled to manage their condition, so if individuals can be enabled to manage 827 Articles Articles Acknowledgments This study was funded by the HTA Programme of the NIHR. FDRH is partly funded by NIHR School for Primary Care Research, NIHR Collaborations for Leadership in Applied Health Research and Care Oxford, and NIHR Oxford Biomedical Research Centre. We thank all the patients and health-care professionals who have contributed their time and eff ort and helpful insights to make POWeR+ possible; the Trial Steering Committee for their support and advice throughout the study; David Turner for help in developing the protocol for funding; Jin Zhang, Matthew Taylor, Mark Weal, and Rosie Jones for technological support for POWeR+ and for assistance with the development; Catherine Brant for help in developing the complex intervention; and Weight Concern (London, UK) for their input into the original application and development of the intervention. 19 Neve M, Morgan P, Jones P, Collins C. Eff ectiveness of web-based interventions in achieving weight loss and weight loss maintenance in overweight and obese adults: a systematic review with meta-analysis. Obesity Rev 2010; 11: 306–21. 20 Kodama S, Siato K, Tanaka S, et al. Eff ect of Web-based lifestyle modifi cation on weight control: a meta-analysis. Int J Obes 2012; 36: 675–85. 21 Hartmann-Boyce J, Johns D, Jebb S, Summerbell C, Aveyard P. Behavioural weight management programmes for adults assessed by trials conducted in everyday contexts: systematic review and meta-analysis. Obese Rev 2014; 15: 920–32. References 828 www.thelancet.com/diabetes-endocrinology Vol 4 October 2016

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Issue 25 / Part 5 /view/meit25-05-110 273 2023 25 05 110 Issue 25 / Part 5 /view/meit25-05-110 273 2023 25 05 110 Modern engineering and innovative technologies http://www.moderntechno.de/index.php/meit/article/view/meit25-05-110 DOI: 10.30890/2567-5273.2023-25-05-110 ISSN 2567-5273 УДК 378. 147 SCIENTIFIC APPROACHES TO PROBLEMS OF COMPETENCE AND PROFESSIONAL COMPETENCES OF FUTURE TEACHERS OF MUSIC ARTS IN PEDAGOGICAL SCIENCE НАУКОВІ ПІДХОДИ ДО ПРОБЛЕМИ КОМПЕТЕНТНОСТІ ТА ПРОФЕСІЙНИХ КОМПЕТЕНЦІЙ МАЙБУТНІХ ВЧИТЕЛІВ МУЗИЧНОГО МИСТЕЦТВА У ПЕДАГОГІЧНІЙ НАУЦІ Д Ц Starodub S, / Стародуб С.Л. teacher / викладач Novokhaotska T./ Новахацька Т.С. teacher / викладач Municipal Institution «Uman Taras Shevchenko Professional College of Education and Humanities of Cherkasy Regional Counsil» 33, Nebesna Sotnia, 20 300 КЗ «Уманський гуманітарно-педагогічний фаховий коледж ім. Т.Г. Шевченка Черкаської обласної ради» вул. Небесної Сотні, 33, 20 300 Д Ц Starodub S, / Стародуб С.Л. teacher / викладач Novokhaotska T./ Новахацька Т.С. teacher / викладач Municipal Institution «Uman Taras Shevchenko Professional College of Education and Humanities of Cherkasy Regional Counsil» 33, Nebesna Sotnia, 20 300 КЗ «Уманський гуманітарно-педагогічний фаховий коледж ім. Т.Г. Шевченка Черкаської обласної ради» вул. Небесної Сотні, 33, 20 300 Starodub S, / Стародуб С.Л. teacher / викладач Novokhaotska T./ Новахацька Т.С. Анотація. У статті досліджено феномен професійной компетентності майбутнього вчителя музичного мистецтва. Проведено аналіз професійної компетентності майбутніх вчителів, шляхи та методи її формування та розвитку. Запропоновано перелік компетентностей запропонованих дослідником Дж. Равеном, психологами, педагогами. Проведено аналіз понять компетенція та компетентність майбутнього фахівця музичного мистецтва. Ключові слова: компетентність, майбутній вчитель музичного мистецтва. Ключові слова: компетентність, майбутній вчитель музичного мистецтва. В останні роки в науці активно досліджується феномен професійной компетентності, яка визначається зміною вимог ринку праці до фахівця у музичній діяльності і процесом модернізації вищої освіти. Професійна компетентність, шляхи, методи, формування та розвиток досліджується соціологами, психологами, педагогами. Основний текст. ISSN 2567-5273 Основний текст. У науковій літературі компетентність це інтегральна якість особистості, що виявляється у загальній здатності і готовності її до музичної діяльності, заснованої на знаннях і досвіді, які придбані у процесі навчання і соціалізації у вищій школі і орієнтовані на самостійну та успішну участь студентів у музичній діяльності. Вперше компетентність як поняття було введено у педагогічну термінологію Дж. Равеном. Аналізуючи соціально-псіхологічні проблеми функціонування британського суспільства, вчений дійшов висновків про необхідність зміни неадекватних переконань, очікувань, цінностей і установок в суспільстві, типах мотивації, та потреб сучасного суспільства. У трактуванні Дж. Равена компетентність ¬ це специфічна здатність, яка необхідна для ефективного виконання конкретної дії у музичній галузі і включає вузькоспеціальні знання, музичні навички, способи мислення, а також розуміння відповідальності за свої дії. www.moderntechno.de www.moderntechno.de 144 Issue 25 / Part 5 Modern engineering and innovative technologies Issue 25 / Part 5 Крім того, Дж. Равен ввів поняття вищих компетентностей, які, незалежно від того, в якій конкретній сфері вони проявляються, припускають наявність у людини ініціативи, здатності організувати інших людей для досягнення поставлених цілей, готовність оцінювати і аналізувати соціальні наслідки своїх дій тощо [4]. Суттєвою ознакою природи компетентності вчений вважав її властивість проявлятися в органічній єдності, з цінностями майбутнього фахівця, тобто за умови глибокої особистісної зацікавленості у музичній діяльності. Перелік компетентностей, запропонований дослідником, представляє собою групу ознак, в яких більшість компетентностей відносяться до психологічних особливостей особистості: – тенденція контролювати свою музичну діяльність; – тенденція контролювати свою музичну діяльність; – впевненість у собі; – самоконтроль; – адаптивність; – самостійність мислення; – критичне мислення; – готовність до риску; – наполегливість; – довіра; – відповідальність; – терпимість до інших тощо. – терпимість до інших тощо. Частину компетентностей визначають як установки, наприклад: здібність, готовність до майбутньої професії.. В педагогічній науці дослідження компетентності зявились в останній чверті XX століття (Н.Кузьмина, А.Маркова, Є.Зеєр, А.Хуторський та ін). Необхідно відмітити, что поряд з терміном компетентність у науковій літературі використовується термін компетенція. Ці терміни іноді використовуються як синоніми, які визначають: – розумові дії (процеси, функції); – особистісні якості студента; – мотиваційні тенденції; – ціннісні орієнтації (установки, диспозиції); – особливості міжособистісної взаємодії; – практичні вміння; – навички тощо. Згідно Глосарію терминів, компетенція визначається як: – здатність працювати добре і ефективно; – відповідність вимогам до даної професії; – здібності виконувати творчі функції [2]. А.Хуторський розділяє поняття «компетентність» і «компетенція» як загальне та індивідуальне. Компетентність ¬ особистісна якість фахівця і мінімально необхідний досвід діяльності у музичній сфері. Висновки. У загальному вигляді вчений визначає компетентності як цілісну і систематизовану сукупність узагальнених знань. Є Зеєр виділяє базові компетентності, як комплекс універсальних знань, які відрізняються широким рівнем узагальнення. Інтегральні знання включають загальнонаукові та загальнопрофесійні категорії, поняття, закони, принципи і закономірності функціонування науки, техніки, суспільства. До базових компетентностей Є. Зеєр відносить загальнонаукові поняття, основні закони природи, суспільства та діяльність людини. У практиці професійної діяльності під компетентністю майбутнього вчителя музичного мистецтва розуміємо міру відповідності знань, умінь і досвіду студентів реальному рівню складності виконуваних ними завдань і розв'язання професійних проблем. Основний текст. Під компетенцією вчений розуміє наперед задану вимогу до освітньої підготовки здобувачів освіти з оволодіння сукупністю взаємозвязаних якостей www.moderntechno.de 145 Issue 25 / Part 5 Issue 25 / Part 5 Modern engineering and innovative technologies Issue 25 / Part 5 особистості, знаннями, вміннями, навичками, способами діяльності, які необхідні для якісної роботи [1]. особистості, знаннями, вміннями, навичками, способами діяльності, які необхідні для якісної роботи [1]. Є.Зеєр, інтерпретуючи компетентність як змістове узагальнення теоретичних і емперічних знань у формі понять, принципів, положень, виділяє: – компетентності теоретичного рівня узагальнення, які відображають внутрішні зв’язки і відношения предметів та явищ дійсності. Іх конкре- тизація выражається у поняттях, законах, принципах; – емпирічні компетентності, які відображають зовнішні властивості предметів та явищ, мають прикладной характер. – емпирічні компетентності, які відображають зовнішні властивості предметів та явищ, мають прикладной характер. Конкретизація цього рівня узагальнення полягає у словах-термінах, символах, знаках, процесуальних знаннях, іллюстраціях, прикладах [1]. Конкретизація цього рівня узагальнення полягає у словах-термінах, символах, знаках, процесуальних знаннях, іллюстраціях, прикладах [1]. До базових компетентностей Є. Зеєр відносить: До базових компетентностей Є. Зеєр відносить: 1) загальнонаукові поняття, основні закони природи, суспільства і діяльності студента; 1) загальнонаукові поняття, основні закони природи, суспільства і діяльності студента; 2) особистісне відношення до музичної діяльності. Компетентність – якості особистості майбутнього вчителя музичного мистецтва і мінімально необхідний досвід діяльності у професійній сфері. Компетентність – якості особистості майбутнього вчителя музичного мистецтва і мінімально необхідний досвід діяльності у професійній сфері. у р ф ф р Под компетенцією вчений розуміє наперед задані вимоги до освітньої підготовки студента, які необхідні для якісної професійної діяльності [3]. Література: р ур 1. Носков В.И., Кальянов А. В., Мирошниченко О. В. и др. Инновационные технологии в гуманитарном вузе / Под ред. проф. В. И. Носкова. – Донецк: ООО „Лебедь”, 2002. – 288 с. 1. Носков В.И., Кальянов А. В., Мирошниченко О. В. и др. Инновационные технологии в гуманитарном вузе / Под ред. проф. В. И. Носкова. – Донецк: ООО „Лебедь”, 2002. – 288 с. 2. Розумний М. Соціальна некомпетентність та шляхи її подолання // Сучасна українська політика. Політики і політологи про неї. – Вип. 5. – Київ, Миколаїв, 2004. – С. 210 - 214. 3. Кіпень В., Коржов Г. Викладачі вузів: соціологічний портрет. – Донецьк: Астро, 2001. – 199 с. 3. Кіпень В., Коржов Г. Викладачі вузів: соціологічний портрет. – Донецьк: Астро, 2001. – 199 с. 4. Равен Дж. Компетентность в современном обществе: выявление, развитие и реализация / Пер. с англ. – М.: Когито-Центр, 2002. – 396 с. www.moderntechno.de ISSN 2567-5273 146 Issue 25 / Part 5 Issue 25 / Part 5 Modern engineering and innovative technologies www.moderntechno.de Modern engineering and innovative technologies Issue 25 / Part 5 Literature: Literature: 1. V. I. Noskov, A. V. Kalyanov, O. V. Myroshnychenko, etc. Innovative technologies in the humanities university / Ed. Prof. V. I. Noskova. - Donetsk: Lebed LLC, 2002. - 288 p. 1. V. I. Noskov, A. V. Kalyanov, O. V. Myroshnychenko, etc. Innovative technologies in the humanities university / Ed. Prof. V. I. Noskova. - Donetsk: Lebed LLC, 2002. - 288 p. 2. Rozumny M. Social incompetence and ways to overcome it // Modern Ukrainian politics. Politicians and political scientists about her. - Issue 5. – Kyiv, Mykolaiv, 2004. – P. 210 - 214. 2. Rozumny M. Social incompetence and ways to overcome it // Modern Ukrainian politics. Politicians and political scientists about her. - Issue 5. – Kyiv, Mykolaiv, 2004. – P. 210 - 214. p y y 3. Kipen V., Korzhov G. University teachers: a sociological portrait. - Donetsk: Astro, 2001. - 199 p. 3. Kipen V., Korzhov G. University teachers: a sociological portrait. - Donetsk: Astro, 2001. - 199 p. p 4. Raven J. Competence in modern society: identification, development and realization / Trans. with English - M.: Kogito-Center, 2002. - 396 p. 4. Raven J. Competence in modern society: identification, development and realization / Trans. with English - M.: Kogito-Center, 2002. - 396 p. Abstract. The article examines the phenomenon of professional competence of the future music teacher. An analysis of the professional competence of future teachers, ways and methods of its formation and development was carried out. A list of competencies proposed by researcher J. Raven, psychologists, and teachers is offered. An analysis of the concepts of competence and competence of the future specialist in musical art was carried out. p f f p Key words: competence, future music teacher. ISSN 2567-5273 www.moderntechno.de 147

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The Influence of “Artificial Intelligence + Human–Computer Interaction” on Teachers’ Psychological Changes in Academic Management in Colleges

Frontiers in psychology

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The Influence of “Artificial Intelligence + Human–Computer Interaction” on Teachers’ Psychological Changes in Academic Management in Colleges Honghai Guan1,2, Qingli Chen1,3, Song Han1 and Baoge Zhang4* 1 Faculty of Education, Northeast Normal University, Changchun, China, 2 School of Educational Science, Mudanjiang Normal University, Mudanjiang, China, 3 Provincial Primary and Secondary School Teacher Development Center, Shaoguan University, Shaoguan, China, 4 Department of Humanities and Social Sciences, Ningbo University, Ningbo, China The purpose was to analyze the psychological changes of teaching staff in the academic management of local colleges, and briefly explore the role of teaching staff in the development of the social economy and colleges. In the environment of artificial intelligence and human–computer interaction (HCI), first, the relevant theories of teaching staffs’ psychological status and the characteristics of teaching staff in college academic management were analyzed and expounded. Next, the way of the questionnaire was selected to analyze the psychology of teaching staff in college academic management at different ages, professional titles, academic qualifications, disciplines, and teaching years. The results showed that the mental health level of college teachers was lower than the current national adult standard; the mental health level of female teachers in colleges was higher than that of male teachers; the p value of mental health of college teachers with different ages, professional titles, education, disciplines, and teaching years was greater than 0.05, indicating that there was no significant difference; the p-value of professional academic and mental health was less than 0.01, indicating that there was a significant correlation, that was, teachers’ professional academic exerted a significant impact on teachers’ mental health. In short, under the background of artificial intelligence and HCI’s rapid development, higher education was moving forward with high quality, and more attention should be paid to the psychological changes of college teaching staff. Reviewed by: Reviewed by: Kuan-Yu Lin, Ling Tung University, Taiwan Sabina Valente, University of Évora, Portugal Alireza Souri, Islamic Azad University, Iran *Correspondence: Baoge Zhang [email protected] Reviewed by: Kuan-Yu Lin, Ling Tung University, Taiwan Sabina Valente, University of Évora, Portugal Alireza Souri, I l i A d U i it I *Correspondence: Baoge Zhang [email protected] Specialty section: This article was submitted to Educational Psychology, a section of the journal Frontiers in Psychology Received: 24 June 2021 Accepted: 15 October 2021 Published: 19 November 2021 Keywords: artificial intelligence, human–computer interaction, colleges, academic management, psychological changes Edited by: Chia-Chen Chen, National Chung Hsing University, Taiwan ORIGINAL RESEARCH published: 19 November 2021 doi: 10.3389/fpsyg.2021.730345 Citation: Guan H, Chen Q, Han S and Zhang B (2021) The Influence of “Artificial Guan H, Chen Q, Han S and Zhang B (2021) The Influence of “Artificial Intelligence + Human–Computer Interaction” on Teachers’ Psychological Changes in Academic Management in Colleges. Front. Psychol. 12:730345. doi: 10.3389/fpsyg.2021.730345 College teacher, as a knowledge-intensive and academic profession, bears a crucial responsibility for training high-quality talents for the country. Li (2019) thought that now there were too many things facing college teachers, not only teaching, still they have the same responsibility with primary and middle school teachers to train builders and successors for the motherland although there are some differences between them. The work of college teachers is a kind of complex mental work that differs from that of primary and middle school teachers (Li, 2019; Kholbutaevna, 2021). Colleges must undertake complex and onerous teaching tasks, as well as more difficult and creative academic November 2021 | Volume 12 | Article 730345 Frontiers in Psychology | www.frontiersin.org Psychological Changes of Teaching Staff Guan et al. tasks (Mccaffrey and Spector, 2018; Park et al., 2021). At present, a series of reforms in the overall education system and management system has been conducted in China’s higher education system to meet the needs of modern development, the internationalization of higher education, the concept of information-based learning and foreign advanced educational technology (Qian et al., 2018; Rogoza et al., 2018). Fischer (2020) thought that the researches on teacher creativity had focused more on values and resources and the reform not only provided a broad space and opportunities for the development of college teachers, but also brought great challenges to teachers. responsibility and interest in work. For school management, Carroll and Dahlstrom (2021) pointed out that schools should not blindly quote the management methods of enterprises due to their particularity. First, the research progress in relevant fields and the shortcomings of existing theoretical research are realized through the research background and the research of existing relevant achievements in the world. Then, the existing important concepts and research background related to the subject are analyzed. Finally, in the AI and HCI environment, the questionnaire is selected to analyze the psychological change indicators of teaching staff. The research innovation is to combine AI and HCI with the psychological changes of college staff, in order to provide reference coping strategies for college staffin academic management (Deng et al., 2021). Citation: There is an increasingly closer relationship between machines and humans with the development of technology and the arrival of the era of artificial intelligence (AI). Wang and Cheng (2019) thought that the man-machine relationship has also undergone corresponding development and changes with the gradual social characterization of intelligent machines with feedback functions such as man-machine dialogue, action, and emotion. The machine has developed from a functional tool attribute to a social role attribute. Bian et al. (2019) pointed out that the impact of intelligent machines on people is not only reflected in the changes of man-machine relations, but also reflected in the impact on people’s needs. Martínez et al. (2021) pointed out that, in general, it dealt with the design and development of machines and computers that best serves the user needs, and the intervention of AI leads to deeper interaction and greater uncertainty in the process of human– computer interaction (HCI). Colleges have more and more requirements, and teachers’ work tasks become heavier and heavier. This is because teachers face greater academic challenges, which affect their psychological status (Brusco et al., 2021). Ji (2020) thought that as a higher education group, college teachers have higher requirements for themselves. With the continuous development of teaching skills and technology, teachers must update their knowledge system and improve their learning and teaching ability, which also brings academic challenges to teachers (Colmenares, 2021). Special attention must be paid to the impact of teachers’ psychological status, teachers’ mental health level must be analyzed, and adaptation strategies must be explored to improve anti stress ability and teachers’ psychological status (Limniou et al., 2019). Based on AI and HCI environment, questionnaire method is selected to analyze teachers’ psychological changes, so as to provide coping strategies for academic management and teaching personnel in colleges and universities. First, the theoretical basis of academic management and psychological characteristics of college teaching staffis expounded. Then, the corresponding questionnaire is designed. Based on this method, the psychological changes of teachers in academic management in colleges are analyzed from multiple angles, and finally the corresponding conclusions are drawn. These conclusions and viewpoints are brand-new, which can be used for reference for subsequent related research. Frontiers in Psychology | www.frontiersin.org Related Theories of the Psychological Status of Teaching Staff g Teachers are a professional group different from other professions. The interpretation of their psychological status should be professional enough to reflect the particularity of their profession (Abascal et al., 2017). Hence, the connotation of teachers’ psychological status should include the psychological status of the general population, and reflect the particularity of teachers’ profession (Babarinde et al., 2021; Baeriswyl et al., 2021). The relationship between the teaching profession and other professions needs to be evaluated to reflect the particularity of teaching profession (Feng and Chen, 2020; Martínez et al., 2021). The media often report accidents that cause personal injury by extreme means such as suicide (Box, 2020; Gibbons et al., 2021). Thus, it is very urgent and crucial to focus on the professional academic and mental health of school teachers, analyze and study the origin of teachers’ mental health problems, and explore the adaptation strategies to maintain teachers’ physical health. The methods of mental health evaluation include self-evaluation, psychological test, pathological evaluation and classification, social adaptability standard, and so on. Common psychological problems are as follows: (1) Depression: it refers to a person’s long-term depression; (2) Paranoia: it often comes from unreasonable ideas about self and the outside world, or even delusions, such as the delusion of being loved, killed, jealous, and worshipped; and (3) Inferiority: people with inferiority tend to have a low evaluation of themselves, and may have other negative emotions or psychological problems, such as depression, excessive shyness, and so on; besides, anxiety, hypersensitivity, jealousy, and cruelty are also common psychological problems. The most prominent feature of the teaching profession is its special object of work – students, which determines the diversity of teachers’ responsibilities. Teachers should not only choose correct education methods and impart knowledge to students, but also exert a positive impact on students and promote their physical and mental development (Ibrahim, 2021). Moreover, teachers must meet the needs of students at different levels or groups (Bentil et al., 2020). The work achievement of teachers lags behind and is difficult to measure due to the particularity of the work object. Teachers’ mental health problems come from their own and external factors. According to the research of relevant literature, there are four kinds of mental health problems in higher vocational teachers, which are emotional problems, interpersonal problems, negative behavior problems, and personality obstacles. Characteristics of Teaching Staff in Academic Management of Colleges College teacher is a highly educated group with an enterprising spirit and strong desire for creation. The academic pressure of higher education reform is unprecedented. Unbearable, excessive and long-term academic work will exert a certain negative impact on teachers’ physical and mental health, leading to an increase of the mental health problems of college teachers in recent years. Artificial Intelligence and Human–Computer Interaction The main research fields of AI include intelligent control, natural language processing, pattern recognition, artificial neural network, machine learning, intelligent robot, and so on. among higher vocational teachers. The problem of interpersonal relationship requires that teachers and students must establish a good interpersonal relationship. Only in this way can they have a good communication with students and effectively transmit knowledge and learning methods to students; for negative behavior problems, teachers with mental health problems will have deviation in behavior, such as negative behavior in work and life. In the future, the two will maintain the current mutually promoting and driven relationship, and will further integrate and develop cooperation. Their integration has reached an unprecedented level (Villegas-Ch et al., 2021; Wang, 2021). The exploration of their integration method makes AI technology more persuasive and HCI technology more natural and practical (Wu et al., 2019; Wu and Song, 2019). The rapid development of China’s higher education provides teachers with broad space and opportunities for development. However, the comprehensive reform of the education system and management system also brings great challenges to teachers. Society has high expectations and requirements for higher education and teachers, and unprecedented vocational academic problems have emerged in the reform of higher education, resulting in some psychological problems of college teachers. The professional academic and psychological status of college teachers have a great practical significance for alleviating their professional academic and improving their psychological status (Treadway et al., 2020). After two major delays in AI and HCI, people no longer imagine that computers are capable of surpassing human beings completely, which is impossible under current technical conditions. People turn to study the most practical and achievable issues, leading to the gradual division of AI into five relatively independent disciplines based on random probability model and calculation, namely, cognitive science, natural language understanding, computer vision, machine learning, and robotics (Zhang et al., 2021). AI and HCI are also reflected in the evaluation of psychological changes of college staff. Wu and Wu (2019) emphasized the application of computers in the study of psychological state. To sum up, AI and HCI gradually run through the academic management of college teaching staff. Artificial Intelligence and Human–Computer Interaction Human–computer interaction and AI are two crucial research fields in the intelligent information age (Horgan et al., 2018). The relationship between them has changed with their development and typical applications based on their deep integration are also reflected in education (Schafer, 2019; Harré, 2021). HCI provides research ideas and application requirements for AI, while AI greatly promotes the transformation and development of HCI technology in the modern world (Vedapradha et al., 2019; Sharma, 2020). AI is a new technology and science for research and development to simulate, extend and expand human intelligence. It simulates human intelligence through machines, such as perception ability (visual perception, auditory perception, and tactile perception) and intelligent behavior (learning ability, memory and thinking ability, and reasoning and planning ability), so that machines can “think and act like people,” and finally machines can do the work that only talents could do in the past. The rapid development of AI has aroused heated discussion. Whether AI can replace people has become the focus of attention. As early as 1993, computer scientist Vernor Vinge put forward the concept of singularity, that is, AI driven computers or robots For the research on teachers’ psychological changes in academic management in colleges, different researchers put forward different coping strategies from different angles. Regarding the research on the current situation and countermeasures of teachers’ mental health, Deng and Chen (2021) pointed out strengthening mental health education, paying attention to teachers’ mental health, respecting teachers’ reasonable needs, and establishing harmonious interpersonal relationships; besides, teachers should learn to adjust themselves. Chen (2019) put forward the countermeasures to solve the psychological problems of college teachers from the three levels of society, school, and individual. Monea (2020) emphasized how to alleviate teachers’ professional pressure and improve teachers’ mental health. Travers and Cooper believed that schools should not only train teachers how to engage in teaching and teach interpersonal skills, but also mobilize teachers’ sense of November 2021 | Volume 12 | Article 730345 Frontiers in Psychology | www.frontiersin.org 2 Psychological Changes of Teaching Staff Guan et al. can design and improve themselves, or design more advanced AI than themselves. In the face of AI, people should not overestimate or underestimate it. It is essential to uphold a rational attitude toward the impact of AI on education. Frontiers in Psychology | www.frontiersin.org Related Theories of the Psychological Status of Teaching Staff Emotional problems involve a wide range, including all bad emotions, so they are also the most common mental health problems The psychological status of teaching staffis affected by multiple factors, including teachers’ personality, cognitive style, interpersonal relationship, coping strategies, and emotions. This exploration focuses on the application of AI in the academic management of teaching staff. The psychological changes of teaching staffwith different ages, education backgrounds, professional titles, disciplines, and teaching years are evaluated. This exploration is based on AI and artificial interactive November 2021 | Volume 12 | Article 730345 3 Psychological Changes of Teaching Staff Guan et al. algorithm to analyze the psychological changes of teaching staff in academic management of colleges in many aspects. Compared with the studies cited, the data research degree of this exploration is deeper and wider, and some new conclusions are found, which has far-reaching significance for the research on the mental health problems of academic management staffin colleges. The feasibility of the scale is tested, and Table 2 displays the results. Fifty subjects are randomly tested. The data of the prediction scale are tested by discrimination test, standard deviation test, and exploratory factor analysis. Then, some invalid items are deleted to form a formal scale. In Table 2, the sphericity test of teacher academic scale is 9299.812, with a high value, and the corresponding probability is 0.000, which is very significant, indicating that there may be sharing factors among elements. Meanwhile, the test statistic value is 0.894, showing that the data is suitable for factor analysis. Exploratory factor analysis is conducted on the teacher academic scale. The scale is extracted by principal component analysis, and the results are rotated orthogonally to the maximum extent. Research Tool SCL-90 is a mental health self-evaluation scale that has been widely used and accepted by Derogatis (1975). It includes 90 evaluation elements with a wider range of content, such as feeling, emotion, sleep, consciousness, and thought. Ten factors, namely somatization (A), compulsion (B), interpersonal sensitivity (C), depression (D), anxiety (E), hostility (F), terror (G), paranoia (H), psychosis (I) and other (J), are adopted to reflect the 10 aspects of psychological symptoms. Questionnaire Design Stratified random sampling is adopted to select teachers from 15 faculties of a college. A total of 300 questionnaires are distributed, and 252 valid questionnaires are finally obtained. The questionnaire consists of teachers’ basic personal information, including gender, age, discipline category, culture category, and education level. This questionnaire is made and issued on Questionnaire Star to collect data. Invalid questionnaires are eliminated, and SPSS25.0 mathematical statistics software is employed to analyze the data of effective questionnaires. The questionnaire has been approved by the participants. It is an anonymous survey for scientific research, and does not involve ethical factors, so the relevant data obtained are strictly confidential. In exploratory factor analysis, the data are first tested for fitness to test whether they are suitable for factor analysis. Table 3 presents the test results. The scale is evaluated by the internal consistency reliability analysis of each factor. As shown in Table 3, the common factors and the Cronbach coefficients of the total table are 0.757, 0.754, 0.753, 0.766, 0.788, 0.765, 0.789, 0.765, 0.768, 0.754, and 0.757, respectively. The results show that the questionnaire has good reliability. The Cronbach coefficients of the three factors and the total table are all above 0.7, which are acceptable, indicating good credibility of the questionnaire. The Overall Reflection of Teachers’ Psychological Condition Unlike the national adult standard (Figure 1), the scores of SCL-90 among 252 college teachers are higher. Besides, the scores of the factors sensitive to interpersonal relationships are significantly higher than the national standard, and the p-value is less than 0.001. Starting with discriminant analysis, first, the total score of each element is calculated. Then, the data are rearranged from top to bottom according to the total score, and 25% of the subjects are divided into high group and low group, respectively. Then, the T-test of independent samples is used to test the difference of subjects’ scores on each question, which can compare whether the difference between the two averages is significant. Besides, some elements are deleted according to whether the element limit ratio reaches a meaningful judgment standard. Next, the higher the standard deviation is, the greater the difference between individuals is and the wider the data distribution is. On the contrary, the smaller the distribution range of individual scores is, the smaller the difference of individual response is. Finally, it is essential to determine the factor analysis, properly test the data and generate the final results. Shen et al. (2019) analyzed social behavior and proposed concept link mining method for analysis, which strongly confirmed AI’s research on psychological changes of university staff. One of the reasons is that the management system, which is formed by the internal fierce competition among the college teachers with very complex mental work, has been reformed. It makes the academic level of teachers’ profession in colleges higher than that in other industries, thus leading to the psychological academic level of teachers, and affecting the level of teachers’ mental health. Then, the existing national adult standards were formulated in the late 1980s, when the social competition was much smaller than today. It is doubtful whether the normative norms still represent the present mental health level of adults in China, which is one of the reasons why the measurement results differ greatly from the national norms. Comparison of the Psychological Status of Teachers With Different Genders The results of SCL-90 (Figure 2) suggest that male teachers have higher factor scores than female teachers. There are significant differences in somatization, depression, hostility, fear, and other psychological problems between male and female teachers, with Questionnaire Table 1 displays the basic demographic characteristics of the respondents. November 2021 | Volume 12 | Article 730345 Frontiers in Psychology | www.frontiersin.org 4 Psychological Changes of Teaching Staff Guan et al. TABLE 1 | Statistics of demographic data of the investigated teaching staff. Gender Age Subject category Educational level Basic information Male Female <35 years old 35–50 years old >50 years old Science departments Liberal arts Undergraduate Master Doctor Number of students 138 114 79 141 32 151 101 85 79 88 Percentage (%) 54.8 45.2 31.3 56.1 12.6 59.9 40.1 33.7 31.3 35 TABLE 1 | Statistics of demographic data of the investigated teaching staff. p-value less than 0.05. There are significant differences in mental symptoms, with p-value less than 0.01. the old and the children in family life; with the strengthening of higher education reform, middle-aged and old college teachers in China focus more on the stability of employment. The knowledge structure and the old teachers’ education mode no longer meet the current higher education needs due to the advent of the information age and explosive growth of knowledge; the spirit of retired teachers and their best efforts is conducive to their mental state. Figure 2 displays the comparison of male and female teachers’ psychological status in SCL-90 results. The psychological level of male teachers is lower than that of female teachers. Their level of somatization, depression, hostility, terror, and other aspects are obviously lower than that of female teachers. Especially, their level of psychiatric symptoms is very low. The social needs of females are often lower than that of male teachers due to their different roles. Male teachers should not only take care of their children and the elderly, but also undertake complex tasks such as leadership, teaching, and academic research. Male teachers tend to take on more responsibilities at work than female teachers, and they are more eager to obtain a sense of achievement in their profession. This strong desire itself will undoubtedly bring psychological pressure to male teachers. Comparison of the Psychological Status of Teachers With Different Educational Backgrounds The qualifications of college teachers are classified into three groups of bachelor’s degree, master’s degree, and doctor’s degree. The results of SCL-90 (Table 4) show that the scores of most symptoms, such as depression, anxiety, and other factors of teachers with a doctoral degree, are higher than those of other teachers with a bachelor degree, while their scores of somatization factor, hostility, and paranoid degree are higher than those of teachers with bachelor’s degree of the same level. However, there is no significant difference in the scores of different disciplines. Comparison of the Psychological Status of Teachers of Different Ages College teachers are categorized into three age groups: young group, middle-aged group, and old group. The results of the SCL- 90 test (Figure 3) illustrate that the scores of hostile factors of middle-aged and old teachers are higher than those of young teachers. However, there is no significant difference in the scores of different groups of teachers, because the p-value is greater than 0.05. The reason for little difference in the psychological status of teachers with different education levels may be that teachers with different educational levels have different expectations. They have different academic skills in competition. For example, regarding the application of teachers in promoting vocational qualifications, colleges have different educational requirements for different vocational qualifications. Doctors have psychological problems because of the high expectation of academics. The survey shows that there is no significant difference in the psychological status of teachers in different age groups, which may be due to the different academic situations faced by teachers of different ages. The experience of young teachers is less, and the practical problems in the management of old and middle-aged teachers are less; young teachers shoulder more psychological academic work in teaching and research; the difficulties they meet promote the academic title; the life of young teachers is the heaviest, and their burden is higher than that of old and middle- aged teachers, especially those facing serious housing problems. Comparison of the Psychological Status of Teachers With Different Titles: Junior, Intermediate, Deputy Senior, and Senior The results of SCL-90 (Table 5) reveal that besides somatization, senior teachers score higher than middle-level teachers in interpersonal sensitivity and other factors. The level of obsessive- compulsive symptoms, interpersonal sensitivity, depression, anxiety, and mental symptoms of deputy senior teachers is higher than that of other teachers. However, there is no significant difference in factor scores among teachers with different professional titles. Comparison of the Psychological Status of Teachers With Different Titles: Junior, Intermediate, Deputy Senior, and Senior The results of SCL-90 (Table 5) reveal that besides somatization, senior teachers score higher than middle-level teachers in interpersonal sensitivity and other factors. The level of obsessive- compulsive symptoms, interpersonal sensitivity, depression, anxiety, and mental symptoms of deputy senior teachers is higher than that of other teachers. However, there is no significant difference in factor scores among teachers with different professional titles. Nowadays, the gap between housing price and wage income, as well as the reform of housing and medical policies, bring special academics to young teachers who have no economic basis to solve basic problems such as housing. Middle-aged teachers should shoulder academic research and teaching, and look after It is believed that there is no significant difference in the psychological status of teachers with different professional titles, which is closely related to the reform of the professional title evaluation system. Colleges with high standard professional title promotion require teachers with different professional titles to have a certain amount of documentation and writing related to education, teaching and scientific research promotion and other November 2021 | Volume 12 | Article 730345 5 Psychological Changes of Teaching Staff Guan et al. TABLE 3 | Reliability analysis. Compulsion Somatization Interpersonal sensitivity Depression Anxiety Hostility Terror Paranoia Other General table Cronbach coefficient 0.757 0.754 0.753 0.766 0.788 0.765 0.789 0.765 0.768 0.754 FIGURE 1 | Comparison of the survey results of college teachers with the national norm (∗p < 0.05, ∗∗∗p < 0.001). FIGURE 2 | Comparison of survey results of male and female college teachers (∗p < 0.05, ∗∗p < 0.01). Frontiers in Psychology | www.frontiersin.org 6 November 2021 | Volume 12 | Article 730345 TABLE 3 | Reliability analysis. TABLE 3 | Reliability analysis. Compulsion Somatization Interpersonal sensitivity Depression Anxiety Hostility Terror Paranoia Other General table Cronbach coefficient 0.757 0.754 0.753 0.766 0.788 0.765 0.789 0.765 0.768 0.754 FIGURE 1 | Comparison of the survey results of college teachers with the national norm (∗p < 0.05, ∗∗∗p < 0.001). Compulsion Somatization Interpersonal sensitivity Depression Anxiety Hostility Terror Paranoia Other General table Cronbach coefficient 0.757 0.754 0.753 0.766 0.788 0.765 0.789 0.765 0.768 0.754 FIGURE 1 | Comparison of the survey results of college teachers with the national norm (∗p < 0.05, ∗∗∗p < 0.001). FIGURE 1 | Comparison of the survey results of college teachers with the national norm (∗p < 0.05, ∗∗∗p < 0.001). Comparison of the Psychological Status of Teachers With Different Titles: Junior, Intermediate, Deputy Senior, and Senior FIGURE 2 | Comparison of survey results of male and female college teachers (∗p < 0.05, ∗∗p < 0.01). FIGURE 2 | Comparison of survey results of male and female college teachers (∗p < 0.05, ∗∗p < 0.01). November 2021 | Volume 12 | Article 730345 Frontiers in Psychology | www.frontiersin.org 6 Guan et al. Psychological Changes of Teaching Staff FIGURE 3 | Comparison of survey results of teachers in different age groups. FIGURE 3 | Comparison of survey results of teachers in different age groups. related professional courses. This undoubtedly brings certain pressure to teachers of different titles and ages. Table 6 reveals that there is no significant difference in the psychological level of teachers in different grades, because the continuous development of modern education technology requires teachers of different ages to change the traditional educational thought, educational concept, and method education, and establish the concept of lifelong learning. At present, the promotion of higher education reform requires teachers of different grades to improve their abilities constantly, making them invincible in competitive colleges. DISCUSSION The reason for little difference in the psychological status of teachers between different disciplines may be the integration of modern disciplines, and fuzzy boundaries, showing a trend of cross integration. With the coming of the knowledge economy, science, engineering, and art teachers are required to have educational methods and contents suitable for the development of the times, so as to match and adapt to the new situation of the current higher education reform. Teachers engaged in science, engineering, and art should conduct teaching and carry out scientific research, which brings about great work pressure. The Implications for Academic Research For a long time, the research of AI focused on the improvement of algorithms and models step by step, Carroll and Dahlstrom (2021) pointed out focusing on the technical content based on prediction ability, while the attention paid to interpretation ability was slightly insufficient. At present, most HCI studies in the world focus on the technical level, ignoring the social level and psychological factors of HCI. However, the success of technology does not mean the success of products and the harmony of the man-machine relationship. Only when the application of technology adapts to human nature and human development, can it be gradually accepted and developed by the world. Present HCI is far from reaching the ideal degree of natural interaction. More and more in-depth research is needed on people’s own mental model and people’s understanding of the HCI process itself. This psychological model includes not only people’s understanding of the operation mode of the system itself, but also people’s understanding of how the operation mode affects their Comparison of the Psychological Status of Teachers in Different Disciplines Teachers are divided into two categories: teachers engaged in scientific and engineering education research and teachers engaged in liberal arts education research. The results of SCL-90 (Figure 4) prove that the scores of teachers engaged in teaching and scientific research and engineering are higher than those of liberal arts teachers engaged in teaching and research, while the difference is not significant. Comparison of the Psychological Status of Teachers of Different Teaching Ages In interaction design, it is essential to suggested that the interaction between human and AI is gradually Psychological Changes of Teaching Staff Guan et al. TABLE 4 | Comparison of survey results of teachers with different educational backgrounds. TABLE 4 | Comparison of survey results of teachers with different educational backgrounds. Somatization Obsession Interpersonal sensitivity Depression Anxiety Hostility Terror Paranoia Other Undergraduate 1.86 ± 0.78 1.75 ± 0.76 1.87 ± 0.76 1.76 ± 0.81 1.78 ± 0.91 1.52 ± 0.77 1.80 ± 0.80 1.77 ± 0.80 1.78 ± 0.37 Master 1.74 ± 0.67 2.03 ± 0.72 1.75 ± 0.70 1.86 ± 0.75 1.70 ± 0.71 1.73 ± 0.71 1.50 ± 0.68 1.72 ± 0.71 1.60 ± 0.73 Doctor 1.90 ± 0.70 2.3 ± 1.82 1.87 ± 0.74 1.99 ± 0.71 1.82 ± 0.76 1.80 ± 0.71 1.56 ± 0.74 1.79 ± 0.73 1.68 ± 0.70 F 0.50 0.77 0.40 0.44 0.19 0.37 0.57 0.17 0.16 P >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 F is the test result. TABLE 5 | Comparison of survey results of teachers with different professional titles. parison of survey results of teachers with different professional titl FIGURE 4 | Comparison of survey results of teachers in different disciplines. FIGURE 4 | Comparison of survey results of teachers in different disciplines. suggested that the interaction between human and AI is gradually deepening, and HCI is being used in the occasion of multi task cooperation. Therefore, the interpretability of AI has become a necessary factor to achieve full human–computer cooperation. cognition and emotion. In interaction design, it is essential to adhere to the human-centered design principle and understand the human information processing process and its operation mechanism, which is of great value for HCI. Compared with previous studies, this exploration obtains the other assistance system to improve the mental health level of college teachers, strengthen the mental health education of teachers, implement a reasonable and effective incentive mechanism, further improve the performance appraisal system of colleges, and further improve the teacher income distribution system. Monea (2020) Comparison of the Psychological Status of Teachers of Different Teaching Ages Teachers are divided into five groups according to the different teaching years: 1–5 years, 6–10 years, 11–15 years, 16–20 years, and above. The results of SCL-90 (Table 6) reveal that the factor scores of teachers teaching for 11–15 years are higher than those of other teachers, while the difference is not significant. November 2021 | Volume 12 | Article 730345 Frontiers in Psychology | www.frontiersin.org 7 Guan et al. Psychological Changes of Teaching Staff TABLE 4 | Comparison of survey results of teachers with different educational backgrounds. Somatization Obsession Interpersonal sensitivity Depression Anxiety Hostility Terror Paranoia Other Undergraduate 1.86 ± 0.78 1.75 ± 0.76 1.87 ± 0.76 1.76 ± 0.81 1.78 ± 0.91 1.52 ± 0.77 1.80 ± 0.80 1.77 ± 0.80 1.78 ± 0.37 Master 1.74 ± 0.67 2.03 ± 0.72 1.75 ± 0.70 1.86 ± 0.75 1.70 ± 0.71 1.73 ± 0.71 1.50 ± 0.68 1.72 ± 0.71 1.60 ± 0.73 Doctor 1.90 ± 0.70 2.3 ± 1.82 1.87 ± 0.74 1.99 ± 0.71 1.82 ± 0.76 1.80 ± 0.71 1.56 ± 0.74 1.79 ± 0.73 1.68 ± 0.70 F 0.50 0.77 0.40 0.44 0.19 0.37 0.57 0.17 0.16 P >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 F is the test result. TABLE 5 | Comparison of survey results of teachers with different professional titles. Somatization Obsession Interpersonal sensitivity Depression Anxiety Hostility Terror Paranoia Other Junior 1.81 ± 0.80 2.1 ± 0.85 1.79 ± 0.88 1.85 ± 0.72 1.77 ± 0.81 1.80 ± 0.84 1.64 ± 0.84 1.82 ± 0.83 1.67 ± 0.74 Intermediate 1.79 ± 0.72 2.05 ± 0.73 1.75 ± 0.72 1.90 ± 0.72 1.72 ± 0.73 1.53 ± 0.73 1.54 ± 0.73 1.75 ± 0.75 1.76 ± 0.73 Deputy senior 1.74 ± 0.67 2.03 ± 0.72 1.75 ± 0.70 1.86 ± 0.75 1.70 ± 0.71 1.73 ± 0.71 1.50 ± 0.68 1.72 ± 0.71 1.60 ± 0.73 Senior 1.90 ± 0.70 2.3 ± 1.82 1.87 ± 0.74 1.99 ± 0.71 1.82 ± 0.76 1.80 ± 0.71 1.56 ± 0.74 1.79 ± 0.73 1.68 ± 0.70 F 0.50 0.77 0.40 0.44 0.19 0.37 0.57 0.17 0.16 P >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 FIGURE 4 | Comparison of survey results of teachers in different disciplines. cognition and emotion. Frontiers in Psychology | www.frontiersin.org ETHICS STATEMENT The studies involving human participants were reviewed and approved by the Northeast Normal University Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. The Implications for Industry and Practices How to make the machine take the initiative to educate, so that users can fully trust and understand AI and participate in interaction, puts forward higher requirements for the interpretability of the system. Moreover, the management of colleges should be people-oriented. Due to time constraints, the occupational mental health status of college teachers is only investigated for some time, and it fails to track the dynamic changes of occupational mental health status in real-time. Based on the analysis of the causes and the discussion of teachers’ psychological problems, measures are taken to improve their mental health education and mental health status, and empirical research is carried out. Studying the professional pressure and mental health problems of college teachers has crucial practical significance for alleviating the professional pressure of college teachers and improving their mental health level. However, due to the limited SCL-90 item scale, the analysis results of the changes in the mental health level of teaching staffneed to be further verified. In the later research, the samples will continue to be expanded and analyzed, and more factors will be included in the discussion. Zhu (2019) discussed the educational reform being caused by AI technology, the reasons why educational reform needs technical support, and how to use technology to promote educational reform, suggesting that the main research field is how technology affects educational reform. This exploration is to take the teaching staffin academic management of colleges as the research object, and explore the evolution process of college teachers’ psychological change by studying from the perspectives of age, professional title, education background, discipline and teaching year. Therefore, relatively speaking, the research content of this exploration is more in-depth and comprehensive. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The Implications for Industry and Practices With the continuous improvement of the autonomy of machines, AI such as Intel’s adaptive robot has realized the ability of November 2021 | Volume 12 | Article 730345 Frontiers in Psychology | www.frontiersin.org 8 Psychological Changes of Teaching Staff Guan et al. TABLE 6 | Comparison of survey results of teachers with different teaching years. Somatization Obsession Interpersonal sensitivity Depression Anxiety Hostility Terror Paranoia Other 1–5 years 1.87 ± 0.78 1.75 ± 0.76 1.86 ± 0.76 1.76 ± 0.81 1.78 ± 0.91 1.54 ± 0.77 1.80 ± 0.80 1.77 ± 0.80 1.78 ± 0.37 6–10 years 1.74 ± 0.67 2.03 ± 0.72 1.76 ± 0.70 1.86 ± 0.75 1.70 ± 0.71 1.76 ± 0.71 1.50 ± 0.68 1.72 ± 0.71 1.60 ± 0.73 11–15 years 1.90 ± 0.70 2.3 ± 1.83 1.87 ± 0.74 1.99 ± 0.71 1.82 ± 0.76 1.80 ± 0.71 1.46 ± 0.74 1.79 ± 0.73 1.69 ± 0.70 16–20 years 1.90 ± 0.80 2.11 ± 0.82 1.88 ± 0.78 1.98 ± 0.77 1.86 ± 0.85 1.83 ± 0.83 1.67 ± 0.86 1.81 ± 0.80 1.77 ± 0.83 Over 20 years 1.85 ± 0.76 2.01 ± 0.75 1.71 ± 0.75 1.83 ± 0.76 1.68 ± 0.78 1.69 ± 0.70 1.47 ± 0.66 1.76 ± 0.74 1.74 ± 0.71 F 0.50 0.77 0.40 0.44 0.19 0.37 0.57 0.17 0.16 P >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 TABLE 6 | Comparison of survey results of teachers with different teaching years. titles, education backgrounds, disciplines, and teaching years; there is a significant correlation between professional academic and mental health, indicating a significant impact of teachers’ professional academic on teachers’ mental health. The research still has limitations. The research is based on the evaluation practice of AI on interactive environment management, ignoring the evaluation of interpretation quality. Meantime, the design based on evaluation from the perspective of developers also has some limitations in index selection. In the future research, a multi-level and multi-angle evaluation system will be built from the perspectives of AI itself, developers, and users. In short, for local colleges and universities, the mental health problems of teaching staffcannot be ignored. A healthy and positive mental state is not only conducive to the improvement of teachers’ academic level and teaching ability, but also promote the high- quality development of the school. autonomous interaction and repair. CONCLUSION HG: conceptualization and writing – original draft. QC: software and resources. SH: methodology. BZ: data curation and supervision. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The theory of teaching staffs’ psychological status, and the characteristics of staffin academic management of colleges are analyzed under the background of AI and HCI. Then, the psychological changes of staffunder different conditions are analyzed through the method of questionnaire and model test. The results show that the mental health level of college teachers is lower than that of the national adult norm; the mental health level of female teachers is higher than that of male teachers; there is no significant difference in the mental health status of college teachers with different ages, professional Frontiers in Psychology | www.frontiersin.org REFERENCES doi: 10.1016/j.chb.2021.106795 Brusco, M., Davis-Stober, C. P., and Steinle, Y. D. (2021). Ising formulations of some graph-theoretic problems in psychological research: models and methods. J. 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Use of offensive language in human- artificial intelligence chatbot interaction: the effects of ethical ideology, social competence, and perceived human-likeness. Comput. Hum. Behav. 121:106795. FUNDING This article is funded by the National Education Science “13th Five-Year Plan” 2019 National Major Topics: Research November 2021 | Volume 12 | Article 730345 Frontiers in Psychology | www.frontiersin.org 9 Psychological Changes of Teaching Staff Guan et al. SUPPLEMENTARY MATERIAL on the Path and Countermeasures of High-quality education development in China in the New Era (No. VFA190004) Phased achievements; Major Tendering project of Key Humanities and Social Science Projects of Colleges and Universities in Zhejiang Province (No. 2016ZB004). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.730345/full#supplementary-material REFERENCES Think. 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Frontiers in Psychology | www.frontiersin.org November 2021 | Volume 12 | Article 730345 REFERENCES Artificial intelligence in cognitive psychology — influence of literature based on artificial intelligence on children’s mental disorders. Aggress Violent Beh. 2021:101590. doi: 10.1016/ j.avb.2021.101590 Copyright © 2021 Guan, Chen, Han and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhu, Z. (2019). Reflection on evaluating the teaching quality of college teachers in China. J. Wuxi Inst. Technol. Curr. Opin. Behav. 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Potential for Hydroclimatically Driven Shifts in Infectious Disease Outbreaks: The Case of Tularemia in High-Latitude Regions

International journal of environmental research and public health/International journal of environmental research and public health

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Received: 28 June 2019; Accepted: 27 September 2019; Published: 2 October 2019 Received: 28 June 2019; Accepted: 27 September 2019; Published: 2 October 2019 Abstract: Hydroclimatic changes may be particularly pronounced in high-latitude regions and can influence infectious diseases, jeopardizing regional human and animal health. In this study, we consider the example of tularemia, one of the most studied diseases in high-latitude regions, which is likely to be impacted by large regional hydroclimatic changes. For this disease case, we use a validated statistical model and develop a method for quantifying possible hydroclimatically driven shifts in outbreak conditions. The results show high sensitivity of tularemia outbreaks to certain combinations of hydroclimatic variable values. These values are within the range of past regional observations and may represent just mildly shifted conditions from current hydroclimatic averages. The methodology developed also facilitates relatively simple identification of possible critical hydroclimatic thresholds, beyond which unacceptable endemic disease levels may be reached. These results call for further research on how projected hydroclimatic changes may affect future outbreaks of tularemia and other infectious diseases in high-latitude and other world regions, with particular focus on critical thresholds to high-risk conditions. More research is also needed on the generality and spatiotemporal transferability of statistical disease models. Keywords: hydroclimatic change; infectious disease; tularemia; critical thresholds; high-latitude regions; Arctic International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health www.mdpi.com/journal/ijerph Potential for Hydroclimatically Driven Shifts in Infectious Disease Outbreaks: The Case of Tularemia in High-Latitude Regions Yan Ma 1,2,* , Arvid Bring 1,2, Zahra Kalantari 1,2 and Georgia Destouni 1,2 1 Stockholm University, Department of Physical Geography, 106 91 Stockholm, Sweden; [email protected] (A.B.); [email protected] (Z.K.); [email protected] (G.D.) 2 Bolin Centre for Climate Research, Stockholm University, 114 19 Stockholm, Sweden * Correspondence: [email protected]; Tel.: +46-73-267-2685 Yan Ma 1,2,* , Arvid Bring 1,2, Zahra Kalantari 1,2 and Georgia Destouni 1,2 1 Stockholm University, Department of Physical Geography, 106 91 Stockholm, Sweden; [email protected] (A.B.); [email protected] (Z.K.); [email protected] (G.D.) 2 Bolin Centre for Climate Research, Stockholm University, 114 19 Stockholm, Sweden * Correspondence: [email protected]; Tel.: +46-73-267-2685 Int. J. Environ. Res. Public Health 2019, 16, 3717; doi:10.3390/ijerph16193717 1. Introduction Hydroclimatic changes in the landscape, such as in runoff[1], evapotranspiration [2], and often both [3], are being observed and projected regionally and globally, adding to, or occasionally opposing, atmospheric changes in precipitation [4]. Such changes have serious implications for terrestrial water cycling and availability, with particular availability impacts in cold regions, where the water cycle also depends on snow and ice conditions [1]. Hydroclimatic changes can also cause profound and complex shifts in the geographical range, prevalence, and/or severity of some infectious diseases [5–9]. The mechanisms of such disease impacts are wide-ranging and may include direct or indirect hydroclimatic influences on abundance of vectors, such as mosquitoes and ticks [10], pathogen survival outside the host [11], host-pathogen interactions that are closely related to community ecology and biodiversity [12,13], dampening of host immunity [14], exposure to water-borne pollution and associated infections [15], including from remobilization of previously frozen pathogens through permafrost thaw under warming [16], and deterioration in health status, e.g., malnutrition and disruption of health systems associated with extreme hydroclimatic events, such as droughts and floods [17]. High-latitude and Arctic regions, where the diversity of animal, plant, and microbial Int. J. Environ. Res. Public Health 2019, 16, 3717; doi:10.3390/ijerph16193717 www.mdpi.com/journal/ijerph 2 of 11 Int. J. Environ. Res. Public Health 2019, 16, 3717 species is low, and where surface temperatures are increasing faster than the global average [18], are subject to particular and in some respects (considering glacier melting and permafrost thaw) more severe and uncertain hydroclimatic changes [19–21] linked to health and infectious diseases [16,22,23]. These particularities and disease links make it essential to understand how such changes can affect infectious diseases, and how to identify and quantify future risk shifts due to changing hydroclimate in these high-latitude regions. Many models have been established to reveal the relations between diseases and hydroclimatic factors. However, the contributions of hydroclimatic changes to predicted disease cases are not directly quantified in these models. In this study, we address this knowledge gap by developing a method for identifying and quantifying hydroclimatic changes and their implications for disease outbreak levels, and possible thresholds to such critical/unacceptable levels, based on a previously established statistical model for the example disease of tularemia in high-latitude areas [24]. Tularemia, caused by the arthropod-borne pathogen Francisella tularensis, affects wild animals and humans, and is one of the most researched high-latitude vector-borne diseases likely to be impacted by hydroclimatic change [25]. 1. Introduction It s prevalence is verified in Europe, Asia, and America [26] and its natural foci includes high-latitude areas in, e.g., Russia [27], Finland, and Sweden [28]. Climate change is further expected to expand or shift the geographical distribution of tularemia in Russia and the U.S.A. [29,30], and increase the disease burden in high-endemic areas of Sweden [31]. However, contradictory predictions regarding the latter effect have also been published [32]. The tularemia model used in this study is based on mosquitoes as the main disease vector in the large boreal forest regions of Alaska, Sweden, Finland, and Russia [33–35]. A lthough it is highly likely that other factors also affect outbreaks, the model has been used successfully to capture a series of annual outbreaks in Dalarna County, Sweden, predicting six out of seven high-incidence years occurring between 1981 and 2007 in this region [24]. These successes indicate that the model has high potential to successfully represent scenarios of possible future tularemia outbreaks under hydroclimatic change in such high-latitude areas, which is a main reason for its selection and being used in the present study. The study also considers and addresses the approach recommended by the World Health Organization (WHO) for assessing climate change impacts on health, by applying the model for possible identification of driver thresholds beyond which unacceptable health hazards may occur under different future hydroclimatic scenarios [36]. 2. Methods with analytical solution development The blue curve in (a) shows the number of tularemia cases in the current year (Tul) as a function of the number of cases in the preceding year (Tullag); the intersection of the blue curve with the black 1:1 line at Tul=Tullag=N* identifies the expected endemic level for the (arbitrary) considered, unchanging hydroclimatic conditions. The blue and orange curves in (b) illustrate the general convergence over time to the endemic level Tul=N* (dashed line) for any initial Tullag value (N01, N02) under the given unchanging hydroclimatic setting. Conceptual diagram of resulting annual number of tularemia outbreaks under some given, Tullag value (N01, N02) under the given unchanging hydroclimatic setting. Equation (3) implies that, for any given (prevailing/projected) combination of hydroclimat variable values determining the value of A, the value of Tul can be computed from the correspondin The number of tularemia cases (Tul) in any current/projected year thus becomes a function of a single variable: the number of tularemia cases in the preceding year (Tullag) to the power of 0.75, a constant derived from the rearrangement of Equation (1) through Equation (2). g p p value of Tullag for the preceding year. The associated functional relationship Tul=F(Tullag) is illustrate in Figure 1a. The blue curve (passing through the origin and convex upwards) intersects with th black 1:1 line (of Tul=Tullag, drawn from the origin with a slope of 1) at some value of Tul=Tullag=N The black line represents the theoretical case of unchanging number of tularemia outbreaks betwee years (Tul=Tullag) and its intersection with the blue curve Tul=F(Tullag) at Tul=Tullag=N* indicates possible steady state number of outbreaks, N*. Through iteration over succeeding years with an given constant value of A (i.e., constant disease-relevant hydroclimatic conditions) and any initi value of Tullag, either larger (N01) or smaller (N02) than N*, the resulting number of tularem outbreaks Tul will in following years converge towards and stabilize at N* (Figure 1b). The resultin value of N* thus represents an expected endemic level for the hydroclimatic conditions considere (determining the value of A). Intersection at the origin, i.e., N*=0, means that the number of annu tularemia outbreaks will tend to vanish over a series of years under the given hydroclimat conditions. 2. Methods with analytical solution development Public Health 2019, 16, 3717 I E i bli l h 3 of 11 3 f represent all the hydroclimate variables by parameter A (Equation (2)), which is a constant in an unchanging hydroclimate setting: A = G(RMA(Q,T), STlag , SP, CW) (2 represent all the hydroclimate variables by parameter A (Equation (2)), which is a constant in an unchanging hydroclimate setting: A = G(RMA(Q,T), STlag , SP, CW) (2 A = G(RMA(Q,T), STlag, SP, CW) (2) Tul = F(Tullag ) = A * Tullag0.75 (3) Tul = F(Tullag ) = A * Tullag0.75 (3) mia cases (Tul) in any current/projected year thus becomes a function of r of tularemia cases in the preceding year (Tullag) to the power of 0.75, t f E ti (1) th h E ti (2) A = G(RMA(Q,T), STlag, SP, CW) (2) Tul = F(Tullag ) = A * Tullag0.75 (3) Tul = F(Tullag ) = A * Tullag0.75 (3) The number of tularemia cases (Tul) in any current/projected year thus becomes a function of single variable: the number of tularemia cases in the preceding year (Tullag) to the power of 0.75, t t d i d f th t f E ti (1) th h E ti (2) A = G(RMA(Q,T), STlag, SP, CW) (2) T l F(T l ) A * T l 0 75 (3) Tul = F(Tullag ) = A * Tullag0.75 (3 ularemia cases (Tul) in any current/projected year thus becomes a function of A = G(RMA(Q,T), STlag, SP, CW) (2) Tul = F(Tullag ) = A * Tullag0.75 (3) f t l i (T l) i t/ j t d th b f ti f (2)(3 Tul = F(Tullag ) = A * Tullag0.75 The number of tularemia cases (Tul) in any current/projected year thus becomes a funct single variable: the number of tularemia cases in the preceding year (Tullag) to the power o t t d i d f th t f E ti (1) th h E ti (2) (3) 0.75, g q ( ) g q ( ) Figure 1. Conceptual diagram of resulting annual number of tularemia outbreaks under some give unchanging hydroclimatic conditions, as a function of (a) corresponding number of outbreaks in t preceding year and (b) time. 2. Methods with analytical solution development The blue curve in (a) shows the number of tularemia cases in the curre year (Tul) as a function of the number of cases in the preceding year (Tullag); the intersection of t blue curve with the black 1:1 line at Tul=Tullag=N* identifies the expected endemic level for t (arbitrary) considered, unchanging hydroclimatic conditions. The blue and orange curves in ( illustrate the general convergence over time to the endemic level Tul=N* (dashed line) for any init T l l (N N ) d th i h i h d li ti tti Figure 1. Conceptual diagram of resulting annual number of tularemia outbreaks under some given, unchanging hydroclimatic conditions, as a function of (a) corresponding number of outbreaks in the preceding year and (b) time. The blue curve in (a) shows the number of tularemia cases in the current year (Tul) as a function of the number of cases in the preceding year (Tullag); the intersection of the blue curve with the black 1:1 line at Tul=Tullag=N* identifies the expected endemic level for the (arbitrary) considered, unchanging hydroclimatic conditions. The blue and orange curves in (b) illustrate the general convergence over time to the endemic level Tul=N* (dashed line) for any initial Tullag value (N01, N02) under the given unchanging hydroclimatic setting. g q Figure 1. Conceptual diagram of resulting annual number of tularemia outbreaks under some given unchanging hydroclimatic conditions, as a function of (a) corresponding number of outbreaks in th preceding year and (b) time. The blue curve in (a) shows the number of tularemia cases in the curren year (Tul) as a function of the number of cases in the preceding year (Tullag); the intersection of th blue curve with the black 1:1 line at Tul=Tullag=N* identifies the expected endemic level for th (arbitrary) considered, unchanging hydroclimatic conditions. The blue and orange curves in (b illustrate the general convergence over time to the endemic level Tul=N* (dashed line) for any initia Figure 1. Conceptual diagram of resulting annual number of tularemia outbreaks under some given, unchanging hydroclimatic conditions, as a function of (a) corresponding number of outbreaks in the preceding year and (b) time. 2. Methods with analytical solution development The statistical model [24] of annual numbers of humans contracting tularemia is based on identified key variables for mosquito abundance and tularemia-relevant local weather, using available data for the period 1981-2007 in Dalarna County, Sweden. Specifically, the model is as follows: Tul = EXP(−11 + 0.52 log2 Tullag + 0.54 log2 RMA + 0.65 STlag + 0.012 SP −0.15 CW) (1 (1) and it relates the annual number of tularemia cases (Tul) to relative annual mosquito abundance (RMA) (which in turn depends on some periodic hydroclimatic variables of water flow (Q) and temperature conditions (T), as explained further below), summer temperature in the preceding year (STlag), summer precipitation in the same year (SP), winter days with low snow coverage (CW), and number of tularemia cases in the preceding year (Tullag). g To quantify and readily compare the sensitivity of the number of tularemia cases, Tul, for different hydroclimatic conditions, and ultimately identify possible critical condition thresholds, we derived a model-implicit comparative index in terms of expected endemic levels by rearranging the analytical model Equation (1). This comparative index is the expected number of disease cases that each combination of hydroclimatic conditions will tend to drive the number of tularemia cases towards (asymptotically for temporally stable conditions) based on the underlying model equations (Figure 1). To mathematically derive this index of expected endemic level, the model was first rearranged to Int. J. Environ. Res. 2. Methods with analytical solution development Note that the index of expected endemic level N* does not represent the actual tularemia cas Equation (3) implies that, for any given (prevailing/projected) combination of hydroclimatic variable values determining the value of A, the value of Tul can be computed from the corresponding value of Tullag for the preceding year. The associated functional relationship Tul=F(Tullag) is illustrated in Figure 1a. The blue curve (passing through the origin and convex upwards) intersects with the black 1:1 line (of Tul=Tullag, drawn from the origin with a slope of 1) at some value of Tul=Tullag=N*. The black line represents the theoretical case of unchanging number of tularemia outbreaks between years (Tul=Tullag) and its intersection with the blue curve Tul=F(Tullag) at Tul=Tullag=N* indicates a possible steady state number of outbreaks, N*. Through iteration over succeeding years with any given constant value of A (i.e., constant disease-relevant hydroclimatic conditions) and any initial value of Tullag, either larger (N01) or smaller (N02) than N*, the resulting number of tularemia outbreaks Tul will in following years converge towards and stabilize at N* (Figure 1b). The resulting value of N* thus represents an expected endemic level for the hydroclimatic conditions considered (determining the value of A). Intersection at the origin, i.e., N*=0, means that the number of annual tularemia outbreaks will tend to vanish over a series of years under the given hydroclimatic conditions. p p in a specific year with weather conditions as in a considered combination of hydroclimatic mod variables, since weather does not remain constant over consecutive years. However, the level N Note that the index of expected endemic level N* does not represent the actual tularemia cases in a specific year with weather conditions as in a considered combination of hydroclimatic model 4 of 11 Int. J. Environ. Res. Public Health 2019, 16, 3717 variables, since weather does not remain constant over consecutive years. However, the level N* works well as a comparative indicator of how different hydroclimatic factors contribute to disease outbreaks, and how the number of tularemia cases will tend to change driven by changes in long-term average hydroclimate. The latter are much slower than weather changes since, by definition, climate is the long-term average of short-term weather conditions. Int. J. Environ. Res. Public Health 2019, 16, x 4 of 11 term average hydroclimate. 2. Methods with analytical solution development The latter are much slower than weather changes since, by definition, climate is the long term average of short term weather conditions g g After developing and using this analytical solution method, we further evaluated the effects of different hydroclimatic variable conditions on disease outbreaks. For this, we calculated the expected endemic level N* for different values of any individual model variable (RMA, STlag, SP, and CW) within its data-given observation range (0.09–39, 12.2–16.8 ◦C, 143–342 mm, and 0–28 days, respectively) [24], while keeping the other model variables at their median values. For RMA, which is not in itself a hydroclimatic variable but depends on such variables, we also calculated its dependence on the relevant hydroclimatic variable values of maximum standardized river flow for two periods preceding the evaluation time t, denoted Q1 and Q2 (for 36–42 days and 22–28 days, respectively, before time t), and mean temperature (T) over 1–7 days before time t. This calculation was based on meteorological and hydrological data for Q1, Q2, and T, obtained from the Swedish Meteorological and Hydrological Institute (www.smhi.se) for the years 1981–2007. Given that any possible extreme daily values of RMA are filtered out when considering the annual median of this variable for use in Equation (1), we calculated the range of annual median RMA for ranges of Q1 and Q2 between −1 and 1 standard deviation of each variable (Figure 2a). Furthermore, we used the observed summer temperature range (12.3 ◦C to 16.8 ◦C) for T (Figure 2b). Since coefficients of Q1 and Q2 in the model are similar under the same hydroclimate, we only plotted scenarios where Q1 is equal to Q2. For scenarios where Q1 is not equal to Q2, derived values of RMA will fall between the two extreme line boundaries (blue and green lines in Figure 2a,b). For these value ranges, the resulting RMA variation is from 0.02 up to 39.9 (Figure 2), which is similar to the previously reported RMA range (0.09–39) [24]. The observation-based ranges of underlying hydroclimatic variables Q1, Q2, and T considered here thus reproduce a consistent range of annual RMA, as needed for hydroclimatic sensitivity assessment of RMA in the model (Equation (1)). climate is the long-term average of short-term weather conditions. After developing and using this analytical solution method, we further evaluated the effects of different hydroclimatic variable conditions on disease outbreaks. 2. Methods with analytical solution development For this, we calculated the expected endemic level N* for different values of any individual model variable (RMA, STlag, SP, and CW) within its data-given observation range (0.09–39, 12.2–16.8 °C, 143–342 mm, and 0–28 days, respectively) [24], while keeping the other model variables at their median values. For RMA, which is not in itself a hydroclimatic variable but depends on such variables, we also calculated its dependence on the relevant hydroclimatic variable values of maximum standardized river flow for two periods preceding the evaluation time t, denoted Q1 and Q2 (for 36–42 days and 22–28 days, respectively, before time t), and mean temperature (T) over 1–7 days before time t. This calculation was based on meteorological and hydrological data for Q1, Q2, and T, obtained from the Swedish Meteorological and Hydrological Institute (www.smhi.se) for the years 1981–2007. Given that any possible extreme daily values of RMA are filtered out when considering the annual median of this variable for use in Equation (1), we calculated the range of annual median RMA for ranges of Q1 and Q2 between −1 and 1 standard deviation of each variable (Figure 2a). Furthermore, we used the observed summer temperature range (12.3 °C to 16.8 °C) for T (Figure 2b). Since coefficients of Q1 and Q2 in the model are similar under the same hydroclimate, we only plotted scenarios where Q1 is equal to Q2. For scenarios where Q1 is not equal to Q2, derived values of RMA will fall between the two extreme line boundaries (blue and green lines in Figure 2a and 2b). For these value ranges, the resulting RMA variation is from 0.02 up to 39.9 (Figure 2), which is similar to the previously reported RMA range (0.09–39) [24]. The observation-based ranges of underlying hydroclimatic variables Q1, Q2, and T considered here thus reproduce a consistent range of annual RMA, as needed for h d li ti iti it t f RMA i th d l (E ti (1)) y y ( q ( )) (a) (b) Figure 2. Relative mosquito abundance (RMA) as a function of (a) mean temperature (T) 1-7 days before evaluation time t, and (b) maximum standardized river flows (in associated standard deviations) Q1 and Q2 at time periods 36-42 days and 22-28 days, respectively, before time t. 3. Results Figure 3 shows modeled expected endemic levels (crosses in the left-hand panels, dashed lines in the right-hand panels) for different examples of hydroclimatic variable values considered. For all hydroclimatic variable sets, median-range values (indicated by bold black lines in all panels) showed endemic levels close to zero. However, even small variable deviations from these median values can trigger large shifts in endemic levels. Modeled endemic levels are particularly sensitive to the value of RMA, and thereby to the underlying values of Q1, Q2, and T (Figure 2). For example, a value of 4.1 for RMA, which is higher than the median value, but far from the maximum observed value, resulted in endemic level close to 800 annual cases. This was much higher than the near-zero endemic level for median RMA and also higher than, for example, the resulting endemic level of slightly above 400 for the maximum value of 342 mm for SP. For CW, the highest resulting endemic level was less than 100, which is much lower than the maximum levels that other variables could trigger. gg To directly compare result sensitivity across all variables, the endemic level response to variable value shifts were plotted over the same standardized value range for all variables (Figure 4). The most dominant variable was then indeed found to be RMA, for which even values far below its maximum observed value yielded very high endemic levels. The next most dominant variables were STlag and SP, for which variations within the observed range, but close to their respective observed maximum value, raised the endemic level to 1200 and 400 annual cases, respectively. In comparison with the other model variables, CW variation contributed much less to outbreak variation (Figure 4). Endemic level can also be used as an index for identification of hydroclimatic thresholds to critical/unacceptable health hazards (Figure 5). A s a basis for such identification, a highest societally acceptable endemic level (Nacc) needs to be defined (upper boundary of gray area in Figure 5b,c). Combinations of observed or projected hydroclimate variable values leading to endemic levels at or around Nacc can then be identified as critical thresholds (e.g., variable combination number 2, light blue in Figure 5) between hydroclimatic conditions that are acceptable (e.g., yellow combination number 1) and those that are high-risk (green, orange, dark blue combinations 3–5) from a health/disease perspective. 2. Methods with analytical solution development The results in (a) are for different Q1 and Q2 values (in associated standard deviations) and the results in (b) are for different T values. All Q1, Q2, and T values considered are within their respective observation-based value ranges. Figure 2. Relative mosquito abundance (RMA) as a function of (a) mean temperature (T) 1–7 days before evaluation time t, and (b) maximum standardized river flows (in associated standard deviations) Q1 and Q2 at time periods 36–42 days and 22–28 days, respectively, before time t. The results in (a) are for different Q1 and Q2 values (in associated standard deviations) and the results in (b) are for different T values. A ll Q1, Q2, and T values considered are within their respective observation-based value ranges. (b) (a) (b) (a) Figure 2. Relative mosquito abundance (RMA) as a function of (a) mean temperature (T) 1-7 days before evaluation time t, and (b) maximum standardized river flows (in associated standard deviations) Q1 and Q2 at time periods 36-42 days and 22-28 days, respectively, before time t. The results in (a) are for different Q1 and Q2 values (in associated standard deviations) and the results in (b) are for different T values. All Q1, Q2, and T values considered are within their respective observation-based value ranges. Figure 2. Relative mosquito abundance (RMA) as a function of (a) mean temperature (T) 1–7 days before evaluation time t, and (b) maximum standardized river flows (in associated standard deviations) Q1 and Q2 at time periods 36–42 days and 22–28 days, respectively, before time t. The results in (a) are for different Q1 and Q2 values (in associated standard deviations) and the results in (b) are for different T values. A ll Q1, Q2, and T values considered are within their respective observation-based value ranges. 5 of 11 Int. J. Environ. Res. Public Health 2019, 16, 3717 3. Results Resulting number of outbreaks and endemic level of tularemia for different hydroclimatic conditions in terms of (a,b) relative mosquito abundance (RMA) (hydroclimatically determined), (c,d) summer temperature in preceding year (STlag), (e,f) summer precipitation (SP), and (g,h) number of cold winter days with low snow coverage (CW). Panels on the left (right) show tularemia cases (solid colored lines) in current year as a function of cases in the preceding year (time), for different given values of the specific hydroclimate variable considered in each panel, and an initial number of 200 cases (all panels). Dot-dashed lines in the left-hand panels indicate results for observed minimum and maximum hydroclimatic variable values, while the bold line indicates the observed median variable value. The dashed lines in the right-hand panels show the resulting endemic levels (crosses in the left- hand panels). Figure 3. Resulting number of outbreaks and endemic level of tularemia for different hydroclimatic conditions in terms of (a,b) relative mosquito abundance (RMA) (hydroclimatically determined), (c,d) summer temperature in preceding year (STlag), (e,f) summer precipitation (SP), and (g,h) number of cold winter days with low snow coverage (CW). Panels on the left (right) show tularemia cases (solid colored lines) in current year as a function of cases in the preceding year (time), for different given values of the specific hydroclimate variable considered in each panel, and an initial number of 200 cases (all panels). Dot-dashed lines in the left-hand panels indicate results for observed minimum and maximum hydroclimatic variable values, while the bold line indicates the observed median variable value. The dashed lines in the right-hand panels show the resulting endemic levels (crosses in the left-hand panels). Figure 3. Resulting number of outbreaks and endemic level of tularemia for different hydroclimatic conditions in terms of (a,b) relative mosquito abundance (RMA) (hydroclimatically determined), (c,d) summer temperature in preceding year (STlag), (e,f) summer precipitation (SP), and (g,h) number of cold winter days with low snow coverage (CW). Panels on the left (right) show tularemia cases (solid colored lines) in current year as a function of cases in the preceding year (time), for different given values of the specific hydroclimate variable considered in each panel, and an initial number of 200 cases (all panels). Dot-dashed lines in the left-hand panels indicate results for observed minimum and maximum hydroclimatic variable values, while the bold line indicates the observed median variable value. 3. Results A cceptable endemic level here is different from the alert thresholds for epidemic-prone conditions of other diseases defined by WHO for EWARN (Early Warning Alert and Response Network) [37], because it is not the actual cases that will happen in a certain year but a comparative indicator of the tendency of tularemia cases driven by different hydroclimatic conditions. Therefore, the acceptable level of this indicator has to be further studied and defined by relevant agencies, based on estimated health risks associated with disease outbreak numbers approaching or becoming greater than this acceptable endemic level. 6 of 11 6 of 11 Int. J. Environ. Res. Public Health 2019, 16, 3717 Int J Environ Res Public Health 2019 16 x Figure 3. Resulting number of outbreaks and endemic level of tularemia for different hydroclimatic conditions in terms of (a,b) relative mosquito abundance (RMA) (hydroclimatically determined), (c,d) summer temperature in preceding year (STlag), (e,f) summer precipitation (SP), and (g,h) number of cold winter days with low snow coverage (CW). Panels on the left (right) show tularemia cases (solid colored lines) in current year as a function of cases in the preceding year (time), for different given values of the specific hydroclimate variable considered in each panel, and an initial number of 200 cases (all panels). Dot-dashed lines in the left-hand panels indicate results for observed minimum and maximum hydroclimatic variable values, while the bold line indicates the observed median variable l Th d h d li i th i ht h d l h th lti d i l l ( i th l ft Figure 3. Resulting number of outbreaks and endemic level of tularemia for different hydroclimatic conditions in terms of (a,b) relative mosquito abundance (RMA) (hydroclimatically determined), (c,d) summer temperature in preceding year (STlag), (e,f) summer precipitation (SP), and (g,h) number of cold winter days with low snow coverage (CW). Panels on the left (right) show tularemia cases (solid colored lines) in current year as a function of cases in the preceding year (time), for different given values of the specific hydroclimate variable considered in each panel, and an initial number of 200 cases (all panels). Dot-dashed lines in the left-hand panels indicate results for observed minimum and maximum hydroclimatic variable values, while the bold line indicates the observed median variable value The dashed lines in the right-hand panels show the resulting endemic levels (crosses in the Figure 3. 3. Results The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0,1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). cold winter days with low snow coverage (CW). Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of disease can be identified. The colored curves and symbols show (a) examples of hydroclimatic variable value combinations (1–5; different colored lines) within the respective observed/projected value range of each variable; the different points that each line passes through in the four normalized axes represent the values of relevant variables in this hydroclimatic setting; (b) number of disease cases as a function of number of cases in the preceding year (as explained in Methods, this function can be obtained by substituting values of the four relevant variables from panel (a) into the model), and endemic level (filled black squares) for the hydroclimatic combination examples (1–5, with similarly colored lines as) in panel (a), compared with the maximum societally acceptable endemic level (Nacc, upper boundary of gray zone); and (c) endemic levels (filled colored circles) for each hydroclimatic combination example (1–5, with similarly colored symbols as the corresponding curves) in panel (a), plotted in relation to the observed/projected variable range (illustrated here for Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of disease can be identified. 3. Results The colored curves and symbols show (a) examples of hydroclimatic variable value (CW). g ( Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of disease can be identified. The colored curves and symbols show (a) examples of hydroclimatic variable value combinations (1–5; different colored lines) within the respective observed/projected value range of each variable; the different points that each line passes through in the four normalized axes represent the values of relevant variables in this hydroclimatic setting; (b) number of disease cases as a function of number of cases in the preceding year (as explained in Methods, this function can be obtained by substituting values of the four relevant variables from panel (a) into the model), and endemic level (filled black squares) for the hydroclimatic combination examples (1–5, with similarly colored lines as) in panel (a), compared with the maximum societally acceptable endemic level (Nacc, upper boundary of gray zone); and (c) endemic levels (filled colored circles) for each hydroclimatic combination example (1–5, with similarly colored symbols as the corresponding curves) in panel (a) plotted in relation to the observed/projected variable range (illustrated here for Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of disease can be identified. The colored curves and symbols show (a) examples of hydroclimatic variable value combinations (1–5; different colored lines) within the respective observed/projected value range of each variable; the different points that each line passes through in the four normalized axes represent the values of relevant variables in this hydroclimatic setting; (b) number of disease cases as a function of number of cases in the preceding year (as explained in Methods, this function can be obtained by substituting values of the four relevant variables from panel (a) into the model), and endemic level (filled black squares) for the hydroclimatic combination examples (1–5, with similarly colored lines as) in panel (a), compared with the maximum societally acceptable endemic level (Nacc, upper boundary of gray zone); and (c) endemic levels (filled colored circles) for each hydroclimatic combination example (1–5, with similarly colored symbols as the corresponding curves) in panel (a), plotted in relation to the observed/projected variable range (illustrated here for Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of disease can be identified. 3. Results The colored curves and symbols show (a) examples of hydroclimatic variable value combinations (1–5; different colored lines) within the respective observed/projected value range of each variable; the different points that each line passes through in the four normalized axes represent the values of relevant variables in this hydroclimatic setting; (b) number of disease cases as a function of number of cases in the preceding year (as explained in Methods, this function can be obtained by substituting values of the four relevant variables from panel (a) into the model), and endemic level (filled black squares) for the hydroclimatic combination examples (1–5, with similarly colored lines as) in panel (a), compared with the maximum societally acceptable endemic level (Nacc, upper boundary of gray zone); and (c) endemic levels (filled colored circles) for each hydroclimatic combination example (1–5, with similarly colored symbols as the corresponding curves) in panel (a), plotted in relation to the observed/projected variable range (illustrated here for Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of disease can be identified. The colored curves and symbols show (a) examples of hydroclimatic variable value combinations (1–5; different colored lines) within the respective observed/projected value range of each variable; the different points that each line passes through in the four normalized axes represent the values of relevant variables in this hydroclimatic setting; (b) number of disease cases as a function of number of cases in the preceding year (as explained in Methods, this function can be obtained by substituting values of the four relevant variables from panel (a) into the model), and endemic level (filled black squares) for the hydroclimatic combination examples (1–5, with similarly colored lines as) in panel (a), compared with the maximum societally acceptable endemic level (Nacc, upper boundary of gray zone); and (c) endemic levels (filled colored circles) for each hydroclimatic combination example (1–5, with similarly colored symbols as the corresponding curves) in panel (a), plotted in relation to the observed/projected variable range (illustrated here for simplicity in just one dimension, the x-axis, whereas the actual range space is multi-dimensional depending on the number of hydroclimatic variables considered). cold winter days with low snow coverage cold winter days with low snow coverage (CW). Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of Figure 5. Schematic illustration of how hydroclimatic thresholds to hazardous endemic levels of disease can be identified. 3. Results The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0,1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0,1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. 3. Results The dashed lines in the right-hand panels show the resulting endemic levels (crosses in the left- hand panels). Figure 3. Resulting number of outbreaks and endemic level of tularemia for different hydroclimatic conditions in terms of (a,b) relative mosquito abundance (RMA) (hydroclimatically determined), (c,d) summer temperature in preceding year (STlag), (e,f) summer precipitation (SP), and (g,h) number of cold winter days with low snow coverage (CW). Panels on the left (right) show tularemia cases (solid colored lines) in current year as a function of cases in the preceding year (time), for different given values of the specific hydroclimate variable considered in each panel, and an initial number of 200 cases (all panels). Dot-dashed lines in the left-hand panels indicate results for observed minimum and maximum hydroclimatic variable values, while the bold line indicates the observed median variable value. The dashed lines in the right-hand panels show the resulting endemic levels (crosses in the left-hand panels). 7 of 11 7 of 11 Int. J. Environ. Res. Public Health 2019, 16, 3717 Int. J. Environ. Res. Public Health 2019, 16, x Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag) summer precipitation in current year (SP) and number of Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0,1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. The values of all variables are normalized to the same range [0–1], shown on the x-axis. The variables are: relative mosquito abundance (RMA), summer temperature in the preceding year (STlag), summer precipitation in current year (SP), and number of cold winter days with low snow coverage (CW). Figure 4. Sensitivity of endemic levels of tularemia to shifts in individual hydroclimate variable values within the range of past observations. 4. Discussion Using a previously tested and validated statistical disease model, we computed model-implicit expected endemic levels of tularemia with hydroclimatic variable values within their respective ranges of observations. We demonstrated that the computed expected endemic level can be used as a comparative index for analyzing the sensitivity of tularemia outbreaks to hydroclimatic conditions and for identifying critical hydroclimatic thresholds to high-risk outbreak conditions. Our analysis revealed that tularemia outbreaks are most sensitive to relative mosquito abundance (RMA) and associated underlying periodic hydroclimatic conditions of flow (Q1 and Q2) and mean temperature (T), followed by summer temperature in the preceding year (STlag) and summer precipitation in the current year (SP). Number of cold winter days with low snow coverage (CW) has the least influence on outbreaks and is the only hydroclimatic variable with a negative relationship with outbreaks, i.e., number of outbreaks and endemic level decreased with increasing CW (Figure 3g,h, Figure 4 ). Although only 370 people were diagnosed with tularemia in the study area of Dalarna County over the 27-year study period (1981–2007) used for development of the statistical model [24], our analysis revealed that certain combinations of relevant hydroclimatic variables, with values within the historically observed ranges, have high potential to trigger many more outbreaks. Projections of future climate suggest that the range of hydroclimatic variations may move outside the historical range for many locations [38], implying even more threatening future conditions. Previous studies have also investigated methods for determining the sensitivity of infectious diseases to hydroclimate change. Time-series studies, especially correlation or regression analyses [39,40], and geographical comparisons [41], have been used for this purpose. A recent study [41] reports advantages of geographical comparisons over time-series analysis, such as the possibility of using short data time series. However, that study used relative sensitivity as a statistical indicator of climate-sensitive infectious diseases, revealing the impact of only a single variable without considering temporal autocorrelation effects, whereby high incidence in one month or year might increase transmission potential in subsequent months or years. In contrast, the use of endemic level as an indicator, based on a validated statistical disease model, considers both interactions among several variables and temporal autocorrelations. Moreover, endemic level may be an effective comparative indicator across different regions and/or different infectious diseases, if validated statistical models are available for identifying endemic levels in comparable ways. 3. Results The colored curves and symbols show (a) examples of hydroclimatic variable value combinations (1–5; different colored lines) within the respective observed/projected value range of each variable; the different points that each line passes through in the four normalized axes represent the values of relevant variables in this hydroclimatic setting; (b) number of disease cases as a function of number of cases in the preceding year (as explained in Methods, this function can be obtained by substituting values of the four relevant variables from panel (a) into the model), and endemic level (filled black squares) for the hydroclimatic combination examples (1–5, with similarly colored lines as) in panel (a), compared with the maximum societally acceptable endemic level (Nacc, upper boundary of gray zone); and (c) endemic levels (filled colored circles) for each hydroclimatic combination example (1–5, with similarly colored symbols as the corresponding curves) in panel (a), plotted in relation to the observed/projected variable range (illustrated here for simplicity in just one dimension, the x-axis, whereas the actual range space is multi-dimensional depending on the number of hydroclimatic variables considered). Int. J. Environ. Res. Public Health 2019, 16, 3717 8 of 11 4. Discussion Here we only considered historic variable observation ranges, but the method for identifying endemic level and using it as an index for further identification of threshold hydroclimatic conditions can also be used with projected possible future values of the relevant hydroclimatic variables. Endemic levels and probability of exceeding hydroclimatic thresholds can then be compared between past conditions and projected future scenarios. Such comparisons will be useful for studying whether, how, and to what extent future hydroclimate changes may affect disease outbreaks and associated risks. The WHO has performed a quantitative risk assessment of the effects of climate change on mortality caused by diarrhea, malaria, and dengue fever, using outcome-specific models to estimate future infectious disease-induced mortality with and without climate change [42]. The differences between the two scenarios are then considered to be climate-driven impacts. A s in the present study, the aim of the WHO assessment is to predict health impacts of climate change, but its approach requires large amounts of data and introduces uncertainty through choices of baseline climate and socioeconomic development, education, and technology scenarios for the future. In comparison, the method developed here only requires data on relevant hydroclimatic conditions for indicating likely trends in tularemia cases under different future situations. Limitations of the method presented here include various constraints in the underlying statistical model, such as not representing changes in human behavior. For example, incorrect prediction by the model of tularemia occurrence in year 1987 may have been caused by reduction in forest berry picking and hunting due to the Chernobyl disaster in the preceding year, leading to minimized exposure to tularemia [24]. Moreover, the method assumes independence of the considered hydroclimatic variables 9 of 11 Int. J. Environ. Res. Public Health 2019, 16, 3717 from societal factors that, in reality, may affect both hydroclimate and the tularemia outbreaks, such as climate mitigation-adaptation measures. The time that it may take for the number of outbreaks to approach expected endemic levels, driven by long-term hydroclimatic changes, is also not considered, and may vary for different hydroclimatic conditions. This modeling limitation can be overcome by considering some convergence rate, although validation of such assumed rates will be difficult under long-term hydroclimatic variability and change. Furthermore, regional statistics-based disease models, such as that used here for tularemia, may not be directly transferable to other geographical regions and/or scales. 5. Conclusions We developed and tested a method that analyzes the sensitivity of infectious diseases to hydroclimatic changes and can assist in identifying critical hydroclimatic thresholds to unacceptable health hazards. A statistical model of tularemia was used as an example. A key contribution of our approach is in identifying comparative endemic levels to which disease outbreak numbers will tend to converge under different hydroclimate conditions. The case results showed high sensitivity of tularemia outbreaks to certain threshold combinations of hydroclimatic variable values within the range of past regional observations, although average conditions may shift under ongoing climate change, increasing the risk. Critical hydroclimatic thresholds can be identified, beyond which the associated expected endemic levels are not societally acceptable. Further research is needed on how such acceptable levels can be defined and whether projected climate change tends to drive hydroclimatic conditions towards and beyond such critical thresholds. The generality and spatiotemporal transferability of statistical disease models, such as that for tularemia used in this study, also need to be investigated in further research. Author Contributions: Conceptualization, Y.M.; methodology, Y.M., A.B., Z.K., and G.D.; analysis and interpretation of data, Y.M., A.B., Z.K., and G.D.; writing—original draft preparation, Y.M.; writing—review and editing, Y.M., A.B., Z.K. and G.D.; visualization, Y.M., A.B., Z.K., and G.D.; supervision, A.B., Z.K., and G.D.; project administration, A.B., Z.K., and G.D.; funding acquisition, G.D. Funding: This work was supported by the Nordforsk Centre of Excellence CLINF (grant number 76413). A cknowledges funding from the Swedish Research Council VR (project no. 2013-7448). Conflicts of Interest: The authors declare no conflict of interest. 4. Discussion One way forward is then to use specifically calibrated models for different regions/scales. For example, region-specific models for seven high-risk parts of Sweden have been established and show high capability in predicting regional outbreaks from 1984 to 2012, with potential of using such region/scale-specific models to analyze hydroclimatically driven shifts from the past to the future in each region and compare model coefficients and results among the regions [43]. The purpose of the present work was to develop a method for interpreting the effect of hydroclimatic factors from statistical models, by which hydroclimatic data can provide a straightforward picture of comparative risk levels. The method has the flexibility to be applied to different statistical disease models, but the extent to which the risk can be analyzed relies on the availability of such validated models. 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Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion

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Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion Cong Li  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Qiping Zhao  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Shunhai Zhu  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Qingjie Wang  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Haixia Wang  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Shuilan Yu  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Yu Yu  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Shashan Liang  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Huanzhi Zhao  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Bing Huang  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Hui Dong  Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Hongyu Han  (  [email protected] ) Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences Version of Record: A version of this preprint was published at Parasites & Vectors on July 25th, 2020. See the published version at https://doi.org/10.1186/s13071-020-04229-5. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Abstract Background: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several species of the protozoan parasite Eimeria. Among these species, E. tenella causes hemorrhagic pathologies and high mortality. These parasites have complex and diverse lifestyles that require the invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp). Background: Avian coccidiosis is a widespread, economically significa several species of the protozoan parasite Eimeria. Among these specie pathologies and high mortality. These parasites have complex and div invasion of their host cells. This is mediated by various proteins secret organelles. Apical membrane antigen 1 (AMA1), which is released from across all apicomplexans, plays a central role in the host cell invasion. EtAMA1-interacting proteins of E. tenella were screened. In this study, w EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp). Methods: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C.The localization of EtEsp was analyzed with an immunofluorescence assay. An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites. Results: The interaction between EtAMA1 and EtEsp was confirmed with BiFC in vivo and by GST pull- down in vitro. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life cycle. An immunofluorescence analysis showed that the EtEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells in vitro. Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Parasites & Vectors on July 25th, 2020. See the published version at https://doi.org/10.1186/s13071-020-04229-5. Page 2/25 Abstract Conclusion: In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in the sporozoite invasion of host cells and maybe involved in  the growth of the parasite in the host. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis. Background Avian coccidiosis is a widespread, economically significant disease of poultry that results in annual global economic losses of approximately $2.4 billion, including both production losses and disease prevention and treatment costs [1]. It is an enteric disease caused by several species of the protozoan parasite Eimeria, predominantly E. brunetti, E. necatrix, E. tenella, E. acervulina, E. maxima, E. mitis, and E. praecox [2]. Of these, E. tenella is one of the species that causes hemorrhagic pathologies and high mortality. Eimeria belongs to the phylum Apicomplexa, which includes important pathogens of humans and domestic animals, such as the causative agents of malaria (Plasmodium spp.), toxoplasmosis Page 3/25 (Toxoplasma gondii), babesiosis (Babesia spp.), and coccidiosis (Eimeria spp.). Most apicomplexans are obligate intracellular parasites and are characterized by their apical complexes of specialized secretory organelles (micronemes, rhoptries, and dense granules) [3]. They use actin-based motility coupled to regulated protein secretion from their apical organelles to actively invade host cells [4]. These parasites have complex and diverse lifestyles that involve the invasion of many different cell types, including erythrocytes, lymphocytes, macrophages, and digestive-tract cells. Despite the diversity of their target host cells, they maintain a highly conserved mechanism for this active invasion process [5]. The host-cell invasion mechanism involves the following steps: attachment, apical reorientation, moving junction formation, and the formation of a protective parasitophorous vacuole. Each invasion step is mediated by various proteins, which are secreted from apical secretory organelles [6]. Apical membrane antigen 1 (AMA1), a type I transmembrane protein, is one of a number of proteins released from micronemes that are conserved across all apicomplexans. It is known to play several important roles during host-cell penetration [7]. For instance, previous reports have shown that antibodies against AMA1 or small specific AMA1-binding peptides inhibit the invasion of host cells by Toxoplasma, E. tenella, Babesia, Neospora, and Plasmodium [3,8–11]. AMA1 is also a long-standing effective candidate vaccine for some apicomplexans, including N. caninum, T. gondii and Plasmodium [11–13]. In Toxoplasma and Plasmodium, AMA1 is reportedly involved in apical reorientation [14], host-cell attachment [7, 15], invasion and establishment of the moving-junction[16], , and the provision of a signal that initiates intracellular replication [17]. In contrast to the functions of AMA1 in other apicomplexan parasites, there are only a few reports of this conserved protein in Eimeria. Background In a previous in vitro study, AMA1 antibodies or specific EtAMA1-binding peptides inhibited the invasion of host cells by E. tenella sporozoites [10, 18]. EtAMA1 also partially protected host cells against homologous challenge with E. tenella when used as a recombinant protein vaccine and against heterologous challenge with E. maxima when the AMA1 protein from E. maxima was expressed as a live vectored vaccine [19]. Although AMA1 plays an important role in host-cell invasion by E. tenella sporozoites, its precise functions are unknown. Proteins perform a vast number of cellular functions when they interact with one or multiple binding partners. Protein–protein interactions are essential in the mediation of almost all cellular processes, including replication, transcription, translation, and signal transduction [20]. The biochemical analysis of protein complexes and the identification of their components have been fundamental to our understanding of their biological functions in cells [21]. To understand the precise functions of EtAMA1 during host-cell invasion, we screened EtAMA1- interacting proteins with a yeast two-hybrid system and identified 14 putative EtAMA1-interacting proteins in a previous study [22]. E. tenella Eimeria -specific protein (EtEsp) (GenBank accession number: JZ905773) is one of these putative interacting proteins. In this study, we cloned and characterized EtEsp. We systematically analyzed its interaction with EtAMA1 using bimolecular fluorescence complementation (BiFC) in vivo and a glutathione S-transferase (GST) pull-down assay in vitro. Our results show that the Page 4/25 Page 4/25 EtEsp is secreted protein, but not from micronemes, interacts with EtAMA1, and is involved in the invasion of host cells by E. tenella sporozoites. EtEsp is secreted protein, but not from micronemes, interacts with EtAMA1, and is involved in the invasion of host cells by E. tenella sporozoites. Parasite collection E．tenella was obtained from the Key Laboratory of Animal Parasitology of the Ministry of Agriculture, Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences，Shanghai, China. The parasites were maintained and propagated by passage through coccidia-free, 2-week-old chickens, as previously described [23]. Coccidia-free 14-day-old chickens were inoculated with 1 × 104 sporulated oocysts of E. tenella. Unsporulated oocysts (UO) were collected from infected chicken ceca at 7 days postinfection. Sporulated oocysts (SO) were derived from UO that had undergone sporulation in 2% potassium dichromate at a temperature 28–30 °C for 72–120 h, under forced aeration with a suitable pump. When more than 90% of the oocysts had sporulated, the oocysts were collected and purified. The sporozoites (Spz) were purified from cleaned SO with in vitro excystation [24]. Second-generation merozoites (sMrz) were isolated from infected chicken ceca at 115 h postinoculation, as described previously [25]. All parasites were collected and frozen in liquid nitrogen. Chickens and rabbits were fed and used according to a protocol approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The chicken embryo fibroblast cell line, DF-1, a derivative of the East Lansing Line (ELL-0) (Jiang et al. 2012), was used for BiFC and in vitro infection experiments. Molecular cloning and sequence analysis of E. tenella-specific protein Total RNA was extracted from E. tenella sporozoites with TRIzol Reagent (Invitrogen, USA). GeneRacer™ primers (GR5P and GR5N) were provided for the random amplification of PCR ends (RACE) in the GeneRacer™ Kit (Invitrogen) and gene-specific primers (GS5P and GS5N) were designed based on the expressed sequence tag (EST) sequence (GenBank accession number: JZ905773) which is 790 bp in length and contains a poly(A) at the 3¢ end (Table S). The 5¢ end of this gene was determined according to the manufacturer’s protocol. The PCR-amplified fragment was then ligated into the pGEM-T Easy Vector (Promega, USA) and used to transform competent Escherichia coli TOP10 cells. After PCR identification, the plasmid DNA was sequenced. After the resulting sequence was assembled and aligned with the original EST sequence, the full-length cDNA sequence of the gene was determined and submitted to the National Center for Biotechnology Information (NCBI) GenBank (accession number: MN161778). The full-length EtEsp cDNA sequence was used in a BLAST search of GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and the E. tenella genome database (http://www.genedb.org/Homepage/Etenella). The deduced amino acid sequence was obtained with the ORF Finder tool at NCBI. The molecular mass and theoretical isoelectric point were calculated with ProtParam tools (http://web.expasy.org/protparam/). The signal peptide sequence was identified with the Total RNA was extracted from E. tenella sporozoites with TRIzol Reagent (Invitrogen, USA). GeneRacer™ primers (GR5P and GR5N) were provided for the random amplification of PCR ends (RACE) in the GeneRacer™ Kit (Invitrogen) and gene-specific primers (GS5P and GS5N) were designed based on the expressed sequence tag (EST) sequence (GenBank accession number: JZ905773) which is 790 bp in length and contains a poly(A) at the 3¢ end (Table S). The 5¢ end of this gene was determined according to the manufacturer’s protocol. The PCR-amplified fragment was then ligated into the pGEM-T Easy Vector (Promega, USA) and used to transform competent Escherichia coli TOP10 cells. After PCR identification, the plasmid DNA was sequenced. After the resulting sequence was assembled and aligned with the original EST sequence, the full-length cDNA sequence of the gene was determined and submitted to the National Center for Biotechnology Information (NCBI) GenBank (accession number: MN161778). The full-length EtEsp cDNA sequence was used in a BLAST search of GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and the E. tenella genome database (http://www.genedb.org/Homepage/Etenella). The deduced amino acid sequence was obtained with the ORF Finder tool at NCBI. The molecular mass and theoretical isoelectric point were calculated with ProtParam tools (http://web.expasy.org/protparam/). Analysis of EtEsp transcript levels with real-time quantitative PCR (qPCR) The expression profiles of EtEsp mRNA were examined in four developmental stages of E. tenella (UO, SO, Spz, and sMrz) with qPCR. cDNA samples were synthesized from DNaseI-treated total RNAs of the E. tenella developmental stages using SuperScript™ II Reverse Transcriptase (Invitrogen) and random pd(N)6 primer. The housekeeping gene 18S rRNA was used as the internal control. The primers used to amplify the EtEsp cDNA (EtEsp-SP and EtEsp-AP) and the 18S rRNA gene (18S-SP and 18S-AP) were designed with Primer3 v. 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) (Table S). qPCR was performed with the StepOnePlus™ Real-Time PCR System using the SYBR® Premix Ex Taq™ II kit (Takara, Japan). All experiments were performed twice, with separate biological replicates. In each experiment, the reactions were performed in triplicate. A dilution series of cDNA templates of the sporozoites was used to establish standard curves, and all standard curves had correlation coefficients of R2 > 0.99. The comparative 2−ΔΔCt method was used to analyze the relative levels of gene expression. Molecular cloning and sequence analysis of E. tenella-specific protein The signal peptide sequence was identified with the Page 5/25 Page 5/25 Page 5/25 SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/), and transmembrane regions were predicted with the TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Protein motifs were scanned with Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/), and transmembrane regions were predicted with the TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Protein motifs were scanned with Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Recombinant protein expression and polyclonal anti-rEtEsp serum The EtEsp open reading frame (ORF) cDNA was amplified with PCR using primers EtEsp-UP and EtEsp-LP (Table S), which contained BamHI and XhoI restriction sites, respectively. The PCR fragment was then ligated into the prokaryotic expression vector pET28a(+) digested with the same restriction endonucleases, to construct the recombinant expression plasmid pET–EtEsp. The recombinant protein His–EtEsp (rEtEsp) was expressed in Escherichia coli BL21 cells at 37 °C with 1 mM isopropyl-thio-α-d- galactoside. The cell pellet was lysed with sonication and digested with 10 µg/mL lysozyme (Sigma- Aldrich, USA). The lysate was then analyzed with 12% SDS-PAGE to confirm that the recombinant protein was present as a soluble protein or inclusion bodies. rEtEsp was purified with His·Bind® Resin (Merck, USA) and its concentration measured with a BCA Protein Assay Kit (Beyotime, China). Two 2-month-old male rabbits were inoculated with 200 μg of purified rEtEsp emulsified in Freund’s complete adjuvant (Sigma-Aldrich). After 14 days, a booster of 200 μg of purified rEtEsp in Freund’s incomplete adjuvant (Sigma-Aldrich) was administered, followed by a second and third booster on days 28 and 42. One week after the final booster, the rabbit serum was collected and stored at −20 °C until use. BiFC assay The ORF fragments of EtEsp and the EtAMA1 ectodomain, with no stop codon, were amplified from the first-strand cDNA with two pairs of primers (BfEtEsp-UP/BfEtEsp-LP and BfEtAMA1-UP/BfEtAMA1-LP, respectively), which contained EcoRI and BglII restriction sites (EtEsp) or EcoRI and XhoII restriction sites (EtAMA1). The fragments were then digested with the appropriate restriction enzymes and ligated into the pBiFC-VN155 and pBiFC-VC155 vectors digested with the same enzymes, respectively, to construct the recombinant plasmids pBiFC-VN155–EtEsp and pBiFC-VC155–EtAMA1, respectively. Before the BiFC assay, the uptake of the expression vectors by the cells was confirmed. DF-1 cells were transfected with the recombinant plasmid pBiFC-VN155–EtEsp or pBiFC-VC155–EtAMA1 using Lipofectamine™ 2000 Transfection Reagent (Invitrogen, USA), according to the manufacturer’s instructions. At 48 h after transfection, the cells were harvested and the proteins were extracted with RIPA Lysis Buffer (Beyotime). Western blots were probed with rabbit anti-rEtEsp antibody and rabbit anti-rEtAMA1 antibody, which were previously produced in our laboratory [10]. After confirmation that the cells had expressed the two constructs, DF-1 cells were cotransfected with pBiFC-VN155–EtEsp and pBiFC-VC155–EtAMA1. DF-1 cells were also cotransfected with pBiFC-bJunVN55 (I152L) and pBiFC-bFosVC155 or pBiFC-VC155– EtAMA1 and pBiFC-VN155 empty vector, or pBiFC-VN155–EtEsp and pBiFC-VC155 empty vector as the positive or negative control, respectively. The DF-1 cells were observed with fluorescence microscopy 24 h after transfection with the different constructs. SDS-PAGE and western blotting Protein samples were prepared from the four E. tenella developmental stages (UO, SO, Spz, and sMrz), and from DF-1 cells transfected with the recombinant plasmids, for western blotting. The protein concentrations were determined with a BCA Protein Assay Kit (Beyotime,China). The purified rEtEsp and protein lysates were separated with SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes. Rabbit antiserum (1:100) against sporozoite proteins, previously produced in our Page 6/25 Page 6/25 laboratory [26], a rabbit anti-rEtEsp antibody (1:100), a mouse monoclonal anti-α-tubulin antibody (1:1000) (Sigma-Aldrich), and a monoclonal anti-His antibody (1:1000) were used as the primary antibodies to detect rEtEsp or native EtEsp. Naïve rabbit serum (1:100) was used as the negative control. IRDye-800CW-labelled goat anti-rabbit IgG antibody (1:25,000) and IRDye-680RD-labeled donkey anti- mouse IgG antibody (1:25,000; LI-Cor, Lincoln, NE, USA) were used as the secondary antibodies. The IRDyes were detected with the Odyssey Infrared Imaging System (LI-Cor). Assay of EtEsp secretion Freshly excysted sporozoites (4 × 106) were incubated in 100 μL of complete medium (CM; Dulbecco’s modified Eagle’s medium [DMEM] supplemented with 10% fetal bovine serum [FBS], 100 U/mL penicillin/streptomycin, 2 mM l-glutamine) for 2 h at 41 °C under 5% CO2 for the secretion experiments. They were then incubated with 5, 10, or 20 μM staurosporine (Sigma; dissolved in dimethylsulfoxide [DMSO]) or an appropriate volume of carrier DMSO, as described previously [28]. The secretion of EtMIC2 and EtGRA(TgGRA7 homologous protein) was used as the control. The sporozoites were then pelleted by centrifugation for 10 min at 6000 × g. The supernatants and sporozoites were recovered and analyzed with western blotting using a rabbit anti-rEtEsp antibody and rabbit anti-rEtMIC2 antibody generated previously in our laboratory [27] and mouse anti-TgGAR7 antibody which generated previous in other laboratory of our Institute . GST pull-down To confirm the interaction between EtAMA1 and EtEsp317 in vitro, a GST pull-down assay was performed with the Pierce™ GST Protein Interaction Pull-Down Kit (Thermo Scientific, USA), according to the manufacturer’s instructions. The recombinant plasmid pGEX-6P–EtAMA1 was previously constructed in our laboratory [10]. The expression of the recombinant protein GST–EtAMA1 was induced and the protein purified with GST resin for use as the bait protein. The ORF of EtEsp was inserted into the pET-28a vector to express the recombinant protein His–EtEsp (rEtEsp) as the prey protein. GST–EtAMA1 was incubated with equilibrated glutathione-agarose to immobilize the bait protein. rEtEsp was then added to the glutathione-agarose and incubated with the bait protein. The bait and prey proteins were eluted from the glutathione-agarose. E. coli BL21 cells were transformed with recombinant plasmid pET–EtMIC2, constructed previously in our laboratory [27], to express the recombinant protein His–EtMIC2 as the negative control. Another, rEtEsp was loaded in an empty glutathione-agarose column as the negative. All All Page 7/25 Page 7/25 the proteins were then resolved with 12% SDS-PAGE and detected with western blotting using the appropriate antibodies, as described above. Immunofluorescence staining of parasites Purified differentially developed parasites (Spz, sporocysts [Sporo], and sMrz) were transferred to glass slides and air-dried, as previously described [10, 29]. Purified freshly sporozoites were infected DF-1 cells after incubation in CM for 2 h at 41°C. At different time points after infection, the DF-1 cells were collected, washed, transferred to glass slides, and air-dried. The slides were then fixed in 2% Purified differentially developed parasites (Spz, sporocysts [Sporo], and sMrz) were transferred to glass slides and air-dried, as previously described [10, 29]. Purified freshly sporozoites were infected DF-1 cells after incubation in CM for 2 h at 41°C. At different time points after infection, the DF-1 cells were collected, washed, transferred to glass slides, and air-dried. The slides were then fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) and placed in 1% Triton X-100 in PBS for 15 min to increase their permeability. Non permeabilized sporozoites and sporocysts were used as control. The slides were blocked with PBS containing 2% (w/v) bovine serum albumin for overnight at 4 °C. A rabbit anti-rEtEsp antibody (1:100) was added and the cells were incubated for 1 h at 37 °C. A 1:500 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) was then added and the cells incubated for 1 h at 37 °C. The cell nuclei were stained by incubation in 10 μg/mL 4¢,6-diamidino-2-phenylindole (Beyotime) at room temperature for 10 min. After each step, the slides were washed three times for 10 min each with PBS containing 0.05% Tween 20. The slides were finally mounted with 50 μL of Fluoromount Aqueous Mounting Medium (Sigma-Aldrich) before observation with a fluorescence microscope (Olympus, Tokyo, Japan). At the same time, we performed the co-localization of EtEsp and EtAMA1 in sporozoites. Purified sporozoites were treated with mouse anti-rEtAMA1 antibody (1:100) and rabbit anti-rEtEsp antibody(1:100), then goat anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated antibody(1:500) and goat anti-mouse IgG cyanine (Cy3)-conjugated antibody (Sigma- Aldrich)(1:500) were secondary antibody. Cloning and sequence analysis of full-length EtEsp cDNA The 1108-bp full-length cDNA of EtEsp was obtained with RACE. A sequence analysis showed that the full-length cDNA included a 5¢-untranslated region (UTR) of 70 bp, a 3¢-UTR of 542 bp with a poly(A) tail, and an ORF of 501 bp, which encoded 166 amino acids with a calculated molecular weight of 18.1 kDa and a theoretical isoelectric point of 4.2 (Fig. 1). Analysis of the amino acid sequence showed a signal peptide of 19 amino acids at the N-terminus and no transmembrane region. Searches in the Motif Database and the Conserved Domain Database revealed the presence of one N-glycosylation site, five casein kinase II phosphorylation sites, six N-myristoylation sites, one tyrosine kinase phosphorylation site, one intein DOD-type homing endonuclease domain, and no conserved domains (Fig. 1). A BLAST search of the E. tenella genome database showed that the ORF sequence shared 100% sequence identity with ETH_00016590, which encodes an Eimeria-specific protein, on supercontig Eth_scaff124: 9216–10141. The amino acid sequence shared 100% homology with the E. tenella Eimeria-specific protein (XP_013228647.1) and 92% (152/170) identity with the E. necatrix Eimeria-specific protein (XP_013435139.1) in NCBI. Therefore, this gene was designated EtEsp and submitted to NCBI GenBank (GenBank accession no. MN161778). It also shared 68% (106/157) amino acid identity with E. brunetti conserved hypothetical protein (CDJ53027.1), 63% (108/172) identity with E. praecox conserved hypothetical protein (CDI81636.1), 74% (97/131) identity with E. maxima conserved hypothetical protein (XP_013336310.1), and 73% (91/124) identity with E. acervulina conserved hypothetical protein (XP_013248166.1). Also, this protein is not found in other apicomplexa parasites. These results show that the protein is conserved in Eimeria spp. Invasion inhibition assay in vitro The invasion inhibition assay was based on previous reports the invasion of DF-1 cells by E. tenella sporozoites. Antibodies were purified with Protein A+G Agarose (Beyotime). DF-1 cells (2 × 105 cells per well) were cultured in 24-well plates (Corning) in CM for 24 h at 37 °C under 5% CO2. The freshly purified Page 8/25 sporozoites were counted and labeled with carboxyfluorescein diacetate succinimidyl ester (Beyotime). The labeled sporozoites were incubated at 37 °C with 50, 100, 200, 300 or 400 μg/mL purified IgG directed against rEtEsp for 2 h. The same quantity of IgG from naïve rabbit serum (Sigma-Aldrich) was used as the negative control, and an equivalent volume of PBS as the normal control. After they were washed twice with sterile PBS, DF-1 cells (105/well) were infected with the labeled sporozoites (105/well) in 24-well plates and cultured for 16 h at 41 °C under 5% CO2. The cells were then collected and analyzed with flow cytometry on a Cytomics™ FC 500 (Beckman Coulter, Indianapolis, IN, USA). The controls were uninfected DF-1 cells. The infected cells, uninfected cells, and free sporozoites were gated with the CXP software to count the infected (labeled sporozoites) and uninfected (fluorescence-free) cells. All assays were performed in triplicate. The percentages of infected cells in the presence or absence of an anti- rEtEsp polyclonal antibody were used to calculate the inhibition rates, as previously described [10]. Confirmation of the interaction between EtAMA1 and EtEsp To characterize the interaction between EtAMA1 and EtEsp in vivo, a BiFC assay was performed. For the BiFC assay, fragments of the EtEsp ORF and the EtAMA1 ectodomain sequence were cloned into the plasmids pBiFC-VN155 and pBiFC-VC155, respectively, to generate the constructs pBiFC-VN155–EtEsp and pBiFC-VC155–EtAMA1, respectively. The total proteins were extracted from DF-1 cells transfected separately with one or other construct. Western blotting showed that the two constructs were expressed individually in the DF-1 cells at 48 h after transfection (Fig. 4A). Strong green fluorescence was observed in DF-1 cells 48 h after they were cotransfected with both constructs. Green fluorescence was also observed in the positive control. However, there was no visible fluorescence in the DF-1 cells which cotransfected with pBiFC-VC155–EtAMA1 and pBiFC-VN155 empty vector or pBiFC-VN155–EtEsp and pBiFC-VC155 empty vector. These results indicate that EtEsp interacts with EtAMA1 in cells (Fig. 4B). EtEsp mRNA and protein expression at different developmental stages of E. tenella qPCR was used to analyze the UO, SO, Spz, and sMrz stages of E. tenella for the presence of EtEsp mRNA. The levels of EtEsp mRNA were much higher in the sMrz stage than in the other three stages, and EtEsp mRNA was almost undetectable in UO (Fig. 3A). The expression of EtEsp in the four developmental stages was also determined with immunoblotting using rabbit antiserum against rEtEsp. A monoclonal anti-α-tubulin antibody was used as the control. Western blotting showed that the anti-rEtEsp antibody reacted with a band of approximately 18 kDa in the parasite lysates prepared from the four different developmental stages of E. tenella The expression levels of EtEsp were higher in sporozoites than in other three stages(Fig. 3B). Expression and characterization of recombinant EtEsp rEtEsp was expressed as a His6-tagged fusion protein. SDS-PAGE showed that rEtEsp was mainly present in the soluble fraction of the bacterial lysate. After the purification of rEtEsp with Ni-NTA chromatography, Page 9/25 Page 9/25 a protein of approximately 21 kDa was observed with SDS-PAGE. Because 3 kDa of the fusion protein was derived from the vector, the predicted molecular mass of EtEsp was about 18.1 kDa. Western blotting showed that purified rEtEsp was recognized by rabbit serum directed against sporozoites and by a monoclonal anti-His6 antibody. Naïve rabbit serum failed to recognize any protein corresponding to the expected size of rEtEsp (Fig. 2). These results indicate that rEtEsp was recognized specifically by rabbit serum directed against a soluble sporozoite protein and by a monoclonal anti-His antibody. GST pull-down To confirm the interaction between EtAMA1 and EtEsp in vitro, a GST pull-down assay was performed. GST–EtAMA1 and His–EtEsp were expressed individually in E. coli and purified. GST–EtAMA1 was bound to an equilibrated glutathione–agarose column, and then His–EtESP was added to the column. The proteins bound to the glutathione–agarose, and any nonspecifically bound proteins were removed by elution with buffer. The proteins retained on the column were then eluted and detected with immunoblotting using anti-rEtAMA1 and anti-rEtEsp antibodies (Fig. 5). The results clearly indicated a direct interaction between the EtAMA1 and EtEsp proteins. Co-localization of EtAMA1 and EtEsp IFAs were performed to determine the location of EtAMA1 and EtEsp. Purified sporozoites were treated with mouse anti-rEtAMA1 antibody and rabbit anti-rEtEsp antibody. The results showed EtEsp was mainly located on the surface of sporozoites, EtAMA1 was distributed throughout the cytoplasm and the membrane of sporozoites with the exception of refractile bodies (Fig 8). Immunolocalization of EtEsp at different developmental stages of E. tenella To investigate the localization and distribution of the EtEsp protein in different development stages of E. tenella, including sporozoites, second-generation merozoites, immature schizonts, and mature schizonts, it was localized with immunofluorescence in vitro using an antibody against rEtEsp. The EtEsp protein was mainly distributed on the surfaces of the permeabilized parasite sporozoites, sporocysts, and second-generation merozoites (Fig. 7A-a, B-a, K,). The protein were also mainly located on the surface of non permeabilized sporozoites and sporocysts (Fig. 7A-b, B-b). After incubation in CM for 2 h, the fluorescence increased and mainly localized to the anterior and surface of parasites (Fig. 7C). EtEsp protein was also mainly located on the surfaces of parasites 2 h after their invasion of DF-1 cells (Fig. 7D). At 12 h after the sporozoites were added to DF-1 cells, EtEsp also localized to the cytoplasm of the sporozoites, except for the refractile body in the posterior section of the parasites, and the intensity of EtEsp staining had increased (Fig. 7E). At 24–72 h postinfection, the EtEsp protein was uniformly distributed in trophozoites, immature schizonts, and mature schizonts, and the protein’s expression had increased (Fig. 7E–J). EtEsp is not secreted from the microneme Page 10/25 Page 10/25 To examine the secretion of EtEsp, sporozoites were incubated in CM at 41 °C. The supernatant containing the excretory–secretory antigens (ESA) from the incubated sporozoites and sporozoites pellets were analyzed with western blotting. Immunoblots of the ESA samples and sporozoites were probed with an anti-rEtEsp antibody and showed that EtEsp was secreted when the sporozoites were incubated at 41 °C under 5% CO2 in CM. Rabbit serum raised against the micronemal protein EtMIC2 was used as the experimental control [27]. To demonstrate whether EtEsp secretion is dependent on the micronemal pathway, we added staurosporine to the CM because staurosporine is a protein kinase inhibitor known to specifically inhibit microneme secretion [28]. In the parasites treated with 5, 10, or 20 μM staurosporine, the secretion of EtEsp and EtGRA into the supernatant was not affected, but the secretion of EtMIC2 in supernatant was significant reduced compared with their secretion in the presence of the DMSO solvent only (Fig. 6A,B). These results show that EtEsp is a secreted protein, but not a micronemal protein. Discussion In this study, we cloned and characterized the E. tenella Eimeria-specific protein, a putative EtAMA1- interacting protein, using a yeast two-hybrid system in our laboratory [22]. Although the yeast two-hybrid system is a widely used and powerful method for identifying the partners of proteins in regulatory complexes and in the analysis of protein–protein interactions [30], the system has several limitations, including the possibility of isolating very large numbers of clones with no biological relevance [31]. Therefore, the interaction between EtAMA1 and EtEsp required validation with an alternative technique, such as a BiFC assay or GST pull-down assay. The BiFC assay is a versatile technique for investigating protein–protein interactions in living systems, and is based on the reconstitution of a fluorescent protein in vivo [32]. GST pull-down is amenable to more specific investigations of protein–protein interactions in vitro, but relies on purified proteins that may not fully mimic the protein’s native conformation or posttranslational modification, which mediate its interactions [33]. Although these assays have some advantages in identifying protein–protein interactions, each also has its drawbacks. Therefore, in many research fields, these methods are often combined to identify the interactions between two proteins [33– 35]. In this study, the interaction between EtAMA1 and EtEsp was confirmed with a GST pull-down assay in vitro and a BiFC assay in vivo. These results indicated that EtEsp interacts with EtAMA1. Proteins perform a vast number of cellular functions through their interactions with one or multiple binding partners. Moreover, many protein–protein interactions are regulated by posttranscriptional modification (e.g., phosphorylation) of the protein of interest, and these modifications are induced by exposure to certain circumstances [36]. In this study, an amino acid sequence analysis predicted that EtEsp contains one N-glycosylation site, five casein kinase II phosphorylation sites, six N-myristoylation sites, one tyrosine kinase phosphorylation site and one intein DOD-type homing endonuclease domain. Inteins, also called protein introns, are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins[37]. These data suggest that its functions may be regulated by posttranslational modification. We supposed that the interaction of EtAMA1 with EtEsp may be regulated by posttranslational modification. To understand the expression of EtEsp in the different developmental stages of parasite, we examined its expression patterns with qPCR and western blotting. Anti-rEtEsp antibodies inhibit DF-1 cell invasion To evaluate the effect of the EtEsp protein on the invasion of DF-1 cells by E. tenella sporozoites, an invasion inhibition assay of sporozoites was performed in vitro. When the sporozoites were incubated with purified anti-rEtEsp antibody before infection, their capacity to invade the DF-1 cells was significantly reduced. After pretreatment with 50, 100, 200, 300, or 400 μg/mL anti-rEtEsp IgG antibody, their invasion Page 11/25 of cells was highly significantly reduced compared with that of sporozoites treated with naïve rabbit IgG (negative control) (P < 0.01). Under these experimental conditions, an inhibition plateau of 62.9% was reached at an antibody concentration of 300 μg/mL. In a comparative analysis, the same dose of the naïve rabbit serum IgG antibody did not significantly affect invasion (Fig. 9). Discussion This phenomenon is found in other proteins secreted by micronemes, including EtMIC3, which only has a signal peptide, with no GPI anchor or cytoplasmic tail, and a transmembrane region, which may indicate its retention on the parasitic surface [28]. Immunofluorescent localization showed that EtEsp was located on the surface of the sporozoite and concentrated around the anterior of the parasite during its incubation in CM. However, the protein has no transmembrane region or glycophosphatidyl inositol (GPI)-anchor sequence, but has a signal peptide, which is characteristic of micronemal proteins, and six N-myristoylation sites. The presence of a signal peptide is necessary for the translocation of proteins from their ribosomal sites of translation into the lumen of the endoplasmic reticulum, from where they are trafficked in the endomembrane system to their final locations within the cell or beyond [40]. We speculated that EtEsp also undergoes posttranslational modification according to the amino acid sequence analysis, including phosphorylation, myristoylation, and glycosylation. Among these modifications, myristoylation is the key factor in the membrane localization of signal-transducing proteins [41]. This phenomenon is found in other proteins secreted by micronemes, including EtMIC3, which only has a signal peptide, with no GPI anchor or cytoplasmic tail, and a transmembrane region, which may indicate its retention on the parasitic surface [28]. Most surface antigens are involved in the invasion, pathogenesis, and immune evasion of parasites. For example, in Plasmodium, merozoite surface proteins are critical for parasite invasion, and represent attractive targets for antibody-based therapies against clinical malaria [42]. We also found that the expression of EtEsp increased and that the protein mainly localized on the anterior and surface of the parasites after incubation in CM for 2 h. This suggests that the protein is involved in the sporozoite invasion of host cells. To investigate the function of EtEsp in the invasion process, we performed an invasion test in vitro and found that polyclonal rabbit anti-rEtEsp serum efficiently reduced the sporozoite invasion of cultured DF-1 cells. EtAMA1 also localized to the anterior of the sporozoites after their invasion of DF-1 cells [10]. Previous reports have shown that monospecific mouse anti-rEtAMA1 serum or polyclonal rabbit antiserum against rEtAMA1 also blocked the invasion of host cells in vitro [10,19]. Apical membrane antigen 1 is conserved in apicomplexans, previous research showed that apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell penetration in T. Discussion Our results indicated that EtEsp mRNA levels were higher in second-generation merozoites and sporozoites than in sporulated oocysts or unsporulated oocysts. But western blotting showed that the expression of EtEsp was higher in sporozoites than other developmental stages of E.tenella. indicating EtEsp expression underwent posttranslational modification. Immunofluorescent localization showed that the expression of the protein increased with the development of the parasites in DF-1 cells. Previous proteomic and transcriptomic data confirm that Page 12/25 Page 12/25 Page 12/25 EtAMA paralogues are tightly stage-regulated [38, 39]. EtAMA1 is a sporozoite-specific protein involved in the invasion process of sporozoites [10, 19, 38]. While another EtAMA1 paralogues, EtAMA2 is a merozoites-specific protein not involved the parasite invasion. All these finding indicate that E. tenella parasites harbour stage-specific AMA proteins that could be relevant during specific phases of the parasite cycle [19]. In this study, EtEsp is differentially expressed during the distinct phases of the parasite life cycle, and may be very important in the invasion and development of the parasite life cycle. EtAMA paralogues are tightly stage-regulated [38, 39]. EtAMA1 is a sporozoite-specific protein involved in the invasion process of sporozoites [10, 19, 38]. While another EtAMA1 paralogues, EtAMA2 is a merozoites-specific protein not involved the parasite invasion. All these finding indicate that E. tenella parasites harbour stage-specific AMA proteins that could be relevant during specific phases of the parasite cycle [19]. In this study, EtEsp is differentially expressed during the distinct phases of the parasite life cycle, and may be very important in the invasion and development of the parasite life cycle. Immunofluorescent localization showed that EtEsp was located on the surface of the sporozoite and concentrated around the anterior of the parasite during its incubation in CM. However, the protein has no transmembrane region or glycophosphatidyl inositol (GPI)-anchor sequence, but has a signal peptide, which is characteristic of micronemal proteins, and six N-myristoylation sites. The presence of a signal peptide is necessary for the translocation of proteins from their ribosomal sites of translation into the lumen of the endoplasmic reticulum, from where they are trafficked in the endomembrane system to their final locations within the cell or beyond [40]. We speculated that EtEsp also undergoes posttranslational modification according to the amino acid sequence analysis, including phosphorylation, myristoylation, and glycosylation. Among these modifications, myristoylation is the key factor in the membrane localization of signal-transducing proteins [41]. Conclusions In this study, we have shown that interacts with EtAMA1 using a BIFC assay in vivo and a GST pull-down assay in vitro. Using staurosporine, we showed that EtEsp is a secreted protein of sporozoites but not from micronemes. An invasion inhibition assay revealed that an antibody against rEtEsp also blocked the parasite invasion of its host cells by more than 62%. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in therapeutic intervention strategies against avian coccidiosis. Supplementary Information Additional file 1: Table S. Primers sequence used in this study. Discussion gondii tachyzoites and Plasmodium merozoites[7]. So EtAMA1maybe mediate sporozoites attachment but not host cell penetration during the host cell invasion process of E.tenella. In this study, EtEsp is involved in invasion as demonstrated by using antibodies raised against EtEsp in vitro. We tested that EtEsp is an interacting protein with EtAMA1 by using BiFC, GST-pull down and yeast two-hybrid system. So EtEsp maybe also associated with sporozoites adhesion to host cells. Therefore, we speculated that EtEsp mediates sporozoites attachment to host cells by interacting with EtAMA1. The exact function of EtEsp needs further study. Page 13/25 Previous studies have shown that in T. gondii and Plasmodium, AMA1 interacts directly with rhoptry neck protein 2 (RON2), which is secreted from the parasite rhoptries and specifically localizes at the moving junction. The RON2–AMA1 interaction is a critical step in the moving-junction-dependent invasion of host cells by apicomplexan parasites [43, 44]. Although the interaction between AMA1 and RON2 has not been reported in Eimeria spp., E. tenella is an apicomplexan and AMA1 is conserved in this phylum. Therefore, we inferred that EtAMA1 may also interact with EtRON2 and specifically localize to the moving junction during the invasion of host cells by E. tenella. In this study, we have shown that EtEsp is an EtAMA1- interacting protein, but whether it localizes to the moving junction during invasion requires further study. Acknowledgments We would like to thank all organizations which funded this work and all the teachers who cooperated in technical assistance and Dr Wang quan who provided mouse anti-TgGRA7 antibody of T.gondii . Abbreviations EtAMA1: Eimeria tenella apical membrane antigen 1; EtEsp: E. tenella Eimeria-specific protein; BiFC: bimolecular fluorescence complementation; GST pull-down: glutathione S-transferase pull-down; RACE: random amplification of PCR ends; UO: unsporulated oocysts; SO: sporulated oocysts; Spz: sporozoites; sMrz: second-generation merozoites; NCBI: the National Center for Biotechnology Information; ORF: open reading frame; CM: complete medium. Author details 1Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, PR China. 2College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China. Declarations Ethics approval and consent to participate Page 14/25 Page 14/25 Page 14/25 The protocol was approved and authorized by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The protocol was approved and authorized by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Authors’ contributions HYH and HD conceived and designed the study. CL, QPZ, SHZ and QJW performed the experiments. CL, BH and HZZ analyzed the data.CL, QPZ, HXW, SLY, YY and SSL collected parasites. CL, QPZ and HYH wrote the manuscript. All authors read and approved the final manuscript. Availability of data and materials Not applicable. Consent for publication Not applicable. Funding This work was supported by the National Natural Science Foundation of China (Grant Nos. 31572266 and 31672551) and National Key Research and Development Program of China (2018YFD0500302) and National Sharing Service Platform for Parasite Resources (No. TDRC-22) and Shanghai Minhang District talent development special funds. Competing interests The authors declare that they have no competing interests. References Page 15/25 Page 15/25 1. Quiroz-Castaneda RE, Dantan-Gonzalez E. Control of avian coccidiosis: future and present natural alternatives. Biomed Res Int. 2015; 2015:430610. 2. Blake DP, Tomley FM. Securing poultry production from the ever-present Eimeria challenge. Trends Parasitol. 2014; 30: 12-9. 3. Tyler JS, Treeck M, Boothroyd JC. Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication. Trends Parasitol. 2011; 27:410-20. 4. Sibley LD. Invasion strategies of intracellular parasites. Science 2004;304:248-53. 5. Sharma P, Chitnis CE. 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Co-immunoprecipitation from Transfected Cells. Methods Mol Biol. 2015; 1278:381-9. 37. Naor A, Altman-Price N, Soucy SM, Green AG, Mitiagin Y, Turgeman-Grott I, et al.. Impact of a homing intein on recombination frequency and organismal fitness. Proc Natl Acad Sci U S A. 2016; 113. 38. Lal K, Bromley E, Oakes R, Prieto JH, Sanderson SJ, Kurian D, et al. Proteomic comparison of four Eimeria tenella life-cycle stages: unsporulated oocyst, sporulated oocyst,sporozoite and second- generation merozoite. Proteomics. 2009; 9: 4566-76. 39. Reid AJ, Blake DP, Ansari HR, Billington K, Browne HP, Bryant J, et al. Genomic analysis of the causative agents of coccidiosis in domestic chickens. Page 15/25 Genome Res. 2014, 24:1676-85. 40. Marugan-Hernandez V, Long E, Blake D, Crouch C, Tomley F. Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle. Sci Rep. 2017; 7:4557. 41. McCabe JB, Berthiaume N-terminal protein acylation confers localization to cholesterol, sphingolipid- enriched membranes but not to lipid rafts/caveolae. Mol Biol Cell. 2001; 12:3601-17. 41. McCabe JB, Berthiaume N-terminal protein acylation confers localization to cholesterol, sphingolipid- enriched membranes but not to lipid rafts/caveolae. Mol Biol Cell. 2001; 12:3601-17. 42. Babacar M, Fall MM, Varela ML, Loucoubar C, Joos C, Fall B, et al. Analysis of antibody responses to selected Plasmodium falciparum merozoite surface antigens in mild and cerebral malaria and associations with clinical outcomes Clin Exp Immunol. 2018; 23. 43. Tonkin ML, Roques M, Lamarque MH, Pugnière M, Douguet D, Crawford J, et al. Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide. Science. 2011; 333:463-7. 44. Shen B, Sibley The moving junction, a key portal to host cell invasion by apicomplexan parasites. Curr Opin Microbiol. 2012; 15:449-55. 44. Shen B, Sibley The moving junction, a key portal to host cell invasion by apicomplexan parasites. Curr Opin Microbiol. 2012; 15:449-55. Page 18/25 Table 1. Primers sequence used in this study Primer name     Primer sequence GS5P (5' Primer) 5'-ATGGTCTCGGGCCAGTTCTCGTTCA -3' GS5N (5' Nested Primer) 5'-GGAGATGAGACCCAGGCGGATGAAA -3' EtEsp-UP 5'-GCGGATCCATGAAGGGCCTGTTCTTCACCGTCG-3' EtEsp-LP 5'-GCCTCGAGCGAATCTACTTCAAGAAAAGCCACG-3' EtEsp-SP 5'-CCCCGACTACCTCAAGTTCCTCAGC -3' EtEsp-AP 5'-TGGGTCCGTCTCCCCCTCCTTGGTG -3' 18S-SP 5'-TGTAGTGGAGTCTTGGTGATTC-3' 18S-AP BfEtESp-UP BfEtESp-LP BfEtAMA1-UP BfEtAMA1-LP 5'-CCTGCTGCCTTCCTTAGATG-3' 5'-GCGAATTCGGGCCACCATGAAGGGCCTGTTCTT-3' 5'-GCAGATCTGCTGCTCGCGTTGCCAGCAGAT -3' 5'-GCGAATTCGGGCCACCATGCAGCCGCCCTAT-3' 5'-GCCTCGAGGGTATTCCTGGTCCAG-3' Figures Page 19/25 Figure 1 Nucleotide sequence of the full-length cDNA of EtEsp and the deduced amino acid sequence. Start and stop codons are underlined. One putative intein DOD-type homing endonuclease domain is shown with wavy underlining. A putative N-glycosylation site has a double line. Five putative casein kinase II phosphorylation sites are shown in yellow. Six putative N-myristoylation sites are shown in gray. Page 20/25 Figure 2 Immunogenicity of rEtEsp. rEtEsp protein was subjected to western blotting. Lane M: protein marker; lan 1: anti-His-tag monoclonal antibody as the primary antibody; lane 2: rabbit serum against sporozoites a the primary antibody; lane 3: naïve rabbit IgG as the primary antibody. Figure 2 Figure 2 Immunogenicity of rEtEsp. rEtEsp protein was subjected to western blotting. Lane M: protein marker; lane 1: anti-His-tag monoclonal antibody as the primary antibody; lane 2: rabbit serum against sporozoites as the primary antibody; lane 3: naïve rabbit IgG as the primary antibody. Figure 3 EtEsp expression at different developmental stages of E. tenella. UO, unsporulated oocysts; SO, sporulated oocysts; Spz, sporozoites; sMrz, second-generation merozoites. A. qPCR of EtEsp at different developmental stages of E. tenella.. B. Western blot showing EtEsp at different developmental stages, probed with rabbit anti-rEtEsp serum or mouse monoclonal anti-α-tubulin antibody. Figure 3 Figure 3 EtEsp expression at different developmental stages of E. tenella. UO, unsporulated oocysts; SO, sporulated oocysts; Spz, sporozoites; sMrz, second-generation merozoites. A. qPCR of EtEsp at different developmental stages of E. tenella.. B. Western blot showing EtEsp at different developmental stages, probed with rabbit anti-rEtEsp serum or mouse monoclonal anti-α-tubulin antibody. Page 21/25 Page 21/25 Page 21/25 Figure 4 Interaction between EtAMA1 and EtEsp in DF-1 cells assessed with BiFC. A. DF-1 cells were transfected with VC155–EtAMA1 and VN155–EtEsp and the cellular lysates were analyzed with immunoblotting using antisera against EtAMA1 and EtEsp, 1. anti-r EtAMA1 antibody;2. anti-r EtEsp antibody. B. BiFC was performed. a, DF-1 cells were cotransfected with VC155–EtAMA1 and VN155–EtEsp. b, DF-1 cells were cotransfected with positive controls bFos and bJun. c, DF-1 cells were cotransfected with pBiFC-VC155– EtAMA1 and pBiFC-VN155 empty vector. . d, DF-1 cells were cotransfected with pBiFC-VN155–EtEsp and pBiFC-VC155 empty vector. Figure 4 Figure 5 Figure 5 In vitro pull-down assay between EtAMA1 and EtEsp. Lane M: protein marker; lane 1:rEtMIC2 protein incubated with rEtEsp ，detected with anti-rEtAMA1 and anti-rEtMIC2 as negative control; lane 2: rEtAMA1 protein incubated with rEtEsp，detected with anti-rEtAMA1 and anti-rEtEsp; lane 3: rEtEsp loaded in an empty glutathione-agarose column, detected with anti-rEtEsp as negative control. Figure 6 Western blotting analysis of secretion assays (supernantants and sporozoites pellet). (A): the supernants(Sup); (B): sporozoites pellet(Spz). Lane M: protein marker; lanes 1–3: 5, 10, and 20 μM staurosporine dissolved in DMSO.; lanes 4–6: volumes of DMSO solvent corresponding to 5, 10, or 20 μM staurosporine Figure 6 Figure 4 Interaction between EtAMA1 and EtEsp in DF-1 cells assessed with BiFC. A. DF-1 cells were transfected with VC155–EtAMA1 and VN155–EtEsp and the cellular lysates were analyzed with immunoblotting using antisera against EtAMA1 and EtEsp, 1. anti-r EtAMA1 antibody;2. anti-r EtEsp antibody. B. BiFC was performed. a, DF-1 cells were cotransfected with VC155–EtAMA1 and VN155–EtEsp. b, DF-1 cells were cotransfected with positive controls bFos and bJun. c, DF-1 cells were cotransfected with pBiFC-VC155– EtAMA1 and pBiFC-VN155 empty vector. . d, DF-1 cells were cotransfected with pBiFC-VN155–EtEsp and pBiFC-VC155 empty vector. Page 22/25 Page 22/25 Figure 5 In vitro pull-down assay between EtAMA1 and EtEsp. Lane M: protein marker; lane 1:rEtMIC2 protein incubated with rEtEsp ，detected with anti-rEtAMA1 and anti-rEtMIC2 as negative control; lane 2: rEtAMA1 protein incubated with rEtEsp，detected with anti-rEtAMA1 and anti-rEtEsp; lane 3: rEtEsp loaded in an empty glutathione-agarose column, detected with anti-rEtEsp as negative control. Figure 6 Western blotting analysis of secretion assays (supernantants and sporozoites pellet). (A): the supernants(Sup); (B): sporozoites pellet(Spz). Lane M: protein marker; lanes 1–3: 5, 10, and 20 μM staurosporine dissolved in DMSO.; lanes 4–6: volumes of DMSO solvent corresponding to 5, 10, or 20 μM staurosporine Western blotting analysis of secretion assays (supernantants and sporozoites pellet). (A): the supernants(Sup); (B): sporozoites pellet(Spz). Lane M: protein marker; lanes 1–3: 5, 10, and 20 μM staurosporine dissolved in DMSO.; lanes 4–6: volumes of DMSO solvent corresponding to 5, 10, or 20 μM staurosporine Page 23/25 Page 23/25 Figure 7 Immunofluorescent localization of EtEsp at different developmental stages of E. tenella. Parasites were immunostained with anti-rEtEsp antibody. A, Sporozoites (Spz) were incubated in PBS, A-a permeabilized sporozoites, A-b: non permeabilized sporozoites; B, sporocysts (Sporo) were incubated in PBS, B-a: permeabilized sporocysts, B-b: non permeabilized sporocysts; C, sporozoites (Spz) were incubated in complete medium (CM) for 2 h at 41 °C; D, E, intracellular sporozoites (iSpz) at 2 h and 12 h postinfection, respectively; F, trophozoites (iTropho) at 24 h postinfection; G, H, immature schizonts (iSc) at 48 and 60 h postinfection, respectively; I, mature schizonts (mSc) at 68 h postinfection; J, first-generation merozoites (fMrz) at 72 h postinfection; K, second-generation merozoites (sMrz) in PBS. Figure 7 Figure 7 Immunofluorescent localization of EtEsp at different developmental stages of E. tenella. Parasites were immunostained with anti-rEtEsp antibody. A, Sporozoites (Spz) were incubated in PBS, A-a permeabilized sporozoites, A-b: non permeabilized sporozoites; B, sporocysts (Sporo) were incubated in PBS, B-a: permeabilized sporocysts, B-b: non permeabilized sporocysts; C, sporozoites (Spz) were incubated in complete medium (CM) for 2 h at 41 °C; D, E, intracellular sporozoites (iSpz) at 2 h and 12 h postinfection, respectively; F, trophozoites (iTropho) at 24 h postinfection; G, H, immature schizonts (iSc) at 48 and 60 h postinfection, respectively; I, mature schizonts (mSc) at 68 h postinfection; J, first-generation merozoites (fMrz) at 72 h postinfection; K, second-generation merozoites (sMrz) in PBS. Immunofluorescent localization of EtEsp at different developmental stages of E. tenella. Parasites were immunostained with anti-rEtEsp antibody. A, Sporozoites (Spz) were incubated in PBS, A-a permeabilized sporozoites, A-b: non permeabilized sporozoites; B, sporocysts (Sporo) were incubated in PBS, B-a: permeabilized sporocysts, B-b: non permeabilized sporocysts; C, sporozoites (Spz) were incubated in complete medium (CM) for 2 h at 41 °C; D, E, intracellular sporozoites (iSpz) at 2 h and 12 h postinfection, respectively; F, trophozoites (iTropho) at 24 h postinfection; G, H, immature schizonts (iSc) at 48 and 60 h postinfection, respectively; I, mature schizonts (mSc) at 68 h postinfection; J, first-generation merozoites (fMrz) at 72 h postinfection; K, second-generation merozoites (sMrz) in PBS. Page 24/25 ( ) p g ( ) Figure 8 Figure 9 Inhibition of sporozoite invasion in vitro. All the assays were performed in triplicate (anti-rEtEsp, rabbit antiserum generated against recombinant EtESP protein; NI, IgG from naïve rabbit serum). **P < 0.01 for differences between treatment with antibody against rEtEsp or with naïve rabbit serum with the same IgG concentration. Inhibition of sporozoite invasion in vitro. All the assays were performed in triplicate (anti-rEtEsp, rabbit antiserum generated against recombinant EtESP protein; NI, IgG from naïve rabbit serum). **P < 0.01 for differences between treatment with antibody against rEtEsp or with naïve rabbit serum with the same IgG concentration. Figure 8 Figure 8 Page 24/25 Co-localization of EtCDPK4 and EtSerpin in sporozoites using mouse anti-rEtAMA1 antibody and rabbit anti-rEtEsp antibody by IFA. Co-localization of EtCDPK4 and EtSerpin in sporozoites using mouse anti-rEtAMA1 antibody and rabbit anti-rEtEsp antibody by IFA. anti-rEtEsp antibody by IFA. Figure 9 Inhibition of sporozoite invasion in vitro. All the assays were performed in triplicate (anti-rEtEsp, rabbit antiserum generated against recombinant EtESP protein; NI, IgG from naïve rabbit serum). **P < 0.01 for differences between treatment with antibody against rEtEsp or with naïve rabbit serum with the same IgG concentration. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Graphicalabstract.doc Graphicalabstract.doc Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Graphicalabstract.doc Page 25/25

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Exposure to high endotoxin concentration increases wheezing prevalence among laboratory animal workers: a cross-sectional study

BMC pulmonary medicine

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* Correspondence: [email protected] 2Department of Medicine, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil Full list of author information is available at the end of the article Freitas et al. BMC Pulmonary Medicine (2016) 16:69 DOI 10.1186/s12890-016-0233-1 Freitas et al. BMC Pulmonary Medicine (2016) 16:69 DOI 10.1186/s12890-016-0233-1 Open Access Exposure to high endotoxin concentration increases wheezing prevalence among laboratory animal workers: a cross-sectional study Amanda Souza Freitas1, Christian Silva Simoneti2, Erica Ferraz2, Ericson Bagatin3, Izaira Tincani Brandão4, Celio Lopes Silva4, Marcos Carvalho Borges2 and Elcio Oliveira Vianna2* Skin prick test Skin prick tests (SPTs) were carried out following the recommendations of the European Academy of Allergol- ogy and Clinical Immunology [19]. Subjects did not take antihistamine drugs for 15 days prior to SPT. The test was considered positive when a reaction led to a wheal diameter of at least 3 mm in the absence of a reaction to physiological saline solution and in the presence of a positive reaction to histamine. The allergens included common allergens (Dermatophagoides farinae, Dermato- phagoides pteronyssinus, Felis domesticus, Canis famil- iaris, Blomia tropicalis, Blattella germanica, Periplaneta americana, Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, and mixed grass) and extracts from animals (mouse, rat, hamster, guinea pig and rabbit). These were called occupational allergens. Questionnaires and diagnostic procedures Q g p Questionnaires were applied to 751 subjects with questions inquiring about personal history of allergic diseases, pet owning history, smoking, respiratory, nasal, ocular, and skin symptoms. The questionnaire items also included job characteristics such as dur- ation of working with laboratory animals, job titles, job contents, frequency of contact with animals, spe- cies, time spent handling animals, use of protective equipment, asthma and rhinitis symptoms. Subjects also underwent skin prick test, spirometry and bron- chial hyperresponsiveness (BHR) test with mannitol. For most questions about symptoms and risk factors, we used questions from the European Community Respiratory Health Survey questionnaire, translated into Portuguese, adapted to the Brazilian lexicon and previously tested [17, 18]. Researchers and technicians who work with laboratory animals are exposed to both animal allergens and endo- toxin in the workplace. Some studies have identified the agents present in organic dirt in the workplace and their interaction with respiratory and allergic diseases among the workers. These studies have evaluated various envi- ronments and populations [9–12], but few of them have dealt with workers exposed to laboratory animals regard- ing the interaction of endotoxin with respiratory symp- toms [13, 14]. In the workplace, the concentration of endotoxin re- sponsible for triggering respiratory effects (including asthma) is often below the permissible exposure limits (PELs) or occupational exposure limits (OELs) [15], but more studies are needed on the role of endotoxin in the workplace and on the genesis of risks for workers. Al- though several negative effects have been reported, there are also beneficial effects of exposure to endotoxin [7, 9] and more studies are needed to clarify such effects. On this basis, we formulated the hypothesis that exposure to endotoxin may cause asthma symptoms. Thus, the ob- jective of the present study was to determine the associ- ation between the quantity of endotoxin detected in the workplace and the presence of asthma or wheezing. Abstract Background: Endotoxin from Gram-negative bacteria are found in different concentrations in dust and on the ground of laboratories dealing with small animals and animal houses. Methods: Cross-sectional study performed in workplaces of two universities. Dust samples were collected from laboratories and animal facilities housing rats, mice, guinea pigs, rabbits or hamsters and analyzed by the “Limulus amebocyte lysate” (LAL) method. We also sampled workplaces without animals. The concentrations of endotoxin detected in the workplaces were tested for association with wheezing in the last 12 months, asthma defined by self-reported diagnosis and asthma confirmed by bronchial hyperresponsiveness (BHR) to mannitol. Results: Dust samples were obtained at 145 workplaces, 92 with exposure to animals and 53 with no exposure. Exposed group comprised 412 subjects and non-exposed group comprised 339 subjects. Animal-exposed workplaces had higher concentrations of endotoxin, median of 34.2 endotoxin units (EU) per mg of dust (interquartile range, 12.6–65.4), as compared to the non-exposed group, median of 10.2 EU/mg of dust (interquartile range, 2.6–22.2) (p < 0.001). The high concentration of endotoxin (above whole sample median, 20.4 EU/mg) was associated with increased wheezing prevalence (p < 0.001), i.e., 61 % of workers exposed to high endotoxin concentration reported wheezing in the last 12 months compared to 29 % of workers exposed to low endotoxin concentration. The concentration of endotoxin was not associated with asthma report or with BHR confirmed asthma. Conclusion: Exposure to endotoxin is associated with a higher prevalence of wheezing, but not with asthma as defined by the mannitol bronchial challenge test or by self-reported asthma. Preventive measures are necessary for these workers. Keywords: Endotoxin, Wheezing, Occupational disease, Animal handler © 2016 Freitas et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Freitas et al. BMC Pulmonary Medicine (2016) 16:69 Page 2 of 9 Page 2 of 9 Background [16]. Volunteers were workers or students with expos- ure to laboratory animals (exposed group) and with- out exposure (non-exposed group). Endotoxin is a constituent of the outer cell wall of Gram-negative bacteria and its main component is lipo- polysaccharide (LPS) [1]. Thorne et al. [2] showed that exposure to endotoxin in the domestic environment sig- nificantly increases the prevalence of asthma in adults. Endotoxin has also been related to the onset of “Monday morning asthma” among cotton workers, to damp fever, grain fever, toxic pneumonia, and acute systemic effects such as malaise and fever [3]. Adults exposed to high endotoxin levels in house dust (more than 11.2 EU/mg of dust) are more susceptible to worsening of allergic and non-allergic asthma and to the development of asthma [4–6]. p p g p Laboratories and workplaces were randomly selected from the facilities and 145 workplaces were included in this study, 53 workplaces belonged to the non- exposed group (339 subjects) and 92 workplaces to the exposed group (412 subjects). At least 90 % of subjects in every workplace consented to participate; the overall consent and participation rate was 95 %. The study was reviewed and approved by the Ethics Committees of both institutions: Medical School of Ribeirão Preto, University of S. Paulo (Comitê de Ética em Pesquisa do FMRP-USP, protocol number 9428/2009), and the School of Medical Sciences, State University of Campinas (Comitê de Ética em Pesquisa da UNICAMP, protocol number 779/2009). Written informed consent was obtained from all subjects after reading and discussing the protocol individually. Although few studies are available, some authors have shown that exposure to high endotoxin concentrations is associated with an increased risk of wheezing during the first year of life [7, 8]. On the other hand, some stud- ies have suggested that early exposure to endotoxin present in the environment has a protective effect on the risk of atopic sensitization in children and possibly also on the population of workers exposed to high endo- toxin levels [3, 8, 9]. Variables Groups: The exposed group was composed of volunteers exposed to laboratory animal, including: researchers, technicians and students. The non-exposed group con- sisted of secretaries, car drivers, management employees, computer technicians, students and other employees who had no contact with laboratory animals. For more details on characteristics of groups and subjects, refer to Ferraz et al. [16]. Methods This is a cross-sectional study that was conducted at two universities, the University of São Paulo (USP) at Ribeirão Preto and State University of Campinas (UNICAMP), São Paulo State, Brazil. Sample selection and study protocol have been previously described Freitas et al. BMC Pulmonary Medicine (2016) 16:69 Page 3 of 9 Page 3 of 9 Dust samples l d Floor dust samples were collected at both groups work- places. One square meter was sampled for 2 min using a vacuum cleaner (Arno Nitro, São Paulo, Brazil) equipped with a fiberglass filter with a pore size of approximately 1 μm. Filters were pre- and post-weighed. Loaded filters were transported separately, packed and stored in a re- frigerator (2 to 8 °C) until processing within a maximum of 4 weeks. Amount of dust varied in different work- places. This amount was compared between groups and concentration of endotoxin was expressed in relation to dust weight. Endotoxin analysis Endotoxin concentration was measured using the kinetic chromogenic Limulusamebocyte lysate assay (LAL - Bio Whittaker - A CAMBREX Company). Briefly, 100 μl of the sample was incubated for 10 min at 37 °C in a 96-well microtiter plate. The LAL reagent was reconsti- tuted in pyrogen-free water, rapidly added to the samples, and kinetics was recorded with a temperature-controlled microplate reader at 405 nm and 37 °C. Endotoxin levels are reported as concentration [endotoxin unit/mg of col- lected dust (EU/mg)]. Self-reported asthma was defined by positive answer to the question: Have you ever had asthma? Confirmed asthma: A volunteer was considered an asthmatic if he/she had a positive bronchial challenge test and had experienced symptoms of wheezing, dys- pnea during the night or at day in the previous 12 months, or tightness of the chest during the night. Current wheezing was defined by a positive answer to the question: “Have you had wheezing or whistling in your chest any time in the last 12 months?” Bronchial challenge test with mannitol Dry powdered mannitol (Aridol) was supplied in kit form (Pharmaxis Ltd., New South Wales, Australia), which contained 1 empty capsule (0 mg), capsules con- taining 5, 10 and 20 mg, and 15 capsules containing 40 mg (cumulative dose of 635 mg). The challenge began with the empty capsule, followed by inhalation of 5, 10, 20, 40, 80, 160, 160, and 160 mg dry powdered mannitol. Forced expiratory volume in one second (FEV1) was measured 60 s after every inhalation. This procedure was repeated for each dose step until a 15 % fall in FEV1 was achieved or the cumulative dose reached 636 mg. A positive test (BHR) was defined as a 15 % decrease in FEV1 to 635 mg or less [22]. Filters with dust were transferred to 50 ml pyrogen- free tubes. Dust was extracted in 35 ml of pyrogen-free water containing 0.05 % (v/v) Tween-20. After an ultra- sound bath for 20 min at 40 kHz frequency (Ultra Sonic Cleaner, Unique, São Paulo, Brazil), the tubes were cen- trifuged for 10 min at 991xg, 1 ml of the extract was pi- petted and again centrifuged at 1000xg for 10 min at room temperature, and the supernatant was stored in 5 μl aliquots. Spirometry and low endotoxin concentrations means ≤20.4 EU/mg. This cut-off is the median of endotoxin concentration of all the workplaces, i.e., both exposed and non-exposed group workplaces (whole sample median). The lung function was analyzed using a Koko spirometer and software (PDS Instrumentation, Inc., Louisville, Colorado, USA), the spirometer were calibrated every day. Lung function was conducted in the sitting position with the volunteers using a nose clip. We performed at most 8 attempts to obtain at least 3 technically satisfac- tory maneuvers for each volunteer. If after 8 attempts, at least 3 technically satisfactory maneuvers were not obtained, lung function test was interrupted [20]. The reference values of Crapo et al. [21] were used. Smoking: This variable was defined by a positive an- swer to the question “Have you smoked for more than one year?” Atopy: This variable was defined by a positive skin prick test to any allergen. Statistical analysis Univariate analysis with chi-square test was used to compare prevalence estimates and proportions be- tween groups (exposed versus non-exposed group), between different asthma definitions and to compare wheezing prevalence among endotoxin concentration quartiles. Two-tailed Student’s t test was used to compare continuous variables (age, dust weight, and endotoxin concentration) between exposed and non- exposed groups. Past exposure to laboratory animals was defined by a positive answer to the question: “In the past, did you work in places with animals (laboratory or animal rooms)?” Pet ownership: This variable was defined by the ques- tion “In the last 12 months, have you had pets at home?” Endotoxin concentration: High endotoxin concentra- tions included values above the median (20.4 EU/mg) Page 4 of 9 Freitas et al. BMC Pulmonary Medicine (2016) 16:69 Page 4 of 9 Multivariate analysis was performed to test for associa- tions between endotoxin levels and asthma or wheezing by using a modified Poisson regression approach, i.e., Poisson regression with a robust error variance [23]. Endotoxin levels were categorized according to the me- dian in high and low concentration levels. The model was adjusted using the PROC GENMOD procedure of the SAS software version 9.2, which was also used for data analysis. of endotoxin (>20.4 EU/mg) and self-reported asthma (Table 3) or asthma confirmed by BHR (Table 4). We found association between wheezing and high levels of endotoxin (>20.4 EU/mg) (Table 5). When endotoxin concentrations were divided into 4 categories (quartiles), wheezing prevalence increased with increasing endo- toxin concentration in the exposed group (Fig. 1). Among the workers who reported asthma in response to the questionnaire, 62 (72 %) also reported wheezing in the last 12 months (p ≤0.01) and among the workers with confirmed asthma, 64 (91 %) also reported wheez- ing (p ≤0.01). Among individuals who reported wheez- ing and asthma (n = 62), there was no association between wheezing and high endotoxin concentration in dust (p = 0.914). In cases of wheezing excluding asthma (n = 80), there was association of wheezing with high endotoxin concentrations (p = 0.001). Results A total of 751 volunteers participated in this study, with 412 volunteers in the exposed group and 339 volunteers in the non-exposed group. The mean age of the volun- teers was 29 years, without difference between groups. Female predominance was observed (p < 0.001). We col- lected 145 samples of dust, 92 samples in the exposed group and 53 in the non-exposed group. The concen- tration of endotoxin in the dust was significantly dif- ferent between groups. In the exposed group, the median concentration of endotoxin was 34.2 EU/mg of dust; and, in the non-exposed group, it was 10.2 EU/mg of dust (p < 0.001). In the exposed group, there was a higher prevalence of students (p < 0.001) (Table 1). Discussion We confirmed the presence of endotoxin in dust col- lected from workplaces that had laboratory animals. Detectable levels of endotoxin were found also in workplaces without laboratory animals. However, the amount of endotoxin was higher in the exposed group (34.2 EU/mg) vs non-exposed group (10.2 EU/mg) (p < 0.001). There was no difference in the prevalence of sensitization to common allergens between the groups. The prevalence of sensitization to occupa- tional allergens was significantly higher in the ex- posed group (16.5 %) compared to the non-exposed group (2.6 %) (p < 0.001).). The prevalence of self- reported asthma, confirmed asthma and current wheezing were not different between the groups. A significant difference was found for past exposure to laboratory animals: 70 % for the animal exposed group and 30 % for the non-exposed group (p < 0.001) (Table 2). In the exposed group, workplaces with high concen- trations of endotoxin (above whole sample median) were more frequent in those dealing with rats (79 % of the workplaces) than in those dealing with mice (27 %, p < 0.001 - data not shown in Results). Low doses of endotoxin were more frequent in the work- places of the non-exposed group (66 % versus 36 % of the exposed group). This showed that the amount of endotoxin was associated with the presence of ani- mals in the workplaces and probably other animal products that may pose risks to workers. In the multivariate analyses, no associations were found between high levels (above whole sample median) Analysis of the relationship between endotoxin concen- tration and report of respiratory symptoms or diseases Table 1 Subjects and workplaces characteristics Variables Total n = 751 Exposed group n = 412 Non-exposed group n = 339 p value Workplaces 145 92 53 Age (years) 29 (25–40) 29 (25–37) 30 (25–41) 0.14 Female, n (%) 446 (59.3 %) 221 (53.6 %) 225 (66.3 %) <0.001* Dust (g/m2) 0.112 (0.04–0.31) 0.110 (0.02–0.23) 0.117 (0.05–0.31) 0.63 Endotoxin (EU/mg of dust) 20.4 (6.6–45.5) 34.2 (12.6–65.4) 10.2 (2.6–22.2) <0.001* Technicians̸administrators 326 (43.4 %) 140 (34.0 %) 186 (54.9 %) <0.001* Students 360 (48.0 %) 240 (58.2 %) 120 (35.4 %) Researchers 65 (8.6 %) 32 (7.8 %) 33 (9.7 %) Results are expressed as median (interquartile range) or n (%) *chi-square test EU endotoxin unit Freitas et al. Discussion BMC Pulmonary Medicine (2016) 16:69 Page 5 of 9 Table 2 Clinical data and diagnoses Variables Total n = 751 Exposed group n = 412 Non-exposed group n = 339 p value Skin prick test Commom allergen (s) 333 (44.3 %) 176 (42.7 %) 157 (46.3 %) 0.269 Occupational allergen (s) 77 (10.2 %) 68 (16.5 %) 9 (2.6 %) <0.001* Smoking 129 (17.1 %) 72 (17.4 %) 57 (16.8 %) 0.811 Spirometry (normal) 715 (94.7 %) 389 (94.4 %) 326 (96.1 %) 0.265 Self-reported asthma 88 (12.0 %) 48 (11.6 %) 40 (11.8 %) 1.00 Confirmed asthma 73 (10 %) 42 (10 %) 31 (9 %) 0.71 Wheezing 163 (21.7 %) 96 (23.3 %) 67 (19.7 %) 0.24 BHRa 95 (12.9 %) 57 (14.0 %) 38 (11.5 %) 0.31 Past exposure to laboratory animals 240 (32 %) 168 (70 %) 72 (30 %) <0.001* Pet owning history 485 (64.6 %) 258 (53.2 %) 227 (46.8 %) 0.22 Results are expressed as median (interquartile range) or n (%). *chi-square test. BHR bronchial hyperresponsiveness a16 subjects did not undergo bronchial challenge test, 6 in the exposed group and 10 in the non-exposed group Results are expressed as median (interquartile range) or n (%). *chi-square test. BHR bronchial hyperresponsiveness a16 subjects did not undergo bronchial challenge test, 6 in the exposed group and 10 in the non-exposed group constant hyperresponsiveness. In this case, hyperre- sponsiveness would not be detected on the day of examination. among workers showed that high concentrations of endotoxin collected from the floor of laboratories and animal rooms were associated with wheezing in the last 12 months. However, we did not find an associ- ation with either reported asthma or confirmed asthma. Our results showed an increase in reported wheezing with increasing endotoxin concentrations in the animal exposed group, whereas this relationship was not de- tected in the non-exposed group. Oldenburg et al. [25] showed an exposure-response relationship between occupational inhalative endotoxin exposure and ob- structive ventilation patterns in German cotton textile workers. A significant decrease for FEV1/FVC rate was detected in association with increasing exposure to air- borne endotoxin measured in 22 different workplaces of the cotton-spinning mill. Thus, the concentration of endotoxin was associated with wheezing, but not with asthma, leading us to ques- tion if wheezing is due to asthma or to other patho- physiological mechanisms [24]. Discussion The association between wheezing and asthma, very well known in the medical literature, was recognized in the present study. Wheez- ing could be an initial manifestation of an endotoxin- dependent asthma, but not yet diagnosed as asthma (therefore, not reported by the subjects). Wheezing could also be related to intermittent asthma - without However, it is difficult to determine whether endotoxin in environments with animals directly causes wheezing, Table 3 Evaluation of risk factors for self-reported asthma Variables Categories Reported asthma Crude model Adjusted model PR 95 % CI p value PR 95 % CI p value Group Exposed vs Non-exposed 0.98 0.66 1.47 0.94 0.99 0.67 1.47 0.96 Endotoxin levels Low vs High 0.99 0.66 1.47 0.95 1.01 0.68 1.50 0.96 Institutions UNICAMP vs USP 1.01 0.68 1.50 0.97 1.06 0.73 1.56 0.76 Atopy Yes vs No 7.93 4.29 14.68 <0.01 7.69 4.18 14.16 <0.01 Smoking Yes vs No 0.88 0.51 1.54 0.67 0.84 0.49 1.43 0.51 Sex Male vs Female 1.09 0.72 1.63 0.69 1.15 0.77 1.73 0.49 Past exposure to laboratory animals Yes vs No 1.41 0.94 2.11 0.10 1.26 0.86 1.84 0.24 Pet owning history Yes vs No 1.19 0.77 1.82 0.43 1.13 0.76 1.70 0.54 Age Continuous 0.99 0.97 1.01 0.16 1.01 0.99 1.04 0.21 Worker functions Technicians/secretaries vs researchers 7.20 1.01 51,54 0.05 6.75 0.92 49.36 0.06 Students vs researchers 8.83 1.24 62.78 0.03 7.73 1.02 58.47 0.05 PR prevalence ratio, CI confidence interval, UNICAMP State University of Campinas, USP University of São Paulo; Low endotoxin concentration: ≤20.4 EU/mg of dust; High endotoxin concentration: > 20.4 EU/mg of dust Table 3 Evaluation of risk factors for self-reported asthma Variables Categories Table 3 Evaluation of risk factors for self-reported asthma Page 6 of 9 Freitas et al. Discussion BMC Pulmonary Medicine (2016) 16:69 Table 4 Evaluation of risk factors for asthma confirmed Variables Categories Asthma confirmed by BHR Crude model Adjusted model PR 95 % CI p value PR 95 % CI p value Group Exposed vs Non-exposed 1.08 0.69 1.68 0.73 1.12 0.71 1.76 0.63 Endotoxin levels Low vs High 1.21 0.78 1.88 0.40 0.79 0.51 1.22 0.29 Institutions UNICAMP vs USP 1.40 0.89 2.18 0.14 1.25 0.81 1.93 0.31 Atopy Yes vs No 7.07 3.68 13.58 <0.01 6.60 3,42 12.71 <0.01 Smoking Yes vs No 0.89 0.48 1.64 0.70 1.00 0.54 1.86 0.99 Sex Male vs Female 1.39 0.87 2.21 0.17 1.48 0.93 2.37 0.10 Past exposure to laboratory animals Yes vs No 1.36 0.87 2.14 0.17 1.24 0.79 1.94 0.35 Pet owning history Yes vs No 1.17 0.73 1.87 0.52 1.05 0.66 1.66 0.83 Age Continuous 0.96 0.94 0.99 <0.01 0.98 0.95 1.01 0.16 Worker functions Technicians/secretaries vs researchers 1.44 0.52 3.95 0.48 1.35 0.49 3.72 0.56 Students vs researchers 1.74 0.65 4.71 0.27 1.09 0.41 3.72 0.56 BHR bronchial hyperresponsiveness, PR prevalence ratio, CI confidence interval, UNICAMP State University of Campinas, USP University of São Paulo, Low endotoxin concentration: ≤20.4 EU/mg of dust, High endotoxin concentration: > 20.4 EU/mg of dust Table 4 Evaluation of risk factors for asthma confirmed Variables Categories Table 4 Evaluation of risk factors for asthma confirmed esponsiveness, PR prevalence ratio, CI confidence interval, UNICAMP State University of Campinas, USP University of São Paulo, Low ion: ≤20.4 EU/mg of dust, High endotoxin concentration: > 20.4 EU/mg of dust or if there is some interaction with other allergenic pro- teins present in the environment that may cause in- creased wheezing. If allergens or other animal products were directly responsible for the increase of wheezing, endotoxin could be only a marker of the presence of ani- mals. In this work, allergen concentration in dust was measured and no association with symptoms, asthma or BHR has been detected [26]. It seems, thus, that the ob- served association between endotoxin levels and wheeze is not due to laboratory animal allergy rather than to endotoxin. detected in the air of animal research laboratories than in laboratories using no animals and in areas outside the animal facilities. This suggests that mice and cages kept in the laboratories may have been the source of endo- toxin. Discussion *p < 0.01 for the comparison of wheezing prevalence among levels of endotoxin concentration in the animal exposed group. Wheezing was a positive answer to the question: Have you had wheezing or whistling in your chest any time in the last 12 months? Fig. 1 Wheezing prevalence according to endotoxin concentration in both groups. *p < 0.01 for the comparison of wheezing prevalence among levels of endotoxin concentration in the animal exposed group. Wheezing was a positive answer to the question: Have you had wheezing or whistling in your chest any time in the last 12 months? are associated with higher circulating CD14 levels, whereas the T allele (CD14/2550 T) is associated with lower plasma-soluble CD14 levels [28, 29]. Individuals who carry the CD14/-260 C allele are more responsive to endotoxin than those who carry the homozygous T al- lele even in the presence of high occupational levels. The dose-response curve for individuals with CD14/-260 CT and CC demonstrated wheezing in the presence of high occupational levels of endotoxin. Pacheco et al. [30] published a study with similar findings, although regard- ing a population of workers exposed to laboratory ani- mals. Workers carrying the CD14/1619 G allele and exposed to high endotoxin concentrations (fourth quar- tile) had a significantly lower pulmonary function than workers similarly exposed but carrying the AA genotype. the workers’ lungs. Endotoxin inhalation can induce a systemic inflammatory response by macrophage acti- vation and by neutrophil influx into the lungs. An in- crease in IL-6 was observed during exposure and the endotoxin-induced increase in blood leukocytes and D-dimer was reversed one year after the factory was closed. Since endotoxin was found to be present in the workers’ blood, a direct stimulation of circulating cells of the immunological system seems to be pos- sible. Consequently, both mechanisms may result in an increase of acute phase proteins. A peculiarity of the present study was collection of dust from the floor. The sampling of endotoxin in the dust deposited on the floor rather than the sampling of airborne endotoxin has the advantage of easy and stan- dardized collection. Indeed, the sampling of airborne endotoxin is a complex procedure for which, to date, there is no fully standardized protocol [32]. Skogstad et al. [31] analyzed the effects of exposure to bacterial endotoxin in 28 workers employed by a factory that produced bioproteins derived from a bac- terial species (Methylococcus capsulatum). Discussion Concentrations of air-transported endotoxin were detected in 100 % of the samples collected, where as mouse allergens were detected in only 70 % of the samples, probably because there were other sources of endotoxin in addition to mice. The association between exposure to endotoxin and wheezing among farmers differs according to CD14 type (a cell differentiation marker that serves as a link be- tween LPS and macrophages) [27]. G and T alleles (21.619 A/G and 2159 C/T polymorphisms, respectively) Our results are similar to those previously reported re- garding increased endotoxin exposure related to animal laboratories [14]. Higher quantities of endotoxin were Table 5 Evaluation of risk factors for wheezing in the last 12 months Variables Categories Wheezing Crude model Adjusted model PR 95 % CI p value PR 95 % CI p value Group Exposed vs Non-exposed 1.18 0.89 1.55 0.24 1.03 0.78 1.37 0.81 Endotoxin levels Low vs High 1.44 1.09 1.90 <0.01 1.49 1.14 1.96 <0.01 Institutions UNICAMP vs USP 0.97 0.74 1.28 0.85 0.89 0.68 1.16 0.38 Atopy Yes vs No 3.38 2.46 4.65 <0.01 3.20 2.33 4.39 <0.01 Smoking Yes vs No 0.87 0.59 1.29 0.49 0.94 0.65 1.37 0.74 Sex Male vs Female 1.06 0.80 1.39 0.70 1.10 0.84 1.44 0.49 Past exposure to laboratory animals Yes vs No 1.32 1.00 1.73 0.05 1.21 0.93 1.57 1.15 Pet owning history Yes vs No 1.39 1.02 1.88 0.04 1.38 1.03 1.85 0.03 Age Continuous 0.97 0.96 0.99 <0.01 0.98 0.96 0.99 0.03 Worker functions Technicians/secretaries vs researchers 1.34 0.73 2.46 0.35 1.08 0.60 1.94 0.80 Students vs researchers 1.66 0.91 3.01 0.10 0.93 0.52 1.69 0.82 PR prevalence ratio, CI confidence interval, UNICAMP State University of Campinas, USP University of São Paulo, Low endotoxin concentration: ≤20.4 EU/mg of dust, High endotoxin concentration: > 20.4 EU/mg of dust Table 5 Evaluation of risk factors for wheezing in the last 12 months Freitas et al. BMC Pulmonary Medicine (2016) 16:69 Page 7 of 9 Fig. 1 Wheezing prevalence according to endotoxin concentration in both groups. *p < 0.01 for the comparison of wheezing prevalence among levels of endotoxin concentration in the animal exposed group. Wheezing was a positive answer to the question: Have you had wheezing or whistling in your chest any time in the last 12 months? Fig. 1 Wheezing prevalence according to endotoxin concentration in both groups. References 1. Doreswamy V, Peden DB. Modulation of asthma by endotoxin. Clin Exp Allergy. 2011;41(1):9–19. 1. Doreswamy V, Peden DB. Modulation of asthma by endotoxin. Clin Exp Allergy. 2011;41(1):9–19. 1. Doreswamy V, Peden DB. Modulation of asthma by endotoxin. Clin Exp Allergy. 2011;41(1):9–19. 2. Thorne PS, Kulhánková K, Yin M, Cohn R, Arbes Jr SJ, Zeldin DC. Endotoxin exposure is a risk factor for asthma: the national survey o endotoxin in U.S. housing. Am J Respir Crit Care Med. 2005;172(11): 1371–7. Acknowledgements We are grateful to PROESTAT (Consultoria Estatística e Pesquisa de Mercado) for statistical analysis. Competing interests The authors declare that they have no competing interests. 13. Pacheco KA, McCammon C, Liu AH, Thorne PS, O’Neill ME, Martyny J, et al. Airborne endotoxin predicts symptoms in non-mouse-sensitized technicians and research scientists exposed to laboratory mice. Am J Respir Crit Care Med. 2003;167(7):983–90. Discussion contributed with study design and endotoxin measurements supervising. MCB helped with patient care, study design and data collection supervising. EOV conceived study design, funding acquisition, data collection supervising, data analysis and manuscript writing. All authors read and approved the final manuscript. contributed with study design and endotoxin measurements supervising. MCB helped with patient care, study design and data collection supervising. EOV conceived study design, funding acquisition, data collection supervising, data analysis and manuscript writing. All authors read and approved the final manuscript. Funding S l g São Paulo Research Foundation (FAPESP) and National Council of Technological and Scientific Development (CNPq), Brazil. São Paulo Research Foundation (FAPESP) and National Council of Technological and Scientific Development (CNPq), Brazil. Abbreviations CI 95 % CI: 95 % confidence interval; BHR: bronchial hyperresponsiveness; CD14: cell differentiation marker; EU: endotoxin unit; FEV1/FVC: forced expiratory volume in one second/forced vital capacity; IL: interleukin; IPE: individual protection equipment; LAL: limulus amebocyte lysate; LPS: lipopolysaccharide; PR: prevalence ratio; UNICAMP: State University of Campinas; USP: University of São Paulo. 10. Liu AH. Endotoxin exposure in allergy and asthma: reconciling a paradox. J Allergy Clin Immunol. 2002;109(3):379–92. 10. Liu AH. Endotoxin exposure in allergy and asthma: reconciling a paradox. J Allergy Clin Immunol. 2002;109(3):379–92. 11. Elliott L, Heederik D, Marshall S, Peden D, Loomis D. Incidence of allergy and allergy symptoms among workers exposed to laboratory animals. Occup Environ Med. 2005;62(11):766–71. 11. Elliott L, Heederik D, Marshall S, Peden D, Loomis D. Incidence of allergy and allergy symptoms among workers exposed to laboratory animals. Occup Environ Med. 2005;62(11):766–71. 12. Ruoppi P, Koistinen T, Susitaival P, Honkanen J, Soininen H. Frequency of allergic rhinitis to laboratory animals in university employees as confirmed by chamber challenges. Allergy. 2004;59(3):295–301. 12. Ruoppi P, Koistinen T, Susitaival P, Honkanen J, Soininen H. Frequency of allergic rhinitis to laboratory animals in university employees as confirmed by chamber challenges. Allergy. 2004;59(3):295–301. Availability of data and materials 8. Park JH, Gold DR, Spiegelman DL, Burge HA, Milton DK. House dust endotoxin and wheeze in the first year of life. Am J Respir Crit Care Med. 2001;163(2):322–8. Database is available on zenodo.org (https://zenodo.org/ record/49686#.VxS4xPkrLIV). Database is available on zenodo.org (https://zenodo.org/ record/49686#.VxS4xPkrLIV). 9. Spaan S, Heederik DJ, Thorne PS, Wouters IM. Optimization of airborne endotoxin exposure assessment: effects of filter type, transport conditions, extraction solutions, and storage of samples and extracts. Appl Environ Microbiol. 2007;73(19):6134–43. Consent for publication Not applicable. Not applicable. Discussion The study lasted 5 years, 4 of them during exposure and 1 after the cessation of exposure. Forced vital capacity values obtained by spirometry were significantly lower in the group exposed to low endotoxin levels compared to the values obtained in the year without exposure. The values of FEV1 were significantly higher after the ces- sation of exposure than during exposure in the group with low exposure. There was also a significant asso- ciation between exposure to high endotoxin concen- trations and number of leukocytes detected in blood tests, with leukocyte values normalizing after the ces- sation of exposure. These findings reveal the associ- ation between exposure and inflammatory activity in Limitations of this study may be a cross-sectional de- sign instead of longitudinal one, lack of evaluation of air- borme endotoxin and lack of evaluation of other environmental variables, such as room ventilation and cleaning schedule. We believe that the study of components present in the workplace, such as endotoxin, and their role in the triggering of allergic and respiratory diseases among the workers could be the first step of a health care program for these workers. Since exposure to high quantities of endotoxin is associated with a more frequent report of wheezing among workers exposed to laboratory animals, it is necessary to propose intervention measures in order to reduce this exposure. Page 8 of 9 Page 8 of 9 Freitas et al. BMC Pulmonary Medicine (2016) 16:69 Freitas et al. BMC Pulmonary Medicine (2016) 16:69 Page 8 of 9 In the present study, we analyzed the quantity of endotoxin and its relationship with the clinical findings, but we did not analyze the interventions needed to re- duce exposure. However, our group has studied and published data obtained by applying questionnaires re- garding the use of measures for the reduction of this exposure. Questions about the availability of breathing masks, glasses or protective visors, gloves and appropri- ate shoes for the workplace and about the use of this in- dividual protection equipment (IPE) during contact with the animals revealed that the most accessible IPE were gloves (99 %) and the least accessible were appropriate shoes (36 %). Nineteen percent of animal handlers used breathing masks at all times while handling animals or when working in animal rooms, but only 7 % wore pro- tective glasses and 24 % wore specific shoes [16]. Author details 1 1Department of Social Medicine, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. 2Department of Medicine, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. 3Department of Preventive and Social Medicine, State University of Campinas, Campinas, Brazil. 4Department of Biochemistry and Immunology, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. The present results are very important for the under- standing of the presence of endotoxins in the workplace and their interaction with workers’ symptoms, since there still are doubts about the role of endotoxins as a risk factor for respiratory diseases or as a protective fac- tor against the latter. Received: 7 December 2015 Accepted: 27 April 2016 Received: 7 December 2015 Accepted: 27 April 2016 Conclusion We conclude that exposure to endotoxin has an effect on the respiratory system of workers even though it is not directly associated with asthma. High endotoxin concentrations were associated with the presence of wheezing, especially in the group exposed to laboratory animals. This shows that animal house workers and workers who are in direct contact with laboratory ani- mals are more susceptible to wheezing, with the need for preventive measures. 3. Dungan RS. Airborne endotoxinfrom indoor and outdoor environments: effects of sample diluition on the kinetic Limulus amebocyte lysate (LAL) assay. J Occup Environ Hyg. 2011;8(3):147–53. 4. Michel O, Ginanni R, Duchateau J, Vertongen F, Le Bon B, Sergysels R. Domestic endotoxin exposure and clinical severity of asthma. Clin Exp Allergy. 1991;21(4):441–8. 5. Michel O, Kips J, Duchateau J, Vertongen F, Robert L, Collet H, et al. Severity of asthma is related to endotoxin in house dust. Am J Respir Crit Care Med. 1996;154(6 Pt 1):1641–6. 6. Schwartz DA. Does inhalation of endotoxin cause asthma? Am J Respir Crit Care Med. 2001;163(2):305–6. 7. Gehring U, Bolte G, Borte M, Bischof W, Fahlbusch B, Wichmann HE, et al. Lifestyle-related factors on the immune system and the development of allergies in childhood. Exposure to endotoxin decreases the risk of atopic eczema in infancy: a cohort study. J Allergy Clin Immunol. 2001;108(5):847–54. Authors’ contributions ASF was responsible for data analysis, manuscript writing, data and dust collection. CSS was responsible for data and dust collection, manuscript writing and reviewing. EF performed fieldwork planning and clinical data collection. EB supervised patient care, study design and data collection. ITB carried out endotoxin measurements and helped with data analysis. CLS 14. Pacheco KA, McCammon C, Thorne PS, O’Neill ME, Liu AH, Martyny JW, et al. Characterization of endotoxin and mouse allergen exposures in mouse facilities and research laboratories. Ann Occup Hyg. 2006;50(6):563–72. Page 9 of 9 15. Gautrin D, Bernstein IL, Brooks SM, Henneberger PK. Reactive airways dysfunction syndrome and irritant-induced asthma. In: Bernstein IL, Chan-Yeung M, Malo JL, Bernstein DI, editors. Asthma in the workplace. New York: Taylor & Francis Group; 2006. p. 581–629. 16. Ferraz E, Arruda LK, Bagatin E, Martinez EZ, Cetlin AA, Simoneti CS, et al. Laboratory animals and respiratory allergies: the prevalence of allergies among laboratory animal workers and the need for prophylaxis. Clinics (São Paulo). 2013;68(6):750–9. 17. [No authors listed]. Variations in the prevalence of respiratory symptoms, self-reported asthma attacks, and use of asthma medication in the European Community Respiratory Health Survey (ECRHS). Eur Respir J. 1996; 9(4):687–95. 17. [No authors listed]. Variations in the prevalence of respiratory symptoms, self-reported asthma attacks, and use of asthma medication in the European Community Respiratory Health Survey (ECRHS). Eur Respir J. 1996; 9(4):687–95. 18. Ribeiro M, Angelini L, Robles-Ribeiro PG, Stelmach R, Santos UP, Terra-Filho M. Validation of the Brazilian-Portuguese version of the European Community Respiratory Health Survey in asthma patients. J Asthma. 2007;44(5):371–5. 19. [No authors listed]. Skin tests used in type I allergy testing position paper. Sub-Committee on Skin Tests of the European Academy of Allergology and Clinical Immunology. Allergy. 1989;44(10):1–59. 20. [No authors listed]. Standardization of Spirometry, 1994 Update. American Thoracic Society. Am J Respir Crit Care Med. 1995;152(3):1107–36. 21. Crapo RO, Morris AH, Gardner RM. Reference spirometric values using techniques and equipment that meet ATS recommendations. Am Rev Respir Dis. 1981;123(6):659–64. 22. Brannan JD, Anderson SD, Perry CP, Freed-Martens R, Lassig AR, Charlton B. The safety and efficacy of inhaled dry powder mannitol as a bronchial provocation test for airway hyperresponsiveness: a phase 3 comparison study with hypertonic (4.5%) saline. Respir Res. 2005;6(1):144. 23. Zou G. A modified poisson regression approach to prospective studies with binary data. Am J Epidemiol. 2004;159(7):702–6. 24. Bakehe P, Vellguth H. Freitas et al. BMC Pulmonary Medicine (2016) 16:69 Authors’ contributions Bronchial asthma and COPD due to irritants in the workplace - an evidence-based approach. J Occup Med Toxicol. 2012;7(1):19. 25. Oldenburg M, Latza U, Baur X. Exposure–response relationship between endotoxin exposure and lung function impairment in cotton textile. Int Arch Occup Environ Health. 2007;80(5):388–95. 26. Simoneti CS, Freitas AS, Barbosa MCR, Ferraz E, Menezes MB, Bagatin E, et al. Study of risk factors for atopic sensitization, asthma, and bronchial hyperresponsiveness in animal laboratory workers. J Occup Health. 2016;58:7–15. 27. Smit LA, Heederik D, Doekes G, Koppelman GH, Bottema RWB, Postma DS, et al. Endotoxin exposure, CD 14 and wheeze among farmers: a gene- environment interaction. Occup Environ Med. 2011;68(11):826–31. 28. Martinez FD. CD14, endotoxin, and asthma risk: actions and interactions. Proc Am Thorac. 2007;4(3):221–5. 29. Levan TD, Michel O, Dentener M, Torn J, Vertongen F, Beijer L, et al. Association between CD14 polymorphisms and serum soluble CD14 levels: effect of atopy and endotoxin inhalation. J Allergy Clin Immunol. 2008;121(2):434–40. 30. Pacheco KA, Rose CS, Silveira LJ, Dyke MVV, Goelz K, MacPhail K, et al. Gene- environment interactions influence airways function in laboratory animal workers. J Allergy Clin Immunol. 2010;126(2):232–40. 31. Skogstad M, Sikkeland LI, Øvstebø R, HAug KBF, Heldal KK, Skare Ø, et al. Long-term occupational outcomes of endotoxin exposure and the effects of exposure cessation. Occup Environ Med. 2012;69(2):107–12. 32. Schram D, Doekes G, Boeve M, Douwes J, Riedler J, Ublagger E, et al. Bacterial and fungal components in house dust of farm children, Rudolf Steiner school children and reference children – the PARSIFAL Study. Allergy. 2005;60(5):611–8. 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English

Notes on Spraying Experiments for the Oyster Shell Scale in Montana

Journal of economic entomology

public-domain

COOLEY: OYSTER SHELL SCALE COOLEY: OYSTER SHELL SCALE February, '10] February, '10] 57 tween treatment and the time the examinations were made. It is a very slow process to make the n-ecessary counts. In regard to moisture, I consider that we have more to investigate in this dirction, but Wehave not found as yet that moisture has any- where near the same importance as temperature. MR, R. 1. SMITH: The results of Professor Hinds' experiments would indicate that the fumigation with carbon bi-sulphide in the case cited was not successful. _The corn only slightly infested with weevils in September showed after a second treatment in October 38 per cent of weevil stages alive. -Most farmers think that the fumigation is worthless unless they secure better results than this. In case a fumi- gation kills 90 or 95 per cent of the weevils, and then two months later the few remaining alive increase to considerable numbers, it gives the farmers the impression that the treatment was worthless; by guest on June 6, 2016 http://jee.oxfordjournals.org/ Downloaded from My reason for making this statement is not to reflect on Profeslilor Hinds' statement, but simply to explain the farmers' point of view. by guest on June 6, 2016 http://jee.oxfordjournals.org/ ded from PRESIDENTBRITTON: We will now hear a paper by Prof. R. A. Cooley, Bozeman, Mont., on "Notes on the Oyster Shell Scale in Montana." By R. .8.. COOLEY, Montana Agricultural Experiment Station By R. .8.. COOLEY, Montana Agricultural Experiment Station During the past ten years the oyster shell scale (Lepidosaphes uZmi L.) has been gradually increasing in the apple orchards in the river valleys in the western part of Montana. By the year 1907 it had come to be regarded by the apple growers as rather a serious pest and perhaps a menace to the orchard industry in the Bitter Root valley and around Flathead lake. In some orchards, particularly in those that have been more or less.neglected, the scales now occur in notable numbers, encrus~ing the limbs and branches almost completely, and even extending down on to the main trunk, where great numbers be- come fastened under the loose scales of bark. Much fruit has been blemished and rendered unsalable by the insects attaching to it, and the stems of the apples are often more or less completely covered. We have repeatedly recommended ilie use of kerosene emulsion, applied as a spray at the time of hatching, but growers have reported that no success followed the treatment. We also recommended li:me- sulfur solution as a winter treatment. Several years ago, in 1903, on April 21 and. 22, Jots of seven to nine apple trees in the orchard of Mr. Delaney, at Lo Lo, Montana, were .JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 3 sprayea withdi4ferent'lime-sul£ur solutions. Two subsequent visits were made to the orchard, one before the hatching of the eggs and one after, and we were unable to detect that any good had been done. During the' past three years we have received several reports from practical fruit growers that attempts to k:ill the scale by winter appli- cations of lime-sulfur solution have not been successful. However, we also have received reports of success. It was, therefore, apparent that an investigation and spraying tests were necessary. Accordingly a series of tests was arranged and car- ried out in 1909. The orchard that was selected for the experiments is located at Lo La and owned by Mr. Fred Gilbert. It is in sod, composed of old trees, and with a number of varieties. The trees had been cut back and pruned, and as the scale was abundant and fairly evenly distributed, the orchard was quite satisfactory for our purposes. By R. .8.. COOLEY, Montana Agricultural Experiment Station by guest on June 6, 2016 http://jee.oxfordjournals.org/ Downloaded from by guest on June 6, 2016 http://jee.oxfordjournals.org/ oaded from In spraying the trees we desired not only to find an· insecticide that would kill and be generally satisfactory, but also to discover just how and when the insects or the eggs were killed. We hoped to find an explanation for the apparent lack of uniformity of results with the use of the lime-sulfur solutions, and remedies for use before the opening of the buds as well as after the hatching of the eggs in June. We therefore conducted a part of our tests on April 17 and 19, before the leaf buds had opened, and then waited for the appearance of the young. Hatching began on June 10, was well under way by June 14, and practically completed by June 20. The spraying for the hatched insects was divided into two senes, the first being applied in the early part of the hatching period and the second in the latter part, for we desired to know whether the summer treatment, to be successful, should be applied at any particular time during the hatch- ing period. Certain lots of trees were therefore sprayed on June 14 to 17, and others on June 21 to 23. For convenience the various tests are here tabulated, with the re- sults, as follows: I First series applied before hatching and before the buds had opened, and intended to kiUthe insect in the egg stage. Spraying Qone..ApriI17-19. 1. Linseed oil emulsion, one gallon to nine of water. 1. Linseed oil emulsion, one gallon to nine of water. Raw linseed oil . Hard soap . Water to make . 1 gal. % lb. 10 gal. COOLEY: OYSTER SHIDLL SCALID 59 February, '10] The emulsion was made as with kerosene emulsion, excepting that a larger volume of hot water was used. The churning was done with the power sprayer by shutting the valve into the supply pipe and forcing the mixture through the overflow pipe back into the supply tank. A violent churning was so produced. Several microscopical examinations made up to the time of hatch- ing showed no apparent results. Examinations made while hatching was under way showed that many eggs had turned light brown and were shriveled and stuck one upon another and upon the bark and sides of the parent scale. Living larvre could be seen vainly attempt- ing to extricate themselves from the adhering mass of eggs. Prac- tically no young attached to the bark and formed scales. Subsequent examinatlons showed that the treatment had been very effective. No injury was done to the trees. by guest on June 6, 20 http://jee.oxfordjournals.org/ Downloaded from by guest on June 6, 2016 http://jee.oxfordjournals.org/ wnloaded from 4. "Rex" lime-sulfur solution, one part to eight of water. See results under No.3. 5. "Rex" lime-sulfur solution, one part to ten ot water. :Seere- sults under No.3. 9. Home-made lime-sulfur solution. See results under NO.3.' by guest on June 6, 2 http://jee.oxfordjournals.org/ Downloaded from by guest on June 6, 2016 http://jee.oxfordjournals.org/ Downloaded from 11. Lye-sulfur solution. Prepared as with lime-sulfur solution; cooked until the sulfur was all dissolved. The results were not con- vincing; some good was done. 11. Lye-sulfur solution. Prepared as with lime-sulfur solutio cooked until the sulfur was all dissolved. The results were not c vincing; some good was done. 12. Pratt's" Scalecide, " one part to ten of water. Before hatching time the eggs showed an oily appearance but hatched normally. Some scales were loosened and dropped off, though a smaller proportion than with undiluted kerosene, as reported under No.2. Not a satisfactory treatment. 13. Whal~-oil soap solution, one pound to one gallon of water, ap- plied hot. Apparently this treatlp.ent had no effect. 2. Undiluted kerosene. 2. Undiluted kerosene. Designed to test the ultimate killing power of kerosene and not thought of as a practical remedy. A considerable number of scales were loosened and dropped to the ground-probably about one third-but the eggs under those left on the tree hatched and the young developed normally. The trees were late in putting out foliage and were injured. 3. "Rex" lime-sulfur solution, one part to six of water. Repeated microscopic examinations made up to the time of hatch- ing showed no visible effects on the eggs. Moreover, the eggs hatched in a normal manner, and the empty egg shells could be found under the old scales later in the summer, but very few of the young ever attached to the bark, or, if they did attach, they soon loosened and dropped. The treatment was, therefore, effective and satis- factory. We were able to find practically no difference in the results ob- tained from the use of "Rex," "Niagara" and home-made lime- sulfur solutions, nor were the greater strengths more effective. All showed good and satisfactory results. 4. "Rex" lime-sulfur solution, one part to eight of water. See results under No.3. 5. "Rex" lime-sulfur solution, one part to ten ot water. :Seere- sults under No.3. 6. "Rex" lime-sulfur solution, one to SIX, with three pounds lye JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. g [Vol. g 60 added to :50 gallons of the mixture. See results under No.3. No advantage was detected from the use of lye. added to :50 gallons of the mixture. See results under No.3. No advantage was detected from the use of lye. 7. "Niagara" lime-sulfur solution, on,e part to six of water. See results under No.3. II Second series applied early in hatching period. Desired to be effective in killing young that had hatched and those about to hatch. June 14-17. 14. Pratt's" Scalecide," at rate of one to :fifty. A small percentage of the young lice were killed. Not a satisfac- COOLEY: OYSTER SHELL SCA.LE February, '10] February, '10] 61 tory treatment. Very distinct, though not extensive InJury to t.hA foliage. tory treatment. Very distinct, though not extensive InJury to t.hA foliage. 15. Pratt's "Scalecide" at rate of one to seventy-five. Same re- sults as with No. 14, but with less injury to the foliage. 1 gal. 1 lb. 12 gal. 16. Linseed oil emulsion, prepared as for test No. 1. Raw linseed oil . Hard soap .. : ......................•........... ViTater to make . Practically all the lice were killed, and the treatment was consid- ered very satisfactory. This and the cottonseed oil emulsion treat- ments were considered to be the most satisfactory for summer use. The trees were not injured. by guest on June 6, 20 http://jee.oxfordjournals.org/ Downloaded from In connection with judging the results of this treatment we should mention that the Board of Horticulture sprayed a neighboring orchard with linseed oil emulsion, independent of the writer's experiments, on June 24 and 25. The scales were all killed and a small amount of injury was done to the foliage. Other trees were sprayed by the board with cottonseed oil emulsion, resulting in a killing of all the young scales, with less injury to the foliage. by guest on June 6, 2016 http://jee.oxfordjournals.org/ aded from 17. Cottonseed oil emulsion. Prepared. as with linseed oil emul- sion. 17. Cottonseed oil emulsion. Prepared. as with linseed oil emul- sion. Cotton seed oil . .Hard soap . Water to make . 1 gal. 1 lb. 12 gal. Results were practically the same as with linseed oil emulsion sum- mer spray. No iojury to foliage. 18. Kerosene emulsion, one part stock emulsion to twelve of water. Very few, if any, of the scales were killed. 19. Whale-oil soap solution at rate of one pound to eight gallons of water, applied warm. A few, possibly 10 to 15 per cent, were killed. 20. "Rex" lime-sulfur solution, one part to :fifteen of water. The treatment was unsatisfactory, very few young being killed. 21. "Self-boiled" lime-sulfur solution. Practically no good was done. Lime '" .........................• Sulfur . Water . 15 lbs. III Third series, applied late in hatching period. Desired to be effective in killing all the living young. Sprayed June 21 to 23. 24. Pratt's "Scalecide," one to fifty, using same trees as test No. 14. by guest on June 6, 20 http://jee.oxfordjournals.org/ Downloaded from by guest on June 6, 2016 http://jee.oxfordjournals.org/ Downloaded from This treatment did very little good. A high percentage" of scales developed on trees sprayed twice during hatchmg time. 25. Pratt's "Scalecide," one to seventy-five, using same trees as test No. 15. Results same as under No. 24. 26. Linseed oil emulsion, using one gallon raw oil to :fifteen gal- lons water. Seven out of the nine trees used in test No. 16 were used. Probably no good was done by the second spraying during the hatch- ing period, as the two trees left Untreated under this number showed practically the same results as under No. 16. Probably no good was done by the second spraying during the hatch- ing period, as the two trees left Untreated under this number showed practically the same results as under No. 16. 27. Cottonseed oil emulsion. Repetition of treatment on trees used in test No. 17. 27. Cottonseed oil emulsion. Repetition of treatment on trees used in test No. 17. It is not clear whether this second treatment was beneficial, as, It is not clear whether this second treatment was beneficial, as, through an error, no trees were left unsprayed, as under No. 26. 28. Kerosene emulsion, one part of stock emulsion to twelve of water. Same trees used as in test No. 18. Very few, if any, were killed. 29. "Rex" lime-sulfur solution, one part to fifteen of water. Same trees used as in test No. 20. There was practically no benefit from this treatment. 30. "Blackleaf" tobacco extract, one part to fifty of water. Same trees used as in test No. 22. Practically no good was accomplished with this treatment, as with No. 22. 31. Linseed oil emulsion, one part to fifteen parts of water. A fresh lot of trees was used in this test. The results under this number are doubtful, as it was later learned that the trees, which were in another orchard, had been sprayed in the spring with another insecticide. 30. "Blackleaf" tobacco extract, one part to fifty of water. Same trees used as in test No. 22. II 10 lbs. 50 gal. 20. "Rex" lime-sulfur solution, one part to :fifteen of water. The treatment was unsatisfactory, very few young being killed. 21. "Self-boiled" lime-sulfur solution. Practically no good was done. Lime '" .........................• Sulfur . Water . 15 lbs. 10 lbs. 50 gal. 20. "Rex" lime-sulfur solution, one part to :fifteen of water. The treatment was unsatisfactory, very few young being killed. 21. "Self-boiled" lime-sulfur solution. Practically no good was 20. "Rex" lime-sulfur solution, one part to :fifteen of water. The treatment was unsatisfactory, very few young being killed. JOURNAL OF ECONOMIC ENTOMOLOGY [Vol. 3 62 22. "Blackleaf" tobacco extract, one part to fifty of water. We were surprised to find that this treatment was of very little benefit. 22. "Blackleaf" tobacco extract, one part to fifty of water. We were surprised to find that this treatment was of very little benefit. 23. "Orwood" tree spray, one part to twelve of water. Appar- ently useless for this purpose. 23. "Orwood" tree spray, one part to twelve of water. Appar- ently useless for this purpose. III We found that certain of the oily insecticides, applied before hatching of the eggs, caused a part of the scales to drop off, but it was impossi- ble to determine what proportion had dropped. It would have been of some value if we had given the various trees a rating designed to indicate the comparative degree of infestation before the treatment, although on trees of which we made microscopical examinations and counts such a rating would have been of but little value, for the ex- aminations were made on small twigs, which naturally would not con- form closely to a tree rating. In examining the scales on the twigs we found a Zeiss binocular microscope of great service. Besides using it as a dissecting microscope we took off the lenses, with the mount- ings, and used the detached part in the hand, as with a field binocular. The apparently conflicting results following the use of lime-sulfur solutions for this insect while the trees are dormant are- striking. It is possible that differing weather conditions may explain the killing at one time and failure to kill at another. It is well understood that the sulfur compounds deposited upon the trees by the spray are acted upon chemically by the carbon dioxide of the air, resulting in the liberation of the gaseous sulfureted hydrogen and leaving on the tree pulverulent deposits of finely divided free sulfur and calcium carbonate. It has been shown on a previous page that this insect is !tilled by the winter application of lime-sulfur solutions only after the eggs hatch. It seems clear that the actual agent in the killing of the young, tender lice is the free sulfur resulting from the decomposition of the sulfur compounds. This decomposition has all taken place long before the hatching of the eggs.' Therefore it seems possible that con- by guest on June 6, 2016 http://jee.oxfordjournals.org/ wnloaded from g p The apparently conflicting results following the use of lime-sulfur solutions for this insect while the trees are dormant are- striking. It is possible that differing weather conditions may explain the killing at one time and failure to kill at another. III Practically no good was accomplished with this treatment, as with No. 22. 31. Linseed oil emulsion, one part to fifteen parts of water. A fresh lot of trees was used in this test. The results under this number are doubtful, as it was later learned that the trees, which were in another orchard, had been sprayed in the spring with another insecticide. 10] COOLEY; OYSTER COOLEY; OYSTER SHELL SCALE February, '10] 63 In all of these tests a small power sprayer owned by the Montana State Board of Horticulture was used. A representative of the board, under the pay of the board, did the actual spraying, under my di- rection. 'l'he board also furnished practically all the material and supplies used in these experiments. It gives me pleasure to express my appreciation of the assistance and courtesy extended by the Board of Horticulture, through Mr. M. L. Dean, state horticultural inspector. In conducting the work twelve trips to the Bitter Root valley were made, as follows: March 25-28, April 17-19, April 24-25, May 6-8, May 18-21, May ~1-June 1, June 10, June 14-17, June 21-23, July 12--16,August 26-27, October 4-7. by guest on June 6, 20 http://jee.oxfordjournals.org/ Downloaded from Even under ideal conditions in orchards selected for such tests, it would be impossible to make entirely reliable statements concern- ing the comparative benefits following different treatments. We found that certain of the oily insecticides, applied before hatching of the eggs, caused a part of the scales to drop off, but it was impossi- ble to determine what proportion had dropped. It would have been of some value if we had given the various trees a rating designed to indicate the comparative degree of infestation before the treatment, although on trees of which we made microscopical examinations and counts such a rating would have been of but little value, for the ex- aminations were made on small twigs, which naturally would not con- form closely to a tree rating. In examining the scales on the twigs we found a Zeiss binocular microscope of great service. Besides using it as a dissecting microscope we took off the lenses, with the mount- ings, and used the detached part in the hand, as with a field binocular. Even under ideal conditions in orchards selected for such tests, it would be impossible to make entirely reliable statements concern- ing the comparative benefits following different treatments. III by guest on June 6, 2016 http://jee.oxfordjournals.org/ Downloaded from by guest on June 6, 2016 http://jee.oxfordjournals.org/ d from MR. BRAUCHER:Some years ago I was engaged in spraying work in Lincoln Park, Chicago, and secured. practically the same results that Professor Cooley has indicated. The eggs of the insect in many cases appear to be perfectly normal up to the time of hatching, but in most cases the young failed to establish themselves, and later in the season I was unable to find living insects on the trees. The home-boiled lime-sulfur wash was used, being applied from November until earIy spring, and gave satisfactory results. MR.. SURFACE:I have found several cases in Pennsylvania where this insect has been practically exterminated by using the lime-sulfur wash. This was used in the orchard of Mr. Robert Beaston, at "Tyrone, Pa., with excellent results. III It is well understood that the sulfur compounds deposited upon the trees by the spray are acted upon chemically by the carbon dioxide of the air, resulting in the liberation of the gaseous sulfureted hydrogen and leaving on the tree pulverulent deposits of finely divided free sulfur and calcium carbonate. It has been shown on a previous page that this insect is !tilled by the winter application of lime-sulfur solutions only after the eggs hatch. It seems clear that the actual agent in the killing of the young, tender lice is the free sulfur resulting from the decomposition of the sulfur compounds. This decomposition has all taken place long before the hatching of the eggs.' Therefore it seems possible that con- JOURNAL OF ftCONOMIC ENTOMOLOGY [Vol. 3 tinued rain storms may so reduce the amount of free sulfur on the bark as to render the treatment harmless to the insects. tinued rain storms may so reduce the amount of free sulfur on the bark as to render the treatment harmless to the insects. Further work on the subject will be done in the season of 1910. From the foregoing two interesting po~ts are apparent: (a) Eggs of the oyster shell scale are unaffected by the applica- tion of lime-sulfur solutions made previous to the opening of the buds. On trees so sprayed the young were killed very SOonafter hatching. The intervention of rain storms before the hatching of the eggs may more or less affect the value of the treatment. (a) Eggs of the oyster shell scale are unaffected by the applica- tion of lime-sulfur solutions made previous to the opening of the buds. On trees so sprayed the young were killed very SOonafter hatching. The intervention of rain storms before the hatching of the eggs may more or less affect the value of the treatment. (h) It is indicated that emulsions of linseed oil and cottonseed oil may be useful for the treatment of this insect while in the egg stage and during the hatching period. (h) It is indicated that emulsions of linseed oil and cottonseed oil may be useful for the treatment of this insect while in the egg stage and during the hatching period. (h) It is indicated that emulsions of linseed oil and cottonseed oil may be useful for the treatment of this insect while in the egg stage and during the hatching period. [The Proceedings will be continued in the next issue.-ED.] AMERICAN ASSOCIATIONOF ECONOMICENTOMOLOGIST In accordance with the provisions of the constitution, the President has appointed the following Committee on Membership for the year 1910: Prof. H. E. Summers, Prof. A. L. Quaintance and Dr. S. A. Forbes. E. D. SANDERSON,President. A. F. BURGESS, Secretary.

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WWP1 germline variants are associated with normocephalic autism spectrum disorder

Cell death and disease

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WWP1 germline variants are associated with normocephalic autism spectrum disorder Giuseppe Novelli 1,2,3, Antonio Novelli4, Paola Borgiani1, Dario Cocciadiferro4, Michela Biancolella5, Emanuele Agolini4, Marco Pietrosanto5, Rosario Casalone6, Manuela Helmer-Citterich5, Emiliano Giardina1,7, Suresh K. Jain8, Wenyi Wei 9,10, Charis Eng 11,12,13,14 and Pier Paolo Pandolfi15,16 the mutation, cannot be ruled out. For each patient (6 males and 4 females; ages 3–26), the clinical data have been reassessed. None of the probands had germline PTEN mutations or other mutations in genes (FMR1, SHANK3, MECP2, CDK19) associated with ASD/intel- lectual disability (ID). We independently confirmed that WWP1 variation does not act as a modifier for ASD phenotypes in PHTS with none of ~600 mainly American PTEN mutation positive research associated with the WWP1 locus. Similarly, routine chromosome studies and FRAXA locus were normal. GnomAD database analysis revealed that the identified WWP1 variants with the exception of R389S, R893H, and M728L (never detected), existed with a cumulative frequency of 0.00085 in ethni- cally matched populations (EUR), indicating that they are very rare variants. Specifically, WWP1 germline variants occurred in 10/396 alleles (allelic freq. = 0.0252) from the 198 unrelated individuals with ASD/ID (Table 1) which is a highly significant difference from European population frequencies from GnomAD (p < 0.00001; OR = 30.6 with 95% CI 16.27 and 57.59). We therefore extended the study to a cohort of 1158 individuals from the Italian general population to establish the frequency of WWP1 variants in this Italian cohort. We detected three WWP1 rare variants (c.1118G>A, p-Arg373Gln; c.1486G>C, p. Glu496Gln; c.2234A>G, p.Asn745S) (3/2316 alleles: allelic freq. = 0.00129). Notably, WWP1 variants were again shown to be over-represented in the ASD/ID series, even when compared with the Italian cohort examined (p < 0.00001; OR = 19.93 with 95% CI 5.47 and 72.90). The variants are found in all functional domains of the protein (the catalytic C-terminal HECT domain; the N-terminal C2 domain and WW domains) with an over- representation in the HECT domain (4/8). To predict the potential impact of the identified variants on the Dear Editor, Autism spectrum disorder (ASD, MIM: 209850) is a group of common but heterogeneous neurodevelop- mental disorders with a prevalence of 4–10 per 10,000 individuals1,2. About 5% of ASD cases are caused by single-gene variants in FMR1 (MIM: 309550), MECP2 (MIM: 300005), or SHANK3 (MIM: 606230); 10% by copy number variants (CNVs)2, while the majority is attributed to polygenic inheritance of common variants3. In addi- tion, germline PTEN mutations have been identified in 2–5% of all ASD patients and ~10% of macrocephalic ASD4. The Author(s) 2020 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. C O M M E N T O p e n A c c e s s Novelli et al. Cell Death and Disease (2020) 11:529 https://doi.org/10.1038/s41419-020-2681-z Novelli et al. Cell Death and Disease (2020) 11:529 https://doi.org/10.1038/s41419-020-2681-z Cell Death & Disease Cell Death & Disease Author details 1 1Department of Biomedicine and Prevention, Tor Vergata University of Rome, 00133 Rome, Italy. 2IRCCS Neuromed, Pozzilli (IS), Italy. 3Department of Pharmacology, School of Medicine, University of Nevada, Reno, NV 89557, USA. 4Laboratory of Medical Genetics, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy. 5Department of Biology, Tor Vergata University of Rome, 00133 Rome, Italy. 6Cytogenetics and Medical Genetics Laboratory, Ospedale di Circolo, ASST Sette Laghi, Varese, Italy. 7Molecular Genetics Laboratory UILDM, Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are grateful to the participating families and Il Ponte del Sorriso Onlus for its support. PPP was supported by the NCI grant R35CA197529 and in part by the PTEN Research Foundation. Received: 18 May 2020 Revised: 26 May 2020 Accepted: 3 June 2020 Received: 18 May 2020 Revised: 26 May 2020 Accepted: 3 June 2020 Received: 18 May 2020 Revised: 26 May 2020 Accepted: 3 June 2020 Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. Our results suggest that germline WWP1 variants iden- tified in ASD/ID/NDDs may contribute to the pathogenesis of ASD/ID/NDDs. In addition, since the enzymatic activity of WWP1 can be inhibited by the natural compound, indole-3-carbinol6, our study identifies a possible ther- apeutic target for individuals with ASD/ID/NDDS. WWP1 germline variants are associated with normocephalic autism spectrum disorder Giuseppe Novelli 1,2,3, Antonio Novelli4, Paola Borgiani1, Dario Cocciadiferro4, Michela Biancolella5, Emanuele Agolini4, Marco Pietrosanto5, Rosario Casalone6, Manuela Helmer-Citterich5, Emiliano Giardina1,7, Suresh K. Jain8, Wenyi Wei 9,10, Charis Eng 11,12,13,14 and Pier Paolo Pandolfi15,16 Ethical declarations The study was conducted in agreement with the principles of the Declaration of Helsinki. Informed written consent was obtained from each patients. As Table 1 Summary of variations in ASD patients carrying WWP1 mutations. Patient ID Sex Exon Position (Hg19) Nucleotide Amino acid Domain GnomAda dbSNP Transmission GM4277 F Int 7 87414243 c.540-5T>C NA 0.0027 rs187132881 Mother GM3474 M 11 87439881 c.1167A>C p.Arg389Ser WW1 0 NA NA A020 M 14 87443954 c.1583G>A p.Arg528His WW4 0.000008 rs554041348 Father GM6802 F 20 87460703 c.2234A>G p.Asn745Ser HECT 0.00003 rs148651938 Mother GM8105 M 20 87460703 c.2234A>G p.Asn745Ser HECT 0.00003 rs148651938 Father GM-1HSL M 20 87460703 c.2234A>G p.Asn745Ser HECT 0.00003 rs148651938 NA GM4098 F 20 87460645 c.2176G>A p.Val726Ile HECT 0.000023 rs144129917 Mother GM8302 F 25 87479031 c.2678G>A p.Arg893His HECT 0 rs755897749 Father A036 M 20 87460651 c.2182A>T p.Met728Leu HECT 0 NA Father A069 M 5 87393781 c.257G>A p.Arg86His C2 0.000023 rs371650373 Mother aEUR. protein we used different tools (PolyPhen2, Mutation Taster, SIFT, MetaLR_pred, and MetaSVM_pred). The recurrent N745S variant has been previously reported by Lee et al.5: it is in the HECT domain and is expected to decrease its binding to the N-terminal domain. Analo- gously, R86H (C2 domain) was also described by Lee et al.5. This variant is functionally relevant since it induces a gain-of-function effect in triggering PTEN poly- ubiquitination5. With regards to the other five coding variants observed in our ASD cases, one is predicted by in silico analysis to be deleterious (R528H), while the others gave conflicting results. Santa Lucia Foundation, 00142 Rome, Italy. 8Intonation Research Laboratories Pvt. Ltd, Hyderabad, Telangana 500076, India. 9Cancer Research Institute, Beth Israel Deaconess Cancer Center, Harvard Medical School, Boston, MA 02215, USA. 10Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. 11Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA. 12Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. 13Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. 14Germline High Risk Cancer Focus Group, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA. 15MBC, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, TO 10126, Italy. 16DRI, Renown Health, Nevada System of Higher Education, Reno, NV 89512, USA Web resources GnomAD, https://gnomad.broadinstitute.org/ PolyPhen2, http://genetics.bwh.harvard.edu/pph2/ Mutation Taster, http://www.mutationtaster.org/ SIFT, https://sift.bii.a-star.edu.sg/ OMIM, https://OMIM.org/. WWP1 germline variants are associated with normocephalic autism spectrum disorder Giuseppe Novelli 1,2,3, Antonio Novelli4, Paola Borgiani1, Dario Cocciadiferro4, Michela Biancolella5, Emanuele Agolini4, Marco Pietrosanto5, Rosario Casalone6, Manuela Helmer-Citterich5, Emiliano Giardina1,7, Suresh K. Jain8, Wenyi Wei 9,10, Charis Eng 11,12,13,14 and Pier Paolo Pandolfi15,16 Patient ID Sex Exon Position (Hg19) Nucleotide Amino acid Domain GnomAda dbSNP Transmission GM4277 F Int 7 87414243 c.540-5T>C NA 0.0027 rs187132881 Mother GM3474 M 11 87439881 c.1167A>C p.Arg389Ser WW1 0 NA NA A020 M 14 87443954 c.1583G>A p.Arg528His WW4 0.000008 rs554041348 Father GM6802 F 20 87460703 c.2234A>G p.Asn745Ser HECT 0.00003 rs148651938 Mother GM8105 M 20 87460703 c.2234A>G p.Asn745Ser HECT 0.00003 rs148651938 Father GM-1HSL M 20 87460703 c.2234A>G p.Asn745Ser HECT 0.00003 rs148651938 NA GM4098 F 20 87460645 c.2176G>A p.Val726Ile HECT 0.000023 rs144129917 Mother GM8302 F 25 87479031 c.2678G>A p.Arg893His HECT 0 rs755897749 Father A036 M 20 87460651 c.2182A>T p.Met728Leu HECT 0 NA Father A069 M 5 87393781 c.257G>A p.Arg86His C2 0.000023 rs371650373 Mother aEUR. Table 1 Summary of variations in ASD patients carrying WWP1 mutations. protein we used different tools (PolyPhen2, Mutation Taster, SIFT, MetaLR_pred, and MetaSVM_pred). The recurrent N745S variant has been previously reported by Lee et al.5: it is in the HECT domain and is expected to decrease its binding to the N-terminal domain. Analo- gously, R86H (C2 domain) was also described by Lee et al.5. This variant is functionally relevant since it induces a gain-of-function effect in triggering PTEN poly- ubiquitination5. With regards to the other five coding variants observed in our ASD cases, one is predicted by in silico analysis to be deleterious (R528H), while the others gave conflicting results. Our results suggest that germline WWP1 variants iden- tified in ASD/ID/NDDs may contribute to the pathogenesis of ASD/ID/NDDs. In addition, since the enzymatic activity of WWP1 can be inhibited b the natural compound Santa Lucia Foundation, 00142 Rome, Italy. 8Intonation Research Laboratories Pvt. Ltd, Hyderabad, Telangana 500076, India. 9Cancer Research Institute, Beth Israel Deaconess Cancer Center, Harvard Medical School, Boston, MA 02215, USA. 10Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. 11Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA. 12Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. 13Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. 14Germline High Risk Cancer Focus Group, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA. 15MBC, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, TO 10126, Italy. 16DRI, Renown Health, Nevada System of Higher Education, Reno, NV 89512, USA Conflict of interest The authors declare that they have no conflict of interest. Ethical declarations The study was conducted in agreement with the principles of the Declaration of Helsinki. Informed written consent was obtained from each patients. As regards the participation of children in the research, consent and authorization were signed by the parents in accordance with the rules laid down by the Ethics Committee of the Bambino Gesù Hospital in Rome (HYPERLINK “https:// urldefense.proofpoint.com/v2/url?u=http-3A__www.ospedalebambinogesu. it_en_home&d=DwMFaQ&c=vh6FgFnduejNhPPD0fl_yRaSfZy8CWbW- nIf4XJhSqx8&r=H8EiHZYdOfzgj3SnkNr1OWfOZuk7IdFteVpx6F9BizvoZAKx_zlI- bLuDzKXrCwF8&m=0dvSb4bLNoeGzXhLeNXyRGhxjEoUL6Qd_oj7- reRTsMg&s=FMPyM3gTbUpOHGox37ytL4D0gGUI3gocQlCNX9p-1IE&e=” MailScanner ha rilevato un possibile tentativo di frode proveniente da “urldefense.proofpoint.com” http://www.ospedalebambinogesu.it/en/home). WWP1 germline variants are associated with normocephalic autism spectrum disorder Giuseppe Novelli 1,2,3, Antonio Novelli4, Paola Borgiani1, Dario Cocciadiferro4, Michela Biancolella5, Emanuele Agolini4, Marco Pietrosanto5, Rosario Casalone6, Manuela Helmer-Citterich5, Emiliano Giardina1,7, Suresh K. Jain8, Wenyi Wei 9,10, Charis Eng 11,12,13,14 and Pier Paolo Pandolfi15,16 Recently, Lee et al.5 identified germline variants within the E3 ubiquitin ligase WWP1 (MIM: 602307) gene in PTEN mutation negative individuals with neoplastic phenotypes found in PHTS (MIM: 158350). To establish whether WWP1 could play a role in ASD and neurodevelopment disorders, we analyzed 198 unre- lated individuals mainly referred for syndromic or non- syndromic developmental delay and/or ASD of unknown genetic etiology. All individuals were clinically diagnosed with ASD on the basis of the fourth edition of the Diag- nostic and Statistical Manual of Mental Disorders (DSM- IV). Whole-exome sequencing, validated by Sanger sequencing, identified eight different heterozygous germline mutations (one recurrent in three unrelated patients) of the WWP1 gene in 10 of 198 unrelated pro- bands via WES (Table 1). None of the variant positive probands had macrocephaly. In two cases, parental origin could not be investigated, therefore, a de novo origin of Correspondence: Giuseppe Novelli ([email protected]) or Pier Paolo Pandolfi(pierpaolo.pandolfi[email protected]) 1Department of Biomedicine and Prevention, Tor Vergata University of Rome, 00133 Rome, Italy 2IRCCS Neuromed, Pozzilli (IS), Italy Full list of author information is available at the end of the article Correspondence: Giuseppe Novelli ([email protected]) or Pier Paolo Pandolfi(pierpaolo.pandolfi[email protected]) 1Department of Biomedicine and Prevention, Tor Vergata University of Rome, 00133 Rome, Italy 2IRCCS Neuromed, Pozzilli (IS), Italy Full list of author information is available at the end of the article © The Author(s) 2020 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Novelli et al. Cell Death and Disease (2020) 11:529 Page 2 of 3 Table 1 Summary of variations in ASD patients carrying WWP1 mutations. e e e ces 1. Charles, J. M. Autism spectrum disorders: an introduction and review of pre- valence data. J. S. C. Med. Assoc. 102, 267–270 (2006). 2. Stefansson, H. et al. CNVs conferring risk of autism or schizophrenia affect cognition in controls. Nature 505, 361–366 (2014). Official journal of the Cell Death Differentiation Association Novelli et al. Cell Death and Disease (2020) 11:529 5. Lee, Y. R. et al. WWP1 gain-of-function inactivates PTEN to drive cancer pre- disposition. N. Eng. J. Med. 382, 2103–2116 (2020). 6. Lee, Y. R. et al. Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway. Science 364, eaau0159 (2019). 3. Clarke, T. K. et al. Common polygenic risk for autism spectrum disorder (ASD) is associated with cognitive ability in the general population. Mol. Psychiatry 21, 419–425 (2016). 4. Yehia, L., Ngeow, J. & Eng, C. PTEN-opathies: from biological insights to evidence-based precision medicine. J. Clin. Invest 129, 452–464 (2019). 3. Clarke, T. K. et al. Common polygenic risk for autism spectrum disorder (ASD) is associated with cognitive ability in the general population. Mol. Psychiatry 21, 419–425 (2016). 4. Yehia, L., Ngeow, J. & Eng, C. PTEN-opathies: from biological insights to evidence-based precision medicine. J. Clin. Invest 129, 452–464 (2019). Official journal of the Cell Death Differentiation Association d spos t o . . g. J. ed. 38 , 03 6 ( 0 0). 6. Lee, Y. R. et al. Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway. Science 364, eaau0159 (2019). References 1. Charles, J. M. Autism spectrum disorders: an introduction and review of pre- valence data. J. S. C. Med. Assoc. 102, 267–270 (2006). 2. Stefansson, H. et al. CNVs conferring risk of autism or schizophrenia affect cognition in controls. Nature 505, 361–366 (2014). Official journal of the Cell Death Differentiation Association Page 3 of 3 Official journal of the Cell Death Differentiation Association

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Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems Using Interval Reduced Kernel PLS

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Received April 5, 2020, accepted April 18, 2020, date of publication April 23, 2020, date of current version May 8, 2020. Received April 5, 2020, accepted April 18, 2020, date of publication April 23, 2020, date of current version May 8, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2989917 Digital Object Identifier 10.1109/ACCESS.2020.2989917 RADHIA FEZAI 1, KAMALELDIN ABODAYEH 2, MAJDI MANSOURI 1, (Member, IEEE), ABDELMALEK KOUADRI 3, MOHAMED-FAOUZI HARKAT 4, HAZEM NOUNOU 1, (Senior Member, IEEE), MOHAMED NOUNOU 5, (Senior Member, IEEE), AND HASSANI MESSAOUD 6 1Electrical and Computer Engineering Program, Texas A&M University at Qatar, Doha 23874, Qatar 2Department of Mathematical Sciences, Prince Sultan University, Riyadh 66833, Saudi Arabia 3Signals and Systems Laboratory, Institute of Electrical and Electronics Engineering, University M Hamed Bougara of Boumerdes, Boumerdes 35000, Algeria 4LASMA, Badji Mokhtar - Annaba University, Annaba 23000, Algeria 5 g g g , y Q , , Q 6Research Laboratory of Automation, Signal Processing and Image (LARATSI), National School of Engineering Monastir, Rue Ibn ELJazzar, Monastir 5019, Tunisia Corresponding author: Majdi Mansouri ([email protected]) This work was made possible by National Priorities Research Program grant NPRP9-330- 2-140 from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors. ABSTRACT Kernel partial least squares (KPLS) models are widely used as nonlinear data-driven methods for faults detection (FD) in industrial processes. However, KPLS models lead to irrelevant performance over long operation periods due to process parameters changes, errors and uncertainties associated with mea- surements. Therefore, in this paper, two different interval reduced KPLS (IRKPLS) models are developed for monitoring large scale nonlinear uncertain systems. The proposed IRKPLS models present an interval versions of the classical KPLS model. The two proposed IRKPLS models are based on the Euclidean distance between interval-valued observations as a dissimilarity metric to keep only the more relevant and informative samples. The first proposed IRKPLS technique uses the centers and ranges of intervals to estimate the interval model, while the second one is based on the upper and lower bounds of intervals for model identification. These obtained models are used to evaluate the monitored interval residuals. The aforementioned interval residuals are fed to the generalized likelihood ratio test (GLRT) chart to detect the faults. In addition to considering the uncertainties in the input-output systems, the new IRKPLS-based GLRT techniques aim to decrease the execution time when ensuring the fault detection performance. The developed IRKPLS-based GLRT approaches are evaluated across various faults of the well-known Tennessee Eastman (TE) process. The performance of the proposed IRKPLS-based GLRT methods is evaluated in terms of missed detection rate, false alarms rate, and execution time. The obtained results demonstrate the efficiency of the proposed approaches, compared with the classical interval KPLS. INDEX TERMS Kernel PLS (KPLS), interval KPLS (IKPLS), interval reduced KPLS (IRKPLS), fault detection (FD), uncertain systems. The associate editor coordinating the review of this manuscript and approving it for publication was Heng Wang . VOLUME 8, 2020 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons I. INTRODUCTION GLRT is considered the most used method for detection of industrial faults [28], [29]. Generally, the quality of the available measurements is essential in the reliability of the identified model as well as in the FD performance. Using the KPLS technique, the mea- sured process data are described by a single value represen- tation. The single-valued data representation is a result of simplification during the data mining procedure. The neces- sity for interval-valued data can arise in connection with the imprecision of measurement devices, process uncertainties, or with the data fluctuations in the case of collected samples during a specific interval of time. Thus, in the proposed IRKPLS-based GLRT approaches, the IRKPLS methods are used for identifying the interval-valued models and the GLRT chart is applied for faults detection. The detection efficiency of the proposed IRKPLS-based GLRT approaches is evaluated using a Tennessee East- man (TE) process. The false alarm rate (FAR), missed detec- tion rate (MDR), and execution time (ET) are used as metrics to evaluate the detection and monitoring abilities. In actual practice, taking into account the minimum and maximum of the collected sample values gives a complete overview of the measured phenomenon than taking into account the average values. Thus, for more accuracy in the representation of real data, the uncertainty of the data may be represented by considering data at intervals representa- tion [19]–[21]. In literature, several linear techniques for interval-valued data have been developed [21]–[27]. How- ever, up to now there is no studies on the problem of nonlinear uncertain systems using interval kernel PLS. The remainder of this paper is structured as follows. In the Preliminaries section, PLS and kernel PLS are briefly introduced. The developed IRKPLS models are presented in Section 3. The fault detection using GLRT is detailed in Section 4. In section 5, the application of the devel- oped IRKPLS-based GLRT approaches to a Tennessee East- man (TE) process is presented. The conclusions are presented in Section 6. y g The interval KPLS (IKPLS) or KPLS-based interval- valued data method is an extension of the single-valued KPLS model in presence of imprecision, variation, and data con- fidence intervals. For example, the IKPLSUL model aims to build two KPLS models on the lower and upper (UL) bounds of interval values [21]. I. INTRODUCTION in modern industrial processes, data-driven FD techniques have been discussed widely by researchers [2]–[9]. Partial least squares (PLS) regression is the most used data-driven technique in modeling and FD of industrial processes, and it has proved excellent performance [10], [11]. The main idea of PLS is to build the correlation between the process and quality variables [12]. By establishing a model through normal data, PLS can be applied for predicting and FD purposes. Fault detection (FD) becomes increasingly important to improve the quality product, ensure process safety and decrease energy and material consumption [1]. As a large amount of process data are gathered in historical databases The associate editor coordinating the review of this manuscript and approving it for publication was Heng Wang . 78343 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems However, the PLS technique assumes that the process data are linear. This assumption makes it inappropriate for nonlinear industrial processes. In order to address this issue, a nonlinear version of PLS model, so-called kernel PLS (KPLS), has been developed in literature [13]–[15]. The KPLS technique aims to map the nonlinear input data into a high-dimensional space in which a linear PLS model is applied. According to the Covers theorem [14], the nonlinear relationship among variables in the original space is most probably linear after a high-dimensional nonlinear mapping. The KPLS can effi- ciently determine the regression coefficients in feature space using nonlinear kernel functions and improve the prediction performance. It has been successfully implemented for mod- eling and FD of many industrial processes [2], [16]–[18]. the detection performance. The proposed IRKPLS schemes do not depend on data outliers and model incompleteness and have the ability to improve both fault detection robustness and sensitivity while maintaining a satisfactory and stable performance over long periods of process operation. Both IRKPLS methods (IRKPLSCR and IRKPLSUL) are based on the Euclidean distance between interval-valued samples as a dissimilarity metric to extract only the non-redundant ones and keep only one informative sample for every small and similar distance. In addition to their abilities to efficiently monitor uncertain processes, the IRKPLS models are able to reduce the execu- tion time and memory space. The two proposed IRKPLSCR and IRKPLSUL models are used to generate the monitored interval residuals. The afore- mentioned interval residuals are fed to generalized likelihood ratio test (GLRT) chart as input for detecting faults. I. INTRODUCTION Another interval technique, so-called Bivariate Center and Range (CR) KPLS (IKPLSCR) consists to create a KPLS model on the new numerical input and output matrices constructed by the concatenation of center and range matrices [21]. However, the major lim- itation of KPLS-based fault detection techniques is their computational complexity. Using KPLS model for FD leads to a high computational cost when the training data set is large since the recorded and stored data are applied for both modeling and monitoring. This is mainly explained by the fact that the kernel methods depend on the collected observations of the whole process variables. To reduce the computational complexity of the IKPLS models, two interval reduced KPLS (IRKPLS) models are proposed in the current work. The objective is to enhance data-driven monitoring methods, especially in experimental industrial applications with imperfect measurements which significantly deteriorate A. PARTIAL LEAST SQUARES Stop when i > ℓ, with ℓbeing the extracted number of latent variables. where ci is the weight vector which is obtained by the opti- mization of the following problem [32], max cT i xT i yiqi ∥ci=1∥∥qi=1∥ (3) (3) 3. Calculate the score vector ti(N ×1) of the latent variable ti of 8i, as Likewise, with t, the score vector ui can be estimated as, Likewise, with t, the score vector ui can be estimated as, ti = Kiui/∥Kiui∥, ∥ti∥= 1. 4. Regress the columns of Yi on ti: ci = Yiti, where ci is a weighting vector. 4. Regress the columns of Yi on ti: ci = Yiti, where ci is a weighting vector. ui = yiqi (4) (4) ui = yiqi The PLS regression model can be represented by using a regression coefficient matrix B and a residual matrix R as, g g 5. Compute the new score-vector: ui = Yici/∥Yici∥, ∥ui∥= 1. 6. Iterate the steps (3) to (5) until the convergence of ti. 7. Deflate the residuals; ( Y = XB + R B = XT U(T T XXT U)−1T T (5) Ki+1 = (I −titT i )Ki(I −titT i ) Yi+1 = Yi −titT i Yi. Ki+1 = (I −titT i )Ki(I −titT i ) Yi+1 = Yi −titT i Yi. (5) i 8. Retain the data in the matrices: T ←ti, U ←ui. B. KERNEL PARTIAL LEAST SQUARES 9. Set i = i + 1, and return to step (2). Stop when i > ℓ, with ℓbeing the extracted number of latent variables. PLS is a linear regression approach which assumes that the process variables are linear. In many applications, this assumption is not realistic. To solve this issue, a nonlinear regression technique so-called kernel PLS (KPLS) is devel- oped [33]–[35]. KPLS is the most widely used nonlinear input-output statistical methods. KPLS consists to map the input data into high-dimensional space. Then, it aims to apply linear PLS in the feature space. Given an input data matrix X =  x1 x2 · · · xN T ∈RN×m with N samples and m process variables (xi ∈Rm), and output matrix Y =  y1 y2 · · · yN T ∈RN×q regrouping N observations with q quality variables (yi ∈Rq). We assume that the original data xi ∈Rm are mapped into the high dimension features space by using a nonlinear projection function φ, so that xi ∈Rm →φ(xi) ∈Rp. Note that p is the dimensionality of the feature space F which can be arbitrarily large or even infinite. The mapped input data matrix 8 is given by After selecting the desired kernel latent variables, the regression coefficient matrix between 8 and Y has the following form [37], [38]: B = 8T U(T T KU)−1T T Y (8) (8) The prediction outputs on training samples is given by The prediction outputs on training samples is given by ˆY = 8B = KU(T T KU)−1T T Y (9) (9) The output prediction of the testing samples x∗with the trained model is written as: The output prediction of the testing samples x∗with the trained model is written as: ˆy = BT φ(x∗) = Y T T h U(T T KU)−1iT 8φ(x∗) = Y T T h U(T T KU)−1iT k(x∗) (10) (10) where k(x∗) is the kernel vector, k(x∗) = 8φ(x∗)=  k(x1, x∗) k(x2, x∗) · · · k(xN, x∗) T (11) where k(x∗) is the kernel vector, k(x∗) = 8φ(x∗)=  k(x1, x∗) k(x2, x∗) · · · k(xN, x∗) T (11) 8 =  φ(x1), · · · , φ(xN) T (6) (6) where PN i=1 φ(xi) = 0. P i=1 φ The KPLS model of (8, Y) is defined as [36]: P i 1 The KPLS model of (8, Y) is defined as [36]: A. PARTIAL LEAST SQUARES Partial least squares (PLS) is a multivariate statistical anal- ysis method. PLS models the relation between input vari- ables (regressors) and output variables (responses). The PLS decomposition of input X ∈RN×m and response Y ∈RN×p have the following form [30], [31]    X = ℓP i=1 tipT i + E = TPT + E Y = ℓP j=1 ujqT j + F = UQT + F, (1) (1) where T =  t1 t2 · · · tℓ  ∈RN×ℓand U =  u1 u2 · · · uℓ  ∈ RN×ℓrepresent the input and output score matrices, respec- tively. P =  p1 p2 · · · pℓ  ∈ Rm×ℓand Q =  q1 q2 · · · qℓ  ∈RN×ℓindicate loading matrices, respec- tively. E ∈RN×m and F ∈RN×p present the residual matrices. 78344 VOLUME 8, 2020 VOLUME 8, 2020 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems it is defined as: k(xi, xj) = exp(−(xi−xj)T (xi−xj) c ). Denote by K = 88T , an N × N kernel matrix. The KPLS algorithm is outlined in Algorithm 1 The score vector ti, which denotes the column of the matrix T, can be determined as, ti = xici (2) (2) Algorithm 1 KPLS Algorithm 1. Set i = 1, K1 = K, and Y1 = Y. 2. Initialize the vector ui(N × 1) of the latent variable ui of Yi, as the maximum-variance column of Yi. 3. Calculate the score vector ti(N ×1) of the latent variable ti of 8i, as Algorithm 1 KPLS Algorithm 1. Set i = 1, K1 = K, and Y1 = Y. 2. Initialize the vector ui(N × 1) of the latent variable ui of Yi, as the maximum-variance column of Yi. 3. Calculate the score vector ti(N ×1) of the latent variable ti of 8i, as ti = Kiui/∥Kiui∥, ∥ti∥= 1. 4. Regress the columns of Yi on ti: ci = Yiti, where ci is a weighting vector. 5. Compute the new score-vector: ui = Yici/∥Yici∥, ∥ui∥= 1. 6. Iterate the steps (3) to (5) until the convergence of ti. 7. Deflate the residuals; Ki+1 = (I −titT i )Ki(I −titT i ) Yi+1 = Yi −titT i Yi. 8. Retain the data in the matrices: T ←ti, U ←ui. 9. Set i = i + 1, and return to step (2). The interval valued data matrix [Y] of outputs is expressed as The interval valued data matrix [Y] of outputs is expressed as [Y] =   h y1(1), y1(1) i . . h yq(1), yq(1) i . . . . . . h y1(N), y1(N) i . . h yq(N), yq(N) i   (13) The reduced kernel matrix for lower bound data is expressed as, (13) K ′ =   k(x′ 1, x′ 1) · · · k(x′ 1, x′ Nr ) ... ... ... k(x′ Nr , x′ 1) · · · k(x′ Nr , x′ Nr )   (19) (19) The Euclidean distance between observations is used as dis- similarity metric. The idea behind dissimilarity metric is adopted to extract a most relevant subset from all avail- able interval-valued samples. To do so, data consisting of measures of dissimilarity between all pairs of interval- valued observations can be designated using a dissimilarity matrix D as In IRKPLS technique, the regression coefficients matrix B′ for lower bound of interval-valued data can be written as, In IRKPLS technique, the regression coefficients matrix B′ for lower bound of interval-valued data can be written as, B′ = 8′T U′(T ′T K ′U′)−1T ′T Y ′ (20) (20) Therefore, the output prediction on reduced training samples for lower bound of interval-valued data is Therefore, the output prediction on reduced training samples for lower bound of interval-valued data is D =   d11 d12 . . . d1N d21 d22 . . . d2N . . . . dN1 dN2 . . . dNN   (14) D =   d11 d12 . . . d1N d21 d22 . . . d2N . . . . dN1 dN2 . . . dNN   (14) ˆY ′ = 8′B′ = K ′U′(T ′T K ′U′)−1T ′T Y ′ (21) (14) (21) The interval output prediction on test data x∗can be made as, where dij is the Euclidean distance between the rows [Xi] and  Xj  of the data matrix [X]. So, dij is given by: where dij is the Euclidean distance between the rows [Xi] and  Xj  of the data matrix [X]. A. EUCLIDEAN DISTANCE METRIC FOR INTERVAL-VALUED DATA Let [Xj] = {[xj(1)], [xj(2)], . . . , [xj(N)]} and [Yj] = {[yj(1)], [yj(2)], . . . , [yj(N)]} be the interval valued data vec- tor of inputs and outputs, where [xj(k)] = [xj(k), xj(k)] ∀k ∈ {1, . . . , N}, with xj(k) ≤xj(k) and [yj(k)] = [yj(k), yj(k)] with yj(k) ≤yj(k). III. INTERVAL REDUCED KERNEL PLS METHOD 8 = TPT + 8r Y = UQT + Yr, (7) This section presents the interval kernel PLS (IKPLS) meth- ods. In effect, interval-valued data presents a method that takes into account the available sensors information with uncertainty and variability. (7) where T ∈RN×A and U ∈RN×A represent the input and output score matrices, respectively. The loading matrices of 8 and Y are denoted by P ∈Rm×A and Q ∈RN×A, respectively. 8r and Yr present the residuals. Denote A as the number of latent variables in high-dimensional space selected by the cumulative percent variance (CPV) criterion [36]. In KPLS, using the kernel function k(xi, xj) = φT (xi)φ(xj), we can avoid to carry out explicit nonlinear mapping and determine dot products in space F using a kernel functions. Radial basis kernel is the most used kernel functions and The dimension of the IKPLS model depends on the number of samples of the training data set. Reducing the structure of the IKPLS model leads to reduce the number of samples. In this paper, therefore, two novel interval reduced kernel PLS (IRKPLS) models are developed to handle large data sets and reduce the computational complexity of the IKPLS models. In the proposed IRKPLS models, first, the Euclidean distance between samples is used as a dissimilarity metric to keep only the more relevant and informative observations in 78345 VOLUME 8, 2020 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems The corresponding reduced interval matrix of outputs is given by the input space. Then, they apply the new reduced data set to build the IRKPLS models. Next, two proposed IRKPLS mod- els based on Euclidean distance (IRKPLSUL and IRKPLSCR) are presented, respectively.  Y ′ =   y′(1)  , · · · ,  y′(Nr)  T (18) (18) Once, the new reduced input and output matrices are eval- uated, the interval-valued data matrices are transformed on a numerical data matrices, then, IKPLS model is applied on the computed matrices. B. IRKPLS BASED ON INTERVAL UPPER AND LOWER BOUNDS (IRKPLSUL) One of the first proposed approaches is IRKPLS based on interval upper and lower bounds IRKPLSUL. It consists to build two KPLS models using the reduced inputs and outputs data matrices formed, respectively, by the lower and upper bounds of interval measurements. Let x′(i) =  x′ 1(i), · · · , x′ m(i)  , with i ∈{1, . . . , Nr}, be the lower interval input vector with lower bound x′ j(i). Let consider y′(i) =  y′ 1(i), · · · , y′ m(i)  the lower bounds of interval output vector. j j The new interval input matrix [X] is given by p [ ] g y [X] =    x1(1), x1(1)  . .  xm(1), xm(1)  . . . . . .  x1(N), x1(N)  . .  xm(N), xm(N)    (12) (12) The interval valued data matrix [Y] of outputs is expressed as The interval valued data matrix [Y] of outputs is expressed as So, dij is given by: ˆy∗= B′T φ(x∗) = Y ′T T ′ h U′(T ′T K ′U′)−1iT k′(x∗) (22) (22) dij = v u u t m X k=1 ([xk(i)] −[xk(j)])2 (15) (15) where k′(x∗) =  k(x′ 1, x∗) · · · k(x′ Nr , x∗) T (23) with with (23) ([xk(i)] −[xk(j)])2 = xk(i) −xk(j)
(xk(i) −xk(j)) (16) Define x′(i) =  x′1(i), · · · x′m(i)  as the upper bound of interval input vector, with upper bound x′j(i). The upper bound of interval output vector can be represented of the form y′(i) =  y′1(i) · · · y′m(i)  . Hence, the dissimilarity matrix D is symmetric and its diag- onal elements are null. Then, all redundancy in observations are eliminated from the input data matrix based on a pick out dissimilarity distance. This plays an important role in reducing the number of observations. In this case, the second reduced kernel matrix K ′ for upper bound of interval valued data is given as The resulting reduced interval matrix of inputs can be defined as: K ′ =   k(x′1, x′1) · · · k(x′1, x′Nr ) ... ... ... k(x′Nr , x′1) · · · k(x′Nr , x′Nr )   (24)  X′ =   x′(1)  , · · · ,  x′(Nr)  T (17) (24) (17) where Nr represents the number of reduced samples. where Nr represents the number of reduced samples. VOLUME 8, 2020 78346 78346 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems In this case, the matrix of regression coefficient B′ for the upper bound in the IRKPLS technique has the following form, where the input center X′c and range X′r matrices, and output center Y ′c and range Y ′r matrices are given by:    X′c = [x′c 1, x′c 2, . . . , x′c Nr]T ∈RNr×m X′r = [x′r 1, x′r 2, . . . , x′r Nr]T ∈RNr×m Y ′c = [y′c 1, y′c 2, . . . , y′c Nr]T ∈RNr×q Y ′r = [y′r 1, y′r 2, . . . IV. FAULT DETECTION BASED ON IRKPLS Once the IRKPLS models are built, the monitored interval residuals are computed and used later for process monitoring purposes. To monitor the behavior of a process, different detection indices are developed in literature [16], [28], [34], [36], [39]–[43]. Among the fault detection charts, the GLRT shows satisfied rapidity for detecting large and small faults in many industrial systems. The GLRT statistic is a hypothesis testing scheme [36], [44]–[47]. Let E ∈RNr is a mea- sured vector follows one of the two Gaussian distributions: N(0, σ 2INr ) or N(θ ̸= 0, σ 2INr ), where θ is the mean vector and σ 2 is the known variance. The hypothesis testing problem is given by, 2. Compute the output estimations ˆy∗and ˆy∗given by equations 22 and 27. 2. Compute the output estimations ˆy∗and ˆy∗given by equations 22 and 27. Algorithm 2 IRKPLSUL Algorithm Algorithm 2 IRKPLSUL Algorithm ˆY ′CR = 8′ CRB′ CR = K ′ CRU′ CR(T ′ CR T K ′ CRU′ CR)−1T ′ CR T Y ′ CR (32) where the new regression coefficient B′ CR is given by B′ CR = 8′ CR T U′ CR(T ′ CR T K ′ CRU′ CR)−1T ′ CR T Y ′ CR (33) ˆY ′CR = 8′ CRB′ CR = K ′ CRU′ CR(T ′ CR T K ′ CRU′ CR)−1T ′ CR T Y ′ CR (32) Training step: Inputs: N × m input data matrices X, X and N × q output data matrices Y, Y. Training step: Inputs: N × m input data matrices X, X and N × q output data matrices Y, Y. (32) where the new regression coefficient B′ CR is given by 1. Calculate the elements of the dissimilarity matrix D B′ CR = 8′ CR T U′ CR(T ′ CR T K ′ CRU′ CR)−1T ′ CR T Y ′ CR (33) (33) 2. Pick the smallest distance from D and extract the new reduced data input matrices X′ ∈RNr×m, X′ ∈RNr×m and output data matrices Y ′ ∈RNr×q, Y ′ ∈RNr×q. 2. Pick the smallest distance from D and extract the new reduced data input matrices X′ ∈RNr×m, X′ ∈RNr×m and output data matrices Y ′ ∈RNr×q, Y ′ ∈RNr×q. For a new observation x∗ CR using the reduced data set, the interval output estimation ˆyCR based on the IRKPLSCR model is represented by, 3. Scale the data matrices to zero mean and unit variance, 3. Scale the data matrices to zero mean and unit variance, 3. Scale the data matrices to zero mean and unit variance, 4. Compute the kernel matrices K ′ and K ′ and scale them, ˆyCR = B′ CR T φ(x∗ CR) = Y ′ CR T T ′ CR h U′ CR(T ′ CR T K ′ CRU′ CR)−1iT k(x∗ CR) (34) 5. Determine the number kernel of latent variables, to be kept in the IRKPLS model, 5. Determine the number kernel of latent variables, to be kept in the IRKPLS model, 6. Compute the regression coefficients matrices B′ and B′ as given by equations 20 and 25, respectively, 6. Testing step: 1. For new testing data vectors x∗, x∗, y∗and y∗, map input data vectors into feature space to get k′(x∗) (equation 23) and k′(x∗) (equation 28) 1. For new testing data vectors x∗, x∗, y∗and y∗, map input data vectors into feature space to get k′(x∗) (equation 23) and k′(x∗) (equation 28) The interval valued data matrix [Y] of outputs is expressed as , y′r Nr]T ∈RNr×q (30) B′ = 8′T U′(T ′T K ′U′)−1T ′T Y ′ (25) (25) (30) The prediction of interval output variables is determined by The prediction of interval output variables is determined by ˆY ′ = 8′B′ = K ′U′(T ′T K ′U′)−1T ′T Y ′ (26) (26) Then, the input reduced data matrix is mapped to a feature space and the reduced kernel matrix K ′CR based on center and range approach is defined as, As a result, the prediction on test subset for upper bound of interval-valued data is written as, As a result, the prediction on test subset for upper bound of interval-valued data is written as, ˆy∗= B′T φ(x∗) = Y ′T T ′ h U′(T ′T K ′U′)−1iT k′(x∗) (27) ˆy∗= B′T φ(x∗) = Y ′T T ′ h U′(T ′T K ′U′)−1iT k′(x∗) (27) where k′(x∗) =  k(x′1, x∗) · · · k(x′Nr , x∗) T (28) K ′CR =   k(x′CR,1, x′CR,1) · · · k(x′CR,1, x′CR,Nr ) ... ... ... k(x′CR,Nr , x′CR,1) · · · k(x′CR,Nr , x′CR,Nr )   (31) where where k′(x∗) =  k(x′1, x∗) · · · k(x′Nr , x∗) T (28) (28) (28)  (31) The main steps of IRKPLSUL algorithm are outlined in Algorithm 2. The prediction of the output variables-based center and range method can be expressed as The prediction of the output variables-based center and range method can be expressed as Algorithm 2 IRKPLSUL Algorithm Compute the regression coefficients matrices B′ and B′ as given by equations 20 and 25, respectively, The IRKPLSCR based center and range of interval algo- rithm is presented in Algorithm 3. FIGURE 1. Diagram of IRKPLS-based GLRT for fault detection. FIGURE 1. Diagram of IRKPLS-based GLRT for fault detection. fθ(E) = 1 (2π) Nr 2 σ Nr exp  −1 2σ 2 ∥E −θ∥2 2  (37) (37) From the IRKPLSCR model and using equation 34, the residual is given by The multivariate GLRT chart is determined for upper and lower bounds of interval-valued data in the feature space as The multivariate GLRT chart is determined for upper and lower bounds of interval-valued data in the feature space as ECR(x∗CR) = yCR −ˆyCR (43) (43) The multivariate fault detection GLRT chart, GCR(x∗CR) of a new sample x∗CR , is computed in the feature space as: ( G = 1 σ 2 ∥E∥2 G = 1 σ 2 ∥E∥2 (38) (38) GCR(x∗CR) = 1 σ 2 ∥ECR(x∗CR)∥2 (44) (44) where σ and σ are the variance of the residuals E and E, repectively. Let GCR,α, be the corresponding control limit. A fault is detected if GCR(x∗CR) > GCR,α. According to equations 22 and 27, the residuals based on the lower and upper bounds of interval-valued data are defined as The IRKPLS-based GLRT statistic algorithm is illustrated schematically in Figure 2. ( E = y −ˆy∗ E = y −ˆy∗ (39) (39) Training step: ( Gα = gχ2 h,α Gα = gχ2 h,α (41) Training step: Inputs: N × m input interval data matrix [X] and N × p output interval data matrix [Y] Training step: Inputs: N × m input interval data matrix [X] and N × p output interval data matrix [Y] Training step: Inputs: N × m input interval data matrix [X] and N × p output interval data matrix [Y] (41) p 1 Compute the center and ranges matrices Xc, Xr, Y c and Y r, respectively, 1 Compute the center and ranges matrices Xc, Xr, Y c and Y r, respectively, where, ( g = b 2a, h = 2a2 b g = b 2a, h = 2a2 b (42) 2 Form the new data matrices XCR = [Xc Xr] and YCR = [Y c Y r], respectively, 2 Form the new data matrices XCR = [Xc Xr] and YCR = [Y c Y r], respectively, (42) 3. Calculate the elements of the dissimilarity matrix D 3. Calculate the elements of the dissimilarity matrix D 4. Pick the smallest distance from D and extract the new reduced data input matrices X′CR ∈RNr×2m and output data matrices Y ′CR ∈RNr×2q, Y ′ ∈RNr×q. 4. Pick the smallest distance from D and extract the new reduced data input matrices X′CR ∈RNr×2m and output data matrices Y ′CR ∈RNr×2q, Y ′ ∈RNr×q. with a and b are the mean and variance of the G index, and a and b are the mean and variance of the G index. The IRKPLSUL-based GLRT fault detection strategy is shown in Figure 1. 5. Standardize the data matrices to zero mean and unit variance, X′ CR and Y ′CR, 6. Determine the kernel matrix K ′CR using data matrix X′ CR, FIGURE 1. Diagram of IRKPLS-based GLRT for fault detection. C 7. Scale the data matrix K ′CR, 8. Compute the number of kernel latent variable, to be kept in the IRKPLS model, 9. Determine the regression coefficients matrix B′CR as given by equation 33, ˆ 10. Compute the estimations of output matrix ˆY ′CR using equation 32, Algorithm 3 IRKPLSCR Algorithm Algorithm 3 IRKPLSCR Algorithm Algorithm 3 IRKPLSCR Algorithm Training step: Inputs: N × m input interval data matrix [X] and N × p output interval data matrix [Y] 1 Compute the center and ranges matrices Xc, Xr, Y c and Y r, respectively, 2 Form the new data matrices XCR = [Xc Xr] and YCR = [Y c Y r], respectively, 3. Calculate the elements of the dissimilarity matrix D 4. Pick the smallest distance from D and extract the new reduced data input matrices X′CR ∈RNr×2m and output data matrices Y ′CR ∈RNr×2q, Y ′ ∈RNr×q. 5. Standardize the data matrices to zero mean and unit variance, X′ CR and Y ′CR, 6. Determine the kernel matrix K ′CR using data matrix X′ CR, 7. Scale the data matrix K ′CR, 8. Compute the number of kernel latent variable, to be kept in the IRKPLS model, 9. Determine the regression coefficients matrix B′CR as given by equation 33, 10. Compute the estimations of output matrix ˆY ′CR using equation 32, Testing step: C. IKPLS BASED ON INTERVAL CENTERS AND RANGES (IRKPLSCR) The proposed IRKPLSCR model aims first to transform the input [X] and output [Y] matrices into numerical matrices based on the interval center and range. Then, it consists to decrease the dimensions of the original data matrices using the Euclidean distance between observations as dissimilarity metric. After that, it uses both the center and the range variables to construct a reduced regression model. In the proposed IRKPLSCR method, new data matrices are built by the concatenation of center and range data matrices, and a KPLS model is applied to this new reduced data matrices. The new reduced input X′CR and output Y ′CR data matrices are defined as, ( H0 = {E ∼N(0, σ 2INr )} (null hypothesis); H1 = {E ∼N(θ, σ 2INr )} (alternative hypothesis). (35) (35) Using the GLRT approach, the unknown parameter θ is changed by its maximum likelihood estimate. The GLRT G(E) decision function is computed as E) = 2log( sup θ∈RNr fθ(E) fθ=0(E) ) (36) (29) G(E) = 2log( sup θ∈RNr fθ(E) fθ=0(E) ) (36) ( X′CR =  X′c X′r ∈RNr×2m Y ′CR =  Y ′c X′r ∈RNr×2q (29) (29) (29) (36) 78347 VOLUME 8, 2020 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems The control limits Gα and Gα are computed as Testing step: 1. For new testing data vectors x∗CR and y∗CR, map the input data vector into feature space to get k(x∗CR) p p g 2. Compute the output estimations ˆyCR using equation 34. where fθ(E) is the probability density function which is given by, where fθ(E) is the probability density function which is given by, A. TE PROCESS DESCRIPTION The TE process has been widely used by the process moni- toring community as a source of data for comparing various methods [48], [52]–[55]. TE process contains, G and H are the main products, A, C, D, and E are reactants (gaseous raw material), F is a by-product, and B is an inert gas. The irreversible exothermic chemical reactions that away place in the reactor are: FIGURE 4. Time evolution of interval-valued variable XMEAS(1). Two conventional and two developed IKPLS models are built using CPV criterion with 95% as an explained variance threshold. The two conventional IKPLS models, referred to IKPLSCR and IKPLSUL are structured by 13 and 12 retained kernel latent variables, respectively. The two IRKPLSCR and IRKPLSUL models are identified by only 585 extracted sam- ples thanks to the Euclidean distance similarity measure from which 12 kernel latent variables are maintained for each of them. The nonlinear uncertain process monitoring is achieved using the different obtained IKPLS models, from which the fault detection index GLRT is assessed for comparison. Its threshold is established at α = 5% as a significance level. A(g) + C(g) + D(g) →G(liq) A(g) + C(g) + E(g) →H(liq) A(g) + E(g) →F(liq) 3D(g) →2F(liq) (45) (45) TE process includes five principal units: a reactor, a com- pressor, a condenser, a stripper, and a vapor/liquid separator. TE process includes five principal units: a reactor, a com- pressor, a condenser, a stripper, and a vapor/liquid separator. TE process includes five principal units: a reactor, a com- pressor, a condenser, a stripper, and a vapor/liquid separator. More information about a process including the reactions are broadly detailed in the literature [56], [57] (Figure 3). TE process includes five principal units: a reactor, a com- pressor, a condenser, a stripper, and a vapor/liquid separator. More information about a process including the reactions are broadly detailed in the literature [56], [57] (Figure 3). More information about a process including the reactions are broadly detailed in the literature [56], [57] (Figure 3). V. CASE STUDY USING THE TENNESSEE EASTMAN (TE) PROCESS A fault is detected using GLRT chart, if both upper and lower indices, G and G, are out of their respective thresholds, Gα and Gα, To evaluate the detection efficiencies of the proposed tech- niques, an industrial benchmark of Tennessee Eastman (TE) process is used. [48]–[51]. The monitoring performance of both schemes is appraised based on false alarms rate (FAR) which is the percentage of false alarms in a healthy set, missed detection rate (MDR) which represents the percentage ( G > Gα G > Gα (40) (40) 78348 78348 VOLUME 8, 2020 VOLUME 8, 2020 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems FIGURE 2. Diagram of IRKPLS-based GLRT statistic for fault detection. FIGURE 3. Tennessee Eastman process. FIGURE 3. Tennessee Eastman process. while 21 different testing datasets, each contains 1024 sam- ples, are generated at a sampling rate of 3 minutes over 21 benchmark faults. Each sample of each variable is asso- ciated with an uncertainty bound of δ = 10% to generate an interval-valued sample for the training and testing datasets. Figure 4 illustrates the time evolution of the interval-valued variable XMEAS(1) during the healthy and faulty statuses. FIGURE 2. Diagram of IRKPLS-based GLRT statistic for fault detection. FIGURE 4. Time evolution of interval-valued variable XMEAS(1). of non-detected faulty observations over abnormal operating conditions of the process, and execution time (ET) as the time consumed in the completion of each of these algorithms which are implemented in the MATLAB computing environ- ment using a PC equipped with Intel Core i5-8600K 3.4 GHz CPU (5 cores), 8 GB of RAM. B. FAULT DETECTION RESULTS The obtained FAR, MDR and ET values contributed by IKPLSCR, IKPLSUL, IRKPLSCR, and IRKPLSUL tech- niques based on GLRT statistic are summarized in Table 1 In this study, a training dataset of 1024 samples is collected from the TE process under normal operating conditions, VOLUME 8, 2020 78349 78349 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems TABLE 1. Summary of FAR, MDR and ET. where the best of them are shown in bold. Whereas the underlined value represents the best value in all techniques for each performance metric. It can clearly seen that the FARs contributed by IRKPLSCR-based GLRT method for almost faults are slightly improved compared to those result- ing from the IKPLSCR-based GLRT technique. Unlike, the IKPLSUL-based GLRT contributes to a fairly acceptable practical FAR and ensures higher robustness compared to all algorithms. While the two developed IRKPLS models provide better results compared to the conventional ones in terms of MDR which is successfully improved for the most faults, ensuring reasonably correct detection. Furthermore, they also give an important ET enhancement in all testing cases. Thus, more than 57% of ET is gained by the IRKPLSCR and IRKPLSUL based GLRT techniques, as a result, memory requirements are satisfactorily reduced to 55%. FIGURE 6. IRKPLSCR-based GLRT statistic in case of fault IDV(6). FIGURE 7. IKPLSUL-based GLRT statistic in case of fault IDV(6). FIGURE 6. IRKPLSCR-based GLRT statistic in case of fault IDV(6). FIGURE 7. IKPLSUL-based GLRT statistic in case of fault IDV(6). TABLE 1. Summary of FAR, MDR and ET. h th b t f th h i b ld Wh th FIGURE 6. IRKPLSCR-based GLRT statistic in case of fault IDV(6). TABLE 1. Summary of FAR, MDR and ET. TABLE 1. Summary of FAR, MDR and ET. FIGURE 6. IRKPLSCR-based GLRT statistic in case of fault IDV(6). FIGURE 7. IKPLSUL-based GLRT statistic in case of fault IDV(6). FIGURE 8. IRKPLSUL-based GLRT statistic in case of fault IDV(6). where the best of them are shown in bold. Whereas the underlined value represents the best value in all techniques for each performance metric. It can clearly seen that the FARs contributed by IRKPLSCR-based GLRT method for almost faults are slightly improved compared to those result- ing from the IKPLSCR-based GLRT technique. Unlike, the IKPLSUL-based GLRT contributes to a fairly acceptable practical FAR and ensures higher robustness compared to all algorithms. B. FAULT DETECTION RESULTS While the two developed IRKPLS models provide better results compared to the conventional ones in terms of MDR which is successfully improved for the most faults, ensuring reasonably correct detection. Furthermore, they also give an important ET enhancement in all testing cases. Thus, more than 57% of ET is gained by the IRKPLSCR and IRKPLSUL based GLRT techniques, as a result, memory requirements are satisfactorily reduced to 55%. FIGURE 6. IRKPLSCR-based GLRT statistic in case of fault IDV(6). FIGURE 7. IKPLSUL-based GLRT statistic in case of fault IDV(6). FIGURE 7. IKPLSUL-based GLRT statistic in case of fault IDV(6). FIGURE 5. IKPLSCR-based GLRT statistic in case of fault IDV(6). FIGURE 8. IRKPLSUL-based GLRT statistic in case of fault IDV(6). FIGURE 5. IKPLSCR-based GLRT statistic in case of fault IDV(6). FIGURE 8. IRKPLSUL-based GLRT statistic in case of fault IDV(6). Figures 5 to 8 show the fault detection results using the four techniques in case of fault IDV6. The threshold associated to the GLRT fault detection index is shown in the red dotted line. 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Sci., vol. 187, pp. 269–279, Sep. 2018. RADHIA FEZAI holds a postdoctoral position at the Electrical Engineering Program, Texas A&M University at Qatar. Her work focuses on the use of applied mathematics and statistics concepts to develop statistical data and model-driven tech- niques and algorithms for modeling, fault detec- tion, and monitoring with the aim of improving industrial systems. HAZEM NOUNOU (Senior Member, IEEE) is currently a Professor of electrical and com- puter engineering with Texas A&M University at Qatar. He has more than 19 years of aca- demic and industrial experience. He has signif- icant experience in research on control systems, databased control, system identification and esti- mation, fault detection, and system biology. He has been awarded several national priorities research program (NPRP) research projects in these areas. He has published more than 200 refereed journal and conference papers and book chapters. He has successfully served as the lead PI and a PI on five QNRF projects, some of which were in collaboration with other PIs in this proposal. He has served as an Associate Editor and on the technical committees of several international journals and conferences. KAMALELDIN ABODAYEH received the M.Sc. degree in functional analysis from University Col- lege Dublin and the Ph.D. degree from University College Cork, Ireland, in 1997. REFERENCES Lammertyn, ‘‘Kernel PLS regres- sion on wavelet transformed NIR spectra for prediction of sugar content of apple,’’ Chemometric Intell. Lab. Syst., vol. 85, no. 2, pp. 243–252, Feb. 2007. [14] R. Rosipal and L. J. Trejo, ‘‘Kernel partial least squares regression in reproducing kernel Hilbert space,’’ J. Mach. Learn. Res., vol. 2, pp. 97–123, Mar. 2001. [39] S. Yin, X. Zhu, and O. Kaynak, ‘‘Improved PLS focused on key- performance-indicator-related fault diagnosis,’’ IEEE Trans. Ind. Elec- tron., vol. 62, no. 3, pp. 1651–1658, Mar. 2015. [15] S. 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MAJDI MANSOURI (Member, IEEE) received the B.E. degree in electrical engineering from the Higher School of Communication of Tunisia (SUPCOM), Tunisia, in 2006, the master’s degree in electrical engineering from the School of Elec- tronic, Informatique and Radiocommunications in Bordeaux (ENSEIRB), France, in 2008, the Ph.D. degree in electrical engineering from the Uni- versity of Technology of Troyes (UTT), France, in 2011, and the H.D.R. degree in electrical engi- neering from the University of Orleans, France, in December 2019. He joined the Electrical Engineering Program, Texas A&M University at Qatar, in 2011, where he is currently an Associate Research Scientist. His work focuses on statistical signal processing. He is the author of more than 150 refereed journal and conference publications and book chapters, and has worked on several projects as a Lead Principal Investigator (LPI) and a Principal Investigator (PI). He served as the technical committee member of several international journals and conferences. g pp [41] S. Wang and F. Xiao, ‘‘AHU sensor fault diagnosis using principal com- ponent analysis method,’’ Energy Buildings, vol. 36, no. 2, pp. 147–160, Feb. 2004. [42] Y. Zhang and C. Ma, ‘‘Fault diagnosis of nonlinear processes using mul- tiscale KPCA and multiscale KPLS,’’ Chem. Eng. Sci., vol. 66, no. 1, pp. 64–72, Jan. 2011. pp [43] M. Mansouri, M. N. Nounou, and H. N. Nounou, ‘‘Multiscale kernel PLS-based exponentially weighted-GLRT and its application to fault detection,’’ IEEE Trans. Emerg. Topics Comput. Intell., vol. 3, no. REFERENCES He had his Post- doctoral at the Department of Process Engineer- ing, University College Cork. Since 2001, he has been with Prince Sultan University, Saudi Arabia. He published more than 40 articles in vari- ous areas of pure and applied mathematics. His research areas include functional analysis, theo- retical physics, discrete potential theory, fixed point theory, and quality monitoring and statistical hypothesis testing. VOLUME 8, 2020 78352 R. Fezai et al.: Reliable Fault Detection and Diagnosis of Large-Scale Nonlinear Uncertain Systems MOHAMED NOUNOU (Senior Member, IEEE) is currently a Professor of chemical engineer- ing with TAMU-Texas A&M University at Qatar. He has more than 19 years of combined academic and industrial experience. He has published more than 200 refereed journal and conference publi- cations and book chapters. His research interests are in the area of systems engineering and control, with emphasis on process modeling, monitoring, and estimation. He has successfully served as the lead PI and a PI on several QNRF projects (six NPRP projects and three UREP projects). He is a Senior Member of the AIChE (American Institute of Chemical Engineers). HASSANI MESSAOUD has prepared the Ph.D. thesis at the University of Nice-Siophia Antipolis, France, in 1993, and the habilitation thesis at the School of Engineers, Tunis, in June 2001. Actu- ally, he is currently a Professor and the Head of the Research Lab LARATSI (Code:LR13ES13), School of Engineers, Monastir, Tunisia. His main majoring is process identification and control as well as signal and image processing. He has pub- lished more than 200 refereed journal and confer- ence publications and book chapters. He served as the technical committee member of several international journals and conferences. 78353 VOLUME 8, 2020

https://openalex.org/W4387119204

https://link.springer.com/content/pdf/10.1007/JHEP09(2023)193.pdf

English

Celestial Berends-Giele current

˜The œJournal of high energy physics/˜The œjournal of high energy physics

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Published for SISSA by Springer Published for SISSA by Springer Open Access, c⃝The Authors. Article funded by SCOAP3. Published for SISSA by Springer Received: August 12, 2023 Accepted: September 18, 2023 Published: September 27, 2023 JHEP09(2023)193 Received: August 12, 2023 Accepted: September 18, 2023 Published: September 27, 2023 E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] Abstract: Celestial amplitude plays an important role in the understanding of hologra- phy. Computing celestial amplitudes by recursion can deepen our understanding of the structure of celestial amplitudes. As an important recursion method, the Berends-Giele (BG) currents on the celestial sphere are worth studying. In this paper, we study the celes- tial BG recursion and utilize this to calculate some typical examples. We also explore the OPE behavior of celestial BG currents. Moreover, we generalize the “sewing procedure” for BG currents to the celestial case. Yi-Xiao Tao Department of Mathematical Sciences, Tsinghua University, Beijing 100084, China Department of Mathematical Sciences, Tsinghua University, Beijing 100084, China Department of Mathematical Sciences, Tsinghua University, Beijing 100084, China Keywords: Scattering Amplitudes, Scale and Conformal Symmetries, Gauge Symmetry Keywords: Scattering Amplitudes, Scale and Conformal Symmetries, Gauge Symmetr ArXiv ePrint: 2307.14772 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP09(2023)193 https://doi.org/10.1007/JHEP09(2023)193 Contents 1 Introduction 1 2 Preliminaries 2 2.1 Celestial amplitude 2 2.2 BG current 4 3 Celestial BG current 4 3.1 Recursion relation 5 3.2 Example: 3-pt gluon celestial amplitude 7 3.3 Soft region 9 3.4 The OPE limit of celestial BG currents 11 4 From tree to loop 12 5 Conclusions and outlooks 15 A 3D BCFW for celestial amplitudes 15 B Wrong conformal weights from integrating the celestial BG currents 17 Contents 1 Introduction 1 2 Preliminaries 2 2.1 Celestial amplitude 2 2.2 BG current 4 3 Celestial BG current 4 3.1 Recursion relation 5 3.2 Example: 3-pt gluon celestial amplitude 7 3.3 Soft region 9 3.4 The OPE limit of celestial BG currents 11 4 From tree to loop 12 5 Conclusions and outlooks 15 A 3D BCFW for celestial amplitudes 15 B Wrong conformal weights from integrating the celestial BG currents 17 1 2 2 4 4 5 7 9 11 12 15 15 17 JHEP09(2023)193 A 3D BCFW for celestial amplitudes B Wrong conformal weights from integrating the celestial BG currents 2 Preliminaries In this section, we will review some basic concepts. For a more comprehensive review of the celestial amplitudes, see [29]. 1 Introduction In section 3, we derive the celestial BG currents for gluon amplitudes and show how to do the recursion. Moreover, we reproduce the result of 3-pt celestial MHV gluon amplitude in the collinear region (in the Klein space with signature (−+ −+) ) [23] and the soft region (in the Minkowski space with signature (−+ ++)) [28]. Finally, we generalize the “sewing procedure” of BG currents to celestial BG currents in section 4. However, the off-shell method on the celestial sphere still needs to be studied. In [26], the authors show an off-shell prescription for scalars. Furthermore, in [27], the author discusses the Feynman rules for celestial scalar amplitudes. In this paper, we will show how to construct BG currents for celestial amplitudes utilizing the gluon case as an example. JHEP09(2023)193 This paper will be organized as follows. In section 2, we will review some basic concepts including celestial amplitudes and BG currents. In section 3, we derive the celestial BG currents for gluon amplitudes and show how to do the recursion. Moreover, we reproduce the result of 3-pt celestial MHV gluon amplitude in the collinear region (in the Klein space with signature (−+ −+) ) [23] and the soft region (in the Minkowski space with signature (−+ ++)) [28]. Finally, we generalize the “sewing procedure” of BG currents to celestial BG currents in section 4. 1 Introduction Recursion methods are very important in amplitudes. We can obtain higher point ampli- tudes from the lower point ones using recursion relations, which means that we can convert the complication calculation of higher point amplitudes to the much easier calculation of lower point ones. In the past several decades, the most popular recursion relation is the Britto-Cachazo-Feng-Witten (BCFW) recursion [1, 2], which is based on the complex-shift method and the residue theorem. Another on-shell recursion, which is raised even before the complex-shift method, is the Cachazo-Svrcek-Witten (CSW) expansion [3–5]. For a more comprehensive introduction, see [6]. Apart from the on-shell recursion, there is also an off-shell, or a semi-on-shell, recursion relation based on the Berend-Giele (BG) cur- rents [7–9]. The definition of the BG currents is amplitudes with one leg off-shell. And the BG recursion can be obtained by evaluating the equation of motion. In other words, the origin of the BG recursion can be regarded as Feynman rules, which means this recursion is very convenient to be generalized to other cases. In recent years, there have been some new developments in the study of BG currents [10–17]. Inspired by the holographic duality of AdS spacetime, we wonder if we can regard the amplitudes as CFT correlators. In fact, after we change the basis of amplitudes, we will obtain CFT correlators on the “celestial sphere” [18]. A superamplitude version can – 1 – be found in [19]. There are also some results about celestial twistor amplitudes [20] and celestial string amplitudes [21, 22]. A natural question is, can we find some recursion relations for the celestial amplitudes? In [23], the authors show an example of BCFW for 4-pt MHV gluon celestial amplitudes. For n-pt MHV gluon celestial amplitudes, a formula based on BCFW is given in [24, 25]. In appendix A, we show a 3D BCFW method for celestial amplitudes as a generalization.f p g However, the off-shell method on the celestial sphere still needs to be studied. In [26], the authors show an off-shell prescription for scalars. Furthermore, in [27], the author discusses the Feynman rules for celestial scalar amplitudes. In this paper, we will show how to construct BG currents for celestial amplitudes utilizing the gluon case as an example. This paper will be organized as follows. In section 2, we will review some basic concepts including celestial amplitudes and BG currents. 2.1 Celestial amplitude Celestial amplitudes can be obtained by expanding position space amplitudes M(Xi) with respect to the conformal primary wave functions ϕ± ∆,s(z, ¯z; X) (or shadow conformal pri- mary wave functions ˜ϕ± ∆,s(z, ¯z; X) instead of plane waves: M∆i si (zi, ¯zi) =   n Y j=1 Z d4Xj     k Y j=1 ϕ+ ∆j,sj(zj, ¯zj; Xj)     n Y j=k+1 ˜ϕ− ∆j,sj(zj, ¯zj; Xj)  M(Xi) (2 (2.1) Here ∆and s are the conformal dimension and the spin (or helicity) respectively, and ± labels whether the particle is outgoing or incoming. The coordinates (zi, ¯zi) on the celestial sphere can be connected to massless on-shell momenta qµ i through Here ∆and s are the conformal dimension and the spin (or helicity) respectively, and ± labels whether the particle is outgoing or incoming. The coordinates (zi, ¯zi) on the celestial sphere can be connected to massless on-shell momenta qµ i through (2.2) qµ = ωˆqµ = ω(1 + z¯z, z + ¯z, −i(z −¯z), 1 −z¯z) (2.2) and for massive on-shell momentum pµ we have pµ = mˆpµ = m 2y(1 + y2 + w ¯w, w + ¯w, −i(w −¯w), 1 −y2 −w ¯w) (2.3) (2.3) Note that ω and m are always positive, which means that whether the particle is incoming or outgoing is very important in this context. Here, we choose to expand the wave functions Note that ω and m are always positive, which means that whether the particle is incoming or outgoing is very important in this context. Here, we choose to expand the wave functions – 2 – of the incoming particles in the conformal primary basis while the outgoing particles in the shadow conformal primary basis [28]. Now we review some wave functions commonly used. For scalars, the conformal primary wave functions are given by For scalars, the conformal primary wave functions are given by ϕ± ∆(z, ¯z; X) = Z ∞ 0 dω ω∆−1e±iωˆq·X−ϵω = (∓i)∆Γ(∆) (−ˆq · X ∓iϵ)∆∝ 1 (−ˆq · X ∓iϵ)∆ (2.4) (2.4) for massless scalars and ϕ± ∆,m(z, ¯z; X) = Z d3ˆp′ ˆp′0 1 (−ˆq · ˆp′)∆e±imˆp′·X = Z dy y3 dwd ¯w( y y2 + |z −w|2 )∆e±imˆp(y,w, ¯w)·X (2 (2.5) JHEP09(2023)193 for scalars with mass m. 1To see this, we need to consider the integral over δ(ω) from the momentum conservation delta functi first when we are calculating a celestial amplitude [28]. 2.2 BG current (2.15) (2.15) Aiµ = ϵiµ. The color-ordered tree-level amplitude can be obtained by the following equation M(P, n + 1) = lim kP →0 k2 P An · AP = lim kP →0 k2 P ϵn · AP . (2.16) M(P, n + 1) = lim kP →0 k2 P An · AP = lim kP →0 k2 P ϵn · AP . (2.16) (2.16) In this way, we can obtain any point color-ordered tree-level amplitudes by the single- particle states (2.15) and the BG recursion (2.14). In this way, we can obtain any point color-ordered tree-level amplitudes by the single- particle states (2.15) and the BG recursion (2.14). 2.1 Celestial amplitude For massive scalars, shadow conformal primary wave functions can be treated as conformal primary wave functions [18]; while for massless scalars, we have ˜ϕ± ∆(z, ¯z; X) ∝(−X2)∆−1ϕ± ∆(z, ¯z; X) (2.6) (2.6) For spin-1 particles, things are more complicated since we need to include the gauge terms. For massless spin-1 particles, the conformal primary wave function is A∆,± µs (ˆq; X) =∆−1 ∆ ∂sˆqµ (−ˆq · X ∓iϵ)∆+ ∂µ ∂sˆq · X ∆(−ˆq · X ± iϵ)∆ =V ∆,± µs (ˆq; X) + ∂µα∆,± s (ˆq; X), (2.7) (2.7) where s = z or ¯z. The choice will depend on the helicity of the particle and 1 √ 2∂sˆqµ can be regarded as the polarization vector. The convention of the polarization vectors will be given in subsection 3.2. Note that we have XµA∆,± µs = 0 and ∂µA∆,± µs = 0. The shadow conformal primary wave function is given by ˜A∆,± µs (ˆq; X) = (−X2)∆−1A∆,± µs (ˆq; X) (2.8) (2.8) For amplitudes, whose legs are all on-shell, using A∆,± µs (ˆq; X) is equivalent to Mellin trans- formation since they are gauge equivalent: A∆,± µs (ˆq; X) = ∆−1 (∓i)∆Γ(∆+ 1) Z dω ω∆−1e±iωˆq·X−ϵω + ∂µα∆,± s (ˆq; X) (2.9) (2.9) However, in the soft region ω = 0, which forces ∆= 1 on the celestial side,1 the conformal wave functions need to be redefined. Alog,± µs (ˆq1; X) = lim ∆→1 ∂∆(A∆,± µs + ˜A2−∆,± µs ) =2Xµ X2 ∂aˆq · X −ˆq · X ∓iϵ + ∂µ  −log(−X2) ∂aˆq · X −ˆq · X ∓iϵ  =V log,± µs + ∂µαlog,± s (2.10) (2.10) It is important to mention that V log,± µs and ∂µαlog,± s do not satisfy the massless Klein- Gordon equation. Only the sum of these two terms can be regarded as an on-shell thing. It is important to mention that V log,± µs and ∂µαlog,± s do not satisfy the massless Klein- Gordon equation. Only the sum of these two terms can be regarded as an on-shell thing. 1To see this, we need to consider the integral over δ(ω) from the momentum conservation delta function first when we are calculating a celestial amplitude [28]. – 3 – 2.2 BG current BG recursion is an off-shell recursion based on the Feynman rules. Using this recursion, one can obtain higher point BG currents from lower point ones. The definition of an n-pt BG current is an (n + 1)-pt amplitude with one leg off-shell. By definition, amplitudes can be generated by BG currents in a rather simple way. Therefore, BG recursion is also a recursion for amplitudes and can be obtained from the equation of motion for an interaction system. In this subsection, we will show how to derive the BG recursion of the Yang-Mills (YM) theory as an example. The equation of motion for YM theory takes the form (we have used the Lorenz gauge here): JHEP09(2023)193 □Aµ = −i[Aν, Fµν] + i[Aν, ∂νAµ]. (2.11)i (2.11) Here, Aµ = Aa µT a denotes the Lie algebra-valued gluon field, with T a being the Lie group generators. The corresponding Lie algebra-valued field strength tensor is given by Fµν = ∂µAν −∂νAµ −i[Aµ, Aν]. Note that the Lorenz gauge is assumed in this case. To obtain the multi-particle solution of (2.11), we assume all particles are outgoing (particles with negative energies are allowed here) and apply the perturbiner expansion ansatz: Here, Aµ = Aa µT a denotes the Lie algebra-valued gluon field, with T a being the Lie group generators. The corresponding Lie algebra-valued field strength tensor is given by Fµν = ∂µAν −∂νAµ −i[Aµ, Aν]. Note that the Lorenz gauge is assumed in this case. To obtain the multi-particle solution of (2.11), we assume all particles are outgoing (particles with negative energies are allowed here) and apply the perturbiner expansion ansatz: Aµ = X P APµeikP ·xT P (2.12) (2.12) where we use the notation k to denote the momentum here, and kP = P i∈P ki. Since the Lie algebra valued Aµ is in the adjoint representation of some gauge group, its coefficients APµ must satisfy the shuffle identity [30–33]: Aµ P
Q = 0, P, Q ̸= ∅ (2.13) (2.13) Substituting the ansatz (2.12) to (2.11), we can obtain the recursion: Substituting the ansatz (2.12) to (2.11), we can obtain the recursion: k2 P APµ = X P=XY (kXµ −kY µ)(AX · AY ) −2AXµ(kX · AY ) + 2AY µ(kY · AX) + X P=XY Z [2AY µ(AX · AZ) −AZµ(AX · AY ) −AXµ(AY · AZ)] (2.14) (2.14) with single-particle states with single-particle states Aiµ = ϵiµ. 3.1 Recursion relation In this subsection, we will consider the Minkowski space. Without loss of generality, we define the n-pt celestial BG currents for n incoming gluons (∆i ̸= 1) and one outgoing off-shell gluon as follows: APµ(∆i, pn+1) = Z n Y i=1 dωiω∆i−1 i APµδ(4) n X i=1 ωiˆqi −pn+1 ! (3.1) (3.1) where P is a permutation of {1, 2, · · · , n} and we set all on-shell legs incoming and the off-shell leg outgoing. Note that the case that all on-shell legs are outgoing and the off-shell leg is incoming has the same delta function as the definition above since the delta function is even. We also need to point out that the factor 1/k2 P in the BG currents is set to 1/p2 n+1 and will not be integrated, where pn+1 = mˆpn+1 with a changeable m is the momentum of the off-shell leg. This definition is equivalent to choosing A∆,± µs (ˆq; X) as the wave functions and setting the pure gauge terms to zero. This will change the expression of the celestial BG currents but will not change the value of the celestial amplitudes. If we want to define the general celestial BG current, we only need to consider δ(4)(Pn i=1 ki +kn+1) with ki = ϵiωiˆqi and kn+1 = ϵn+1mˆpn+1, and then assign the particles with correct conformal primary wave functions or shadow conformal primary wave functions. JHEP09(2023)193 It is important to deal with the delta function. We have the following two identities: δ(4) n X i=1 ωiˆqi −pn+1 ! = Z d4Jd4Kδ(4)(qX −J)δ(4)(qY −K)δ(4)(J + K −pn+1) (3. (3.2) δ(4) n X i=1 ωiˆqi −pn+1 ! Z (3.3) X i=1 + ! = Z d4Jd4Kd4Lδ(4)(qX −J)δ(4)(qY −K)δ(4)(qZ −L)δ(4)(J + K + L −pn+1), (3.3) = Z d4Jd4Kd4Lδ(4)(qX −J)δ(4)(qY −K)δ(4)(qZ −L)δ(4)(J + K + L −pn+1), ( ) for P = XY Z, where qX = P i∈X ωiˆqi. 3 Celestial BG current In this section, we will show how to obtain a celestial BG recursion and how to derive celestial amplitudes by this recursion. We only discuss the gluon case without loss of generality. In this paper, the normalization constant is not very important. Therefore we sometimes ignore these overall constants. – 4 – 3.1 Recursion relation From these two identities, we can obtain the recursion of the celestial BG currents: p2 n+1APµ = − X P=XY Z d4Jd4Kδ(4)(J + K −pn+1)×  X i∈X AY · Ai X ˆqiµ − X j∈Y AX · Aj Y ˆqjµ + 2 X k∈Y AX · ˆqkAk Y µ −2 X l∈X AY · ˆqlAl Xµ  + X P=XY Z Z d4Jd4Kd4Lδ(4)(J + K + L −pn+1)×  2AY µ(AX · AZ) −AZµ(AX · AY ) −AXµ(AZ · AY )  (3 4)  X i∈X AY · Ai X ˆqiµ − X j∈Y AX · Aj Y ˆqjµ + 2 X k∈Y AX · ˆqkAk Y µ −2 X l∈X AY · ˆqlAl Xµ  + X P=XY Z Z d4Jd4Kd4Lδ(4)(J + K + L −pn+1)×  2AY µ(AX · AZ) −AZµ(AX · AY ) −AXµ(AZ · AY )  ( ) (3.4) ( ) where Ai X denotes AX with ∆i →∆i + 1. The minus sign of the first term comes from k = −ωˆq for incoming particles. ( ) where Ai X denotes AX with ∆i →∆i + 1. The minus sign of the first term comes from k = −ωˆq for incoming particles. – 5 – For BG currents, the initial condition is the single-particle state For BG currents, the initial condition is the single-particle state (3.5) Aiµ = ϵiµ. (3.5) However, for celestial BG currents, things are more subtle. In fact, we have Aiµ = Z dωiω∆i−1 i ϵiµδ(4)(ωiˆqi −p). (3.6) (3.6) In this case, p must be null from the momentum conservation. However, let us pretend that p is still timelike first and find out another description of the delta function from the momentum conservation. Recall that p can be written as JHEP09(2023)193 JHEP09(2023)193 pµ = m 2y(1 + y2 + w ¯w, w + ¯w, −i(w −¯w), 1 −y2 −w ¯w) (3.7) (3.7) with a changeable m. The support of the delta function is m 2y = ωi, y = 0, w = z, ¯w = ¯z. (3.8) m 2y = ωi, y = 0, w = z, ¯w = ¯z. (3.8) (3.8) The Jacobian for the transformation from (pµ) →( m 2y, y, w, ¯w) is |J| = m3 y2 (3.9) (3.9) then the delta function is then the delta function is δ(4)(ωiˆqi −p) = y2 m3 δ( m 2y −ωi)δ(y)δ(2)(w −zi). 2Here the helicity is the 4D helicity for an incoming or outgoing particle. Note that the helicity (−−−) will not give a zero since there is an outgoing particle and if we regard it as an incoming particle with minus energy we will get + helicity. 3.1 Recursion relation (3.10) (3.10) Substituting this to (3.6), we can obtain the initial condition for the celestial BG c to (3.6), we can obtain the initial condition for the celestial BG currents: Aiµ = Z dωiω∆i−1 i ϵiµδ(4)(ωiˆqi −p) = 1 4ϵiµ m∆i−4 (2y)∆i−3 δ(y)δ(2)(w −zi) (3.11) Aiµ = Z dωiω∆i−1 i ϵiµδ(4)(ωiˆqi −p) = 1 4ϵiµ m∆i−4 (2y)∆i−3 δ(y)δ(2)(w −zi) (3.11) (3.11) We can also obtain the celestial amplitudes from the celestial BG currents. The celestial amplitude of n incoming gluons and 1 outgoing gluon can be obtained by assigning the shadow conformal primary wave function to the particle n + 1: M(P, n + 1) = Z d3ˆpn+1 ˆp0 n+1 ˜A∆n+1,− µ (zn+1, ¯zn+1; pn+1) lim p2 n+1→0 p2 n+1Aµ P . (3.12) (3.12) the shadow basis can be written as ˜A∆,− µ (z, ¯z; X) = R d4p ˜A∆,− µ (z, ¯z; p)e−ip·X. In µ ( , ; ) R p µ ( , ; p) the Klein space, things are easier. There is no distinction between incoming and outgoing in Klein space since they are connected by a Lorentz transformation [23]. Therefore we can always use Mellin transformation and do not need to consider the directions of the particles. In this case we have M(P, n + 1) = Z dωn+1ω∆n+1−1 n+1 lim p2 n+1→0 p2 n+1ϵn+1 · AP . (3.13) (3.13) The formalism for the celestial BG recursion looks rather simple. However, it is not easy to use this recursion due to lots of delta functions. In the next subsection, we will show an important example, and show how to use this recursion and obtain the celestial amplitudes through this example. – 6 – 3.2 Example: 3-pt gluon celestial amplitude In this subsection, we will show how to obtain the 3-pt gluon celestial amplitude only from (3.11) and the celestial BG recursion. This approach, based on recursion and the massless limit, is quite different from [23]. In this subsection, we will show how to obtain the 3-pt gluon celestial amplitude only from (3.11) and the celestial BG recursion. This approach, based on recursion and the massless limit, is quite different from [23]. First of all, we need to derive the 2-pt celestial BG current A12µ (or equivalently, p2 3A12µ). Here we only show the full calculation of the term proportional to (ϵ1 · ϵ2)ˆq1µ, which corresponds to the term A2 · A1 1ˆqiµ in (3.4). −p2 3A12µ ⊃(ϵ1·ϵ2)ˆq1µ Z d4Jd4K 1 16 2yJy3 m2mJ|z12|2 m∆1−3 J (2yJ)∆1−2 m∆2−4 K (2yK)∆2−3 δ(2)(wJ −z1)δ(2)(wK −z2) ×δ(yJ)δ(yK)δ mJ 2yJ − 2yJm2 4mJ|z12|2 ! δ y− 2mω1|z12|2 m2+4ω2 1|z12|2 ! δ(2) w−m2z2+4ω2 1z1z2 12 m2+4ω2 1|z12|2 ! ( ) Z 1 m3 J m3 K 2yJy3 m∆1−3 J m∆2−4 K ( ) ( ) −p2 3A12µ ⊃(ϵ1·ϵ2)ˆq1µ Z d4Jd4K 1 16 2yJy3 m2mJ|z12|2 m∆1−3 J (2yJ)∆1−2 m∆2−4 K (2yK)∆2−3 δ(2)(wJ −z1)δ(2)(wK −z2) ×δ(yJ)δ(yK)δ mJ 2yJ − 2yJm2 4mJ|z12|2 ! δ y− 2mω1|z12|2 m2+4ω2 1|z12|2 ! δ(2) w−m2z2+4ω2 1z1z2 12 m2+4ω2 1|z12|2 ! = (ϵ1·ϵ2)ˆq1µ Z daJdyJdaKdyK 1 16 m3 J y2 J m3 K y2 K 2yJy3 m2mJ|z12|2 m∆1−3 J (2yJ)∆1−2 m∆2−4 K (2yK)∆2−3 δ(yJ)δ(yK) ×δ mK 2yK − 2yJm2 4mJ|z12|2 ! δ y− 2mω1|z12|2 m2+4ω2 1|z12|2 ! δ(2) w−m2z2+4ω2 1z1z2 12 m2+4ω2 1|z12|2 ! = (ϵ1·ϵ2)ˆq1µ Z daJ y3 m2|z12|2 a∆1−∆2 J m2 4|z12|2 !∆2−1 δ y− 2mω1|z12|2 m2+4ω2 1|z12|2 ! δ(2) w−m2z2+4ω2 1z1z2 12 m2+4ω2 1|z12|2 ! (3 14) JHEP09(2023)193 = (ϵ1·ϵ2)ˆq1µ Z daJdyJdaKdyK 1 16 m3 J y2 J m3 K y2 K 2yJy3 m2mJ|z12|2 m∆1−3 J (2yJ)∆1−2 m∆2−4 K (2yK)∆2−3 δ(yJ)δ(yK) J K ×δ mK 2yK − 2yJm2 4mJ|z12|2 ! δ y− 2mω1|z12|2 m2+4ω2 1|z12|2 ! δ(2) w−m2z2+4ω2 1z1z2 12 m2+4ω2 1|z12|2 ! = (ϵ1·ϵ2)ˆq1µ Z daJ y3 m2|z12|2 a∆1−∆2 J m2 4|z12|2 !∆2−1 δ y− 2mω1|z12|2 m2+4ω2 1|z12|2 ! δ(2) w−m2z2+4ω2 1z1z2 12 m2+4ω2 1|z12|2 ! (3 14 (3.14) ! (3.14) where we have used the well-known parametrization for the delta function with respect to 2 null vectors and 1 timelike vector. We should point out that when we take yJ →0, the variable aJ = mJ 2y remains finite since when yJ →0, mJ →0. 3.2 Example: 3-pt gluon celestial amplitude where we have used the well-known parametrization for the delta function with respect to 2 null vectors and 1 timelike vector. We should point out that when we take yJ →0, the variable aJ = mJ 2y remains finite since when yJ →0, mJ →0. y After replacing the integral variable aJ →ω1, the full 2-pt celestial BG current is −p2 3A12µ = Z dω1ω∆1−1 1 m2 4|z12|2ω1 !∆2−1 1 m2 1 ω1|z12|2 y3 × " ω1ˆq1µ − m2 4ω1|z12|2 ˆq2µ ! (ϵ1 · ϵ2) + 2ϵ2µ m2 4ω1|z12|2 ˆq2 · ϵ1 −2ϵ1µω1ˆq1 · ϵ2 # × δ y − 2mω1|z12|2 m2 + 4ω2 1|z12|2 ! δ(2) w −m2z2 + 4ω2 1z1|z12|2 m2 + 4ω2 1|z12|2 ! (3 15) (3.15)i This result is the same as the Mellin transformation of the 2-pt BG current, which verifies our recursion. The next step is to obtain the 3-pt gluon celestial amplitude. For simplicity, but without loss of generality, we choose the helicity of 3 particles as (−−−)2 and we set particle 2Here the helicity is the 4D helicity for an incoming or outgoing particle. Note that the helicity (−−−) will not give a zero since there is an outgoing particle and if we regard it as an incoming particle with minus energy we will get + helicity. – 7 – 3 to be outgoing.3 Our convention for the polarization vector of incoming particles is 3 to be outgoing.3 Our convention for the polarization vector of incoming particles is ϵµ + = 1 √ 2(¯z, 1, −i, −¯z) ϵµ −= 1 √ 2(z, 1, i, −z), (3.16) (3.16) while for outgoing particles we need to exchange z and ¯z. And we can obtain the 2-pt celestial BG current with particles 1 and 2 “−” helicity: −p2 3A12µ = Z dω1ω∆1−1 1 m2 4|z12|2ω1 !∆2−1 1 m2 1 ω1|z12|2 y3 × " −2 √ 2ϵ2µ m2 4ω1|z12|2 z12 −2 √ 2ϵ1µω1z12 # × δ y − 2mω1|z12|2 m2 + 4ω2 1|z12|2 ! δ(2) w −m2z2 + 4ω2 1z1|z12|2 m2 + 4ω2 1|z12|2 ! . (3.17) JHEP09(2023)193 (3.17) When we take the massless limit, we must keep in mind that ω3 = m 2y. We can rewrite the current so that there is no dependence on y. If we choose zij to be non-vanishing real numbers and also independent of ¯zij, i.e. 3It is worth mentioning that although changing the directions of particles will not change the wavefunc- tions, it will change the constraints of zij as we will see later and finally affect the result. 3.2 Example: 3-pt gluon celestial amplitude The 2-pt celestial BG current now can be written as −p2 3A12µ = − √ 2 Z dω1ω∆1−1 1 z31 z23 ω1 ∆2−1 1 2ω3 1 ω1|z12|2  ϵ2µ z31 z23 ω1z12 + ϵ1µω1z12  × |z12|2 |z13||z23|δ  ω3 −ω1 z21 z23  δ ¯z12 − m2z23 4ω2 1z12z31 ! δ ¯z13 + m2¯z23 4ω2 1z12¯z12 ! . (3 22) JHEP09(2023)193 (3.22) ( ) Now we can take the massless limit m →0 (Note that ϵ+ · ϵ−= 1): Now we can take the massless limit m →0 (Note that ϵ+ · ϵ−= 1): M−−−(123) = − Z dω3ω∆3−1 3 lim p2 3→0 p2 3ϵ3 · A12 = − √ 2 2 Z dω1  ω1 z21 z23 ∆3−2 ω∆1−1 1 z31 z23 ω1 ∆2−1 z2 12 z23 1 |z13||z23|δ(¯z12)δ(¯z13). (3 23) (3.23) ( ) Note that when we take m →0, we have ¯z23/¯z12 →1. Let ωi = 1 + iλi, then from the identity Z ( ) Note that when we take m →0, we have ¯z23/¯z12 →1. Let ωi = 1 + iλi, then from the identity Z Z dωωiλ−1 = 2πδ(λ), (3.24) (3.24) we have we have M−−−(123) = − √ 2πδ(P i λi)δ(¯z12)δ(¯z13) z−∆3 21 z∆2+∆3−2 23 z1−∆2 31 |z13||z23| = √ 2πδ(P i λi)sgn(z12z23z31)δ(¯z12)δ(¯z13) |z12|−∆3|z23|2−∆1|z13|2−∆2 . (3.25) (3.25) This result matches with [23] up to some normalizations and conventions. 3.2 Example: 3-pt gluon celestial amplitude we consider the Klein space here, the support of the delta functions can be written as When we take the massless limit, we must keep in mind that ω3 = m 2y. We can rewrite the current so that there is no dependence on y. If we choose zij to be non-vanishing real numbers and also independent of ¯zij, i.e. we consider the Klein space here, the support of the delta functions can be written as ω3 = ω1 z21 z23 ¯z12 = m2z23 4ω2 1z12z31 ¯z13 = −m2¯z23 4ω2 1|z12|2 , (3.18) (3.18) where we set w = z3. Then we can rewrite the delta functions as follows: δ y − 2mω1|z12|2 m2 + 4ω2 1|z12|2 ! δ(2) w −m2z2 + 4ω2 1z1|z12|2 m2 + 4ω2 1|z12|2 ! = m 2y2 z12¯z12 |z13||z23|δ  ω3 −ω1 z21 z23  δ ¯z12 − m2z23 4ω2 1z12z31 ! δ ¯z13 + m2¯z23 4ω2 1|z12|2 ! , (3.19) (3.19) where |zij| is the absolute value of a real variable. We need to stop and make some comments about these delta functions here. There are some extra constraints on zij. For example, since m, ω1, y are always positive, the sign of z21, z23, z31 must be the same. Moreover, we have integrated a delta function δ(ω2 − m2 4ω1|z12|2 ), which requires that ω2 = m2 4ω1|z12|2 = z31 z23 ω1. (3.20) (3.20) 3It is worth mentioning that although changing the directions of particles will not change the wavefunc- tions, it will change the constraints of zij as we will see later and finally affect the result. 3It is worth mentioning that although changing the directions of particles will not change the wavefunc- tions, it will change the constraints of zij as we will see later and finally affect the result. – 8 – For other cases, say particle 3 outgoing, things are similar: we can always obtain the relations among ωi. Then the condition For other cases, say particle 3 outgoing, things are similar: we can always obtain the relations among ωi. Then the condition 0 ⩽ ωi ω1 + ω2 + ω3 ⩽1 (3.21) (3.21) in fact constrains the value of zij. Only when all these conditions are satisfied, the celestial amplitude does not vanish. Let us continue. The 2-pt celestial BG current now can be written as Let us continue. 3.3 Soft region The delta function δ(4)(q1 + q2 −p3) has another support ω1 = 0 q2 = p3. (3.26) (3.26) This corresponds to the soft region, with ∆1 forced to be 1 [28]. However, the wave function of a soft gluon is not equivalent to the Mellin transformation on plane waves. Therefore, We need to rewrite the celestial BG currents and consider the soft region carefully. In this subsection, we will consider the Minkowski space. For the incoming soft gluon, i.e. particle 1, the 1-pt celestial BG current is Asoft 1µs = Z d4XAlog,− µs (ˆq1; X)eip·X (3.27) (3.27) – 9 – – 9 – while for particle 2, the 1-pt celestial BG current is still while for particle 2, the 1-pt celestial BG current is still while for particle 2, the 1-pt celestial BG current is still while for particle 2, the 1-pt celestial BG current is still A2µs = Z dω2ω∆2−1 2 ϵ2µδ(ω2q2 −p) = 1 4ϵ2µ m∆2−4 (2y)∆2−3 δ(y)δ(2)(w −z2). (3.28) (3.28) We can rewrite this current through a more convenient formalism (up to an overall con- stant): A2µ = Z d4XA∆2,− µs (ˆq2; X)eip·X (3.29) (3.29) Here we only show the concrete calculation of the first term of p2 3A12µ: JHEP09(2023)193 −p2 3A12µ ⊃ Z d4Jd4Kδ(4)(−J −K+p3) Z d4Y A∆2,− s2 (ˆq2;Y )eiK·Y · Z d4XAlog,− s1 (ˆq1;X)eiJ·Xq1µ = Z d4Jd4Kd4Lei(−J−K+p3)·L Z d4Y A∆2,− s2 (ˆq2;Y )eiK·Y · Z d4XAlog,− s1 (ˆq1;X)eiJ·Xq1µ =i Z d4Lδ(4)(L−X)δ(4)(L−Y )eip3·L Z d4Y A∆2,− s2 (ˆq2;Y )· Z d4X∂µAlog,− s1 (ˆq1;X) =i Z d4Leip3·LA∆2,− s2 (ˆq2;L)·∂µAlog,− s1 (ˆq1;L) (3 30) (3.30) (3.30) After some calculations, we can obtain the following result: ( ) After some calculations, we can obtain the following result: −p2 3A12µ = i Z d4X[(∂µAlog,−(ˆq1; X)) · A∆2,−(ˆq2; X)) −(Alog,−(ˆq1; X)) · ∂µA∆2,−(ˆq2; X)) + 2(∂νA∆2,− µ )(ˆq2; X)Alog,− ν (ˆq1; X) −2(∂νAlog,− µ (ˆq1; X))A∆2,− ν (ˆq2; X)]eip·X, (3.31) (3.31) ( ) where we have omitted the label si to simplify the notation. Integrate this integral by part, we have ( ) where we have omitted the label si to simplify the notation. Integrate this integral by part, we have −p2 3A12µ = i Z d4X[(∂µV log,−(ˆq1; X)) · A∆2,−(ˆq2; X)) −(V log,−(ˆq1; X)) · ∂µA∆2,−(ˆq2; X)) + (∂νA∆2,− µ )(ˆq2; X)V log,− ν (ˆq1; X) −ipνA∆2,− µ V log,− ν −2(∂νV log,− µ (ˆq1; X))A∆2,− ν (ˆq2; X) + ipµA∆2,− ν ∂ναlog,−−p2A∆2,− µ αlog,−]eip·X. 3.3 Soft region (3 32) (3.32) (3.32) After some calculation, we find that the sum of the terms (3 3 ) After some calculation, we find that the sum of the terms ( After some calculation, we find that the sum of the terms ∂µV log,−· A∆2,−−V log,−· ∂µA∆2,−= 2ϵ1 · X X2(−ˆq1 · X)A∆2,− µ + 2ϵ1 · X X2(−ˆq1 · X)A∆2,− µ = 4ϵ1 · X X2(−ˆq1 · X)A∆2,− µ (3.33) (3.33) ( q1 ) and and −2∂νV log,− µ A∆2,− ν =− 4ϵ1 · X X2(−ˆq1 · X)A∆2,− µ − 4Xµ X2(−ˆq1 · X)ϵ1 · A∆2,−−4Xµϵ1 · X X2(−ˆq1 · X) ˆq1 · A∆2,− (3.34) −2∂νV log,− µ A∆2,− ν =− 4ϵ1 · X X2(−ˆq1 · X)A∆2,− µ − 4Xµ X2(−ˆq1 · X)ϵ1 · A∆2,−−4Xµϵ1 · X X2(−ˆq1 · X) ˆq1 · A∆2,− (3.34) – 10 – will vanish after we consider the on-shell wave function of particle 3. If fact, all the following terms will vanish after we take the on-shell limit ipµA∆2,− ν ∂ναlog,−−p2A∆2,− µ αlog,−− 4Xµ X2(−ˆq1 · X)ϵ1 · A∆2,−− 4Xµϵ1 · X X2(−ˆq1 · X) ˆq1 · A∆2,−, (3.35) since if we take particle 3 on shell we have p2 = 0 X · ˜A∆3 = 0 and ∂µ ˜A∆3 = 0 for the ipµA∆2,− ν ∂ναlog,−−p2A∆2,− µ αlog,−− 4Xµ X2(−ˆq1 · X)ϵ1 · A∆2,−− 4Xµϵ1 · X X2(−ˆq1 · X) ˆq1 · A∆2,− (3 3 (3.35) since if we take particle 3 on-shell, we have p2 = 0, Xµ · ˜A∆3 = 0 and ∂µ ˜A∆3 µ = 0 for the on-shell wave function of particle 3. If we ignore these terms, we will get p2 3A12µ = −i Z d4X[(∂νA∆2,− µ )V log,− ν −ipνA∆2,− µ V log,− ν ]eip·X (3.36) (3.36) JHEP09(2023)193 and the celestial amplitude is given by M = lim p2 3→0 Z dp3 p2 3A12 · ˜A∆3,+(ˆp3; p3) (3.37) (3.37) where ˜A∆,±(z, ¯z; p) is defined as R dp ˜A∆,±(z, ¯z; p)e±ip·X = ˜A∆,±(z, ¯z; X). This result matches with [28] up to some normalizations after we contract the celestial BG current with the on-shell wave function of particle 3. Assigning the helicity of 3 particles (+ −+), we write down the final result directly: This result matches with [28] up to some normalizations after we contract the celestial BG current with the on-shell wave function of particle 3. 3.3 Soft region Assigning the helicity of 3 particles (+ −+), we write down the final result directly: M1+ soft2−→3+ ∝δ(∆2 −∆3) 1 z∆2−1 23 ¯z∆2+1 23  1 z12 + 1 z31  . (3.38) (3.38) 3.4 The OPE limit of celestial BG currents 3.4 The OPE limit of celestial BG currents In celestial BG currents, we can do the OPE for the legs on the celestial sphere. In this subsection, we will focus on the holomorphic collinear limit z1 →z2. Consider the 2-pt celestial BG current (3.15), we set the helicity to be (++) in order to obtain a singularity of z12: p2 3A12µ = 2 √ 2 Z dω1ω∆1−1 1 m2 4|z12|2ω1 !∆2−1 1 m2 1 ω1z12 y3 " ϵ2µ m2 4ω1|z12|2 + ϵ1µω1 # × δ y − 2mω1|z12|2 m2 + 4ω2 1|z12|2 ! δ(2) w −m2z2 + 4ω2 1z1|z12|2 m2 + 4ω2 1|z12|2 ! (3.39) (3.39) We can solve ω1 from the delta function δ(y − 2mω1|z12|2 m2+4ω2 1|z12|2 ): We can solve ω1 from the delta function δ(y − 2mω1|z12|2 m2+4ω2 1|z12|2 ): ω1 = ω3 2 ± 1 2 s ω2 3 − m2 |z12|2 = ω± > 0 (3.40) (3.40) Then we have Then we have p2 3A12µ = 2ω∆1−1 + (ω−)∆2−1 1 ω3 ω+ 4|ω+ −ω−| 1 ω+z12 [ϵ2µω−+ ϵ1µω+] × δ(2) w −m2z2 + 4ω2 +z1|z12|2 m2 + 4ω2 +|z12|2 ! + (+ ↔−) (3.41) (3.41) – 11 – – 11 – Now we take the holomorphic collinear limit z1 →z2: p2 3A12µ ∼ √ 2ω∆1−1 + ω∆2−1 − 1 2|ω+ −ω−| 1 z12 ϵ2µδ(2)(w −z2) + (+ ↔−) (3.42) Now we take the holomorphic collinear limit z1 →z2: 2A √ ∆1 1 ∆2 1 1 1 δ(2)( ) ( ) ( ) Now we take the holomorphic collinear limit z1 →z2: Now we take the holomorphic collinear limit z1 →z2: p2 3A12µ ∼ √ 2ω∆1−1 + ω∆2−1 − 1 2|ω+ −ω−| 1 z12 ϵ2µδ(2)(w −z2) + (+ ↔−) (3.42) (3.42) Let w− w+ = t, then A12µ ∼−t∆1−1 + t∆2−1 √ 2|1 −t| ω∆1+∆2−3 + m2 1 z12 ϵ2µδ(2)(w −z2) (3.43) (3.43) where we have used p2 3 = −m2. The above calculation implies that the OPE behavior of celestial BG currents is full of subtleties. However, we can still extract some useful information from it. Recall that the 1-pt celestial BG current for particle 2 is given by JHEP09(2023)193 A2µ = 1 4ϵ2µ m∆2−4 (2y)∆2−3 δ(y)δ(2)(w −z2) = 1 2ϵ2µ ω∆2−2 m2 yδ(y)δ(2)(w −z2) (3.44) (3.44) where we set w = m 2y. Note that yδ(y) always vanishes. However, in the 1-pt celestial BG current, the momentum conservation implies ω2 = ω. 3.4 The OPE limit of celestial BG currents If we also impose this condition for the 2-pt celestial BG current, i.e. we impose ω+ = ω3 for (3.43), this condition will correspond to t = 0 and also brings a vanishing factor. This fact tells us that we will find the 1-pt celestial BG current with conformal dimension ∆1 + ∆2 −1 embedded in the 2-pt celestial BG currents after taking z12 →0. This feature from the OPE limit is the same as [34], where they considered the OPE of two gluon operators. 4 From tree to loop For BG currents, there exists a technique called the “sewing procedure” [35]. Using this technique, one can obtain 1-loop integrands from BG currents. In this section, we will gen- eralize this technique to celestial BG currents, and summarize how to get 1-loop integrands from celestial BG currents directly. In this section, we still use k = ϵωq to denote the mo- mentum vector in BG recursion. To make things easier, in this section we will consider the Klein space, where for all on-shell particles we can use the Mellin transformation. In order not to lose the off-shell information, we will use the Feynman gauge in this section. Let us start with the BG recursion (2.14). Now we replace the word P with lP and the word P in lP is still a permutation of {1, 2, · · · , n). In other words, we consider the (n+1)-pt BG current with the first on-shell leg labeled by l. The leg l will be taken to be off-shell later so that we can “sew” it with the original off-shell leg which is labeled by n+1. k2 lP AlPµ = (klµ −kPµ)(Al · AP ) −Alµ(kl · AP ) −Alµ(klP · AP ) + APµ(kP · Al) + APµ(klP · Al) + X P=XY [2AXµ(Al · AY ) −AY µ(Al · AX) −Alµ(AX · AY )] + X P=XY (klXµ −kY µ)(AlX · AY ) −AlXµ(klX · AY ) −AlXµ(klP · AY ) + AY µ(kY · AlX) + AY µ(klP · AlX) + X P=XY Z [2AY µ(AlX · AZ) −AZµ(AlX · AY ) −AlXµ(AZ · AY )]. (4 1) (4.1) – 12 – Now we take the leg l to be off-shell. This operation does nothing on the multi-point BG currents but only tells us that k2 l ̸= 0. The first two lines of (4.1) will correspond to the tadpole terms after the sewing procedure and will vanish finally after integrating the loop momentum. Therefore, we can ignore such terms. Note that for a subset X of P (X ̸= P), we cannot do the same thing for the recursion of AlX since this case does not appear tadpole terms. 4 From tree to loop The remaining terms are k2 lP AlPµ = X P=XY (klXµ −kY µ)(AlX · AY ) −AlXµ(klX · AY ) −AlXµ(klP · AY ) + AY µ(kY · AlX) + AY µ(klP · AlX) + X P=XY Z [2AY µ(AlX · AZ) −AZµ(AlX · AY ) −AlXµ(AZ · AY )]. (4.2) (4.2) JHEP09(2023)193 et AlPµ = Aν l JPµν, then Now we set AlPµ = Aν l JPµν, then Now we set AlPµ = Aν l JPµν, then Now we set AlPµ = Aν l JPµν, then Now we set AlPµ = Aν l JPµν, then k2 lP Aν l JPµν = X P=XY (klXµ −kY µ)(Aν l JXµνAµ Y ) −Aν l JXµν(klX · AY ) + AY µ(kµ Y Aν l JXµν) −Aν l JXµν(klP · AY ) + AY µ(kµ lP Aν l JXµν) + X P=XY Z [2AY µ(Aν l JXµνAµ Z) −AZµ(Aν l JXµνAµ Y ) −Aν l JXµν(AZ · AY )]. (4 3) (4.3) Here JPµν is called the pre-integrand. After setting kl = −kn+1 = l, we have is called the pre-integrand. After setting kl = −kn+1 = l, we have ηµνJPµν = 1 k2 l X P=XY (kν lX −kν Y )(JXµνAµ Y ) −Jµ Xµ(klX · AY ) + Aν Y (kµ Y JXµν) −Jµ Xµ(kl · AY ) + Aν Y (kµ l JXµν) + X P=XY Z [2Aν Y (JXµνAµ Z) −Aν Z(JXµνAµ Y ) −Jµ Xµ(AZ · AY )]. (4 4) (4.4) ( ) Then we have “sewed” the legs l and n + 1. One may find that this operation is equivalent to replace ϵlµϵn+1,ν with ηµν, which is just the Feynman rules. From δ(4)(kX + kY ) = Z dJ4dK4δ(4)(kX −l −J)δ(4)(kY −K)δ(4)(J + K + l), the celestial loop integrand can be written as I1−loop P = 1 l2 X P=[XY ] Z d4Jd4Kδ(4)(J +K+l)×  −2J µ Xµl·AY +lνJXµνAµ Y +lµJXµνAν Y + X i∈X Aµ Y J i Xµνϵiˆqν i − X j∈Y JXµνAjµ Y ϵj ˆqν j + X k∈Y JXµνϵkˆqkµAkν Y − X m∈X AY ·ϵmˆqmJ mµ Xµ  + 1 l2 X P=[XY Z] Z d4Jd4Kd4Lδ(4)(J +K+L+l) ×  2Aν Y (JXµνAµ Z)−Aν Z(JXµνAµ Y )−J µ Xµ(AZ ·AY )  ( ) (4.5) – 13 – Figure 1. The repeated diagram. Figure 1. The repeated diagram. Conclusions and outlooks 5 In this paper, we have shown how to construct celestial BG currents and how to use celestial BG currents to calculate celestial amplitudes. Moreover, we can also calculate the contribution of the soft region by changing the 1-pt celestial BG current. We also explore the OPE behavior of the 2-pt celestial BG current. Finally, we have discussed the “sewing procedure”, a technique that allows us to obtain 1-loop integrands from BG currents. We have generalized this prescription to the celestial case. Celestial BG currents have many advantages. First, we can obtain celestial BG currents by recursion and can obtain celestial amplitudes easily from them, which means that we can calculate celestial amplitudes recursively. Moreover, compared with the BCFW recursion, BG recursion does not need to deal with the possible boundary terms. Secondly, celestial BG currents include one leg off-shell which corresponds to some delta functions involving an effective mass of the off-shell leg. This gives a new way to deal with the delta functions. Finally, since BG currents are linked closely to the Feynman rules, which is the most fundamental thing of amplitudes, celestial BG currents will be helpful in studying the structure of celestial amplitudes from the amplitude perspective. JHEP09(2023)193 However, there are also some problems to solve. The greatest obstacle to evaluating BG currents is the delta function coming from the momentum conservation. At least we need to find out a way to deal with the delta function of 3 massive momenta. As for the gluon case, we also need to know how to deal with the delta function of 4 massive momenta. Another interesting question is can we find an algebra for celestial BG currents just like the case in [12] for BG currents, pretending that we know nothing about the flat case and just starting with the celestial side? This may deepen our understanding of the celestial holography. We leave these problems here and hope that some progress can be made. Acknowledgments I would like to thank Chi-Ming Chang and Wen-Jie Ma for valuable discussions which help me a lot. YT is partly supported by National Key R&D Program of China (NO. 2020YFA0713000). 4 From tree to loop where JPµν = Z Y i∈P dωiω∆i−1 i JPµνδ(4) X i∈P ki −pn+1 −l ! (4.6) (4.6) JHEP09(2023)193 As before, Ajµ Y means Aµ Y with ∆j →∆j + 1. Here [· · · ] means all inequivalent deconcate- nations of cyclic permutations in P. In P = [XY ] and P = 1234, for example, (1, 234) and (234, 1) are equivalent. As for P = [XY Z], all deconcatenations of cyclic permutations in P are inequivalent. However, in this case, different elements in P = [XY Z] may give the same Feynman diagrams even though they are inequivalent since one deconcatenation cor- responds to many Feynman diagrams. For example, say P = 1234, (12, 3, 4) and (34, 1, 2) both give the Feynman diagram in figure 1. When we use this prescription to calculate 1-loop integrands, we must avoid double-counting some Feynman diagrams. From the derivation above, we can summarize how to obtain the celestial loop inte- grands from the celestial BG currents directly. 1. Consider the inverse of Mellin transformation: 1. Consider the inverse of Mellin transformation: 1. Consider the inverse of Mellin transformation: f(w) = Z c+i∞ c−i∞ d∆w−∆˜f(∆). (4.7) (4.7) Then consider AlP and do the inverse Mellin transformation on the leg l: ˜ AlP = Z c+i∞ c−i∞ d∆lω−∆ l AlP (4.8) (4.8) 2. let ˜ AlPµ = ϵν l Jµν, then set the loop momentum l = kl = −kn. 3. consider I1−loop P = ηµνJµν and the modification of the deconcatenation sum P = XY →P = [XY ], this is the celestial loop integrand in the Klein space. 3. consider I1−loop P = ηµνJµν and the modification of the deconcatenation sum P = XY →P = [XY ], this is the celestial loop integrand in the Klein space. 3. consider I1−loop P = ηµνJµν and the modification of the deconcatenation sum P = XY →P = [XY ], this is the celestial loop integrand in the Klein space. We have not considered the ghost loop here. However, things are similar for the ghost loop, the only subtlety is that if our external leg is a ghost, it can only be an off-shell leg. The total 1-loop integrand should be written as I = Igluon −Ighost. (4.9) (4.9) For more about the ghost loop from the sewing procedure, see [35]. – 14 – A 3D BCFW for celestial amplitudes In this appendix, we want to show the 3d generalization of the BCFW recursion to deepen our understanding of the celestial recursion. We will follow some of the notations in [25]. Here we only consider massless scalars and do not consider the shadow basis for simplicity, as in [36]. In the 3D case, the momentum can be written as the following formalism [37]: pµ = ϵω(1 + x2, 2x, 1 −x2) (A.1) (A.1) – 15 – where ϵ is used to label incoming or outgoing. In 3D spinor-helicity formalism, there is no square bracket, which means we have pµ ab = ⟨p|a⟨p|b. We use λa to denote ⟨p|a: where ϵ is used to label incoming or outgoing. In 3D spinor-helicity formalism, there is no square bracket, which means we have pµ ab = ⟨p|a⟨p|b. We use λa to denote ⟨p|a: λa = √ −2ϵω x −1 ! λa = εabλb = − √ −2ϵω 1 x ! (A.2) (A.2) where εab = 0 1 −1 0 ! . By chain rules and ω = −1 2ϵ(λ1)2, x = λ2 λ1 , we also have JHEP09(2023)193 ∂ ∂λ1 = 1 √−2ϵω  x ∂ ∂x −2ω ∂ ∂ω  ∂ ∂λ2 = − 1 √−2ϵω ∂ ∂x (A.3) (A.3) In the celestial case, these operators, which act directly on amplitudes, need to be trans- lated: ∂ ω ∂ ∂ω →−∆ ω →T (A.4) (A.4) erator can add 1 to the ∆in celestial amplitudes. where T operator can add 1 to the ∆in celestial amplitudes. where T operator can add 1 to the ∆in celestial amplitudes. In 3d BCFW, the method of the complex shift is not the same as 4D, since the con- straints (momentum-conservation and on-shell condition) are so strong for 3D that the shift part must be zero. To fix this we do the nonlinear complex shift [6, 38]. The corresponding operator with i, jth particles shifted for flat amplitudes is Di,j = exp " z+z−1 2 −1 ! λi ∂ ∂λi −z−z−1 2i λj ∂ ∂λi + z+z−1 2 −1 ! λj ∂ ∂λj + z−z−1 2i λi ∂ ∂λj # (A.5) j # (A.5) and Di,jM = M(z). Here we use M to denote amplitudes and xij = xi−xj. After translat- ing to the celestial case, we have Di,j = exp h (2−z−z−1)(∆i+∆j) i exp " z−z−1 2i Ωi,j xij ∂ ∂xj −2∆j ! A 3D BCFW for celestial amplitudes −Ωj,i  xji ∂ ∂xi −2∆i !# (A 6) q ϵi ϵj T 1/2 i T −1/2 j . where Ωi,j = q ϵi ϵj T 1/2 i T −1/2 j . where Ωi,j = q ϵi ϵj T 1/2 i T −1/2 j . q j j For 3D BCFW, we have (without loss of generality, we choose i, j = 1, n) Mn = 1 2πi I z=1 D1,nAn z −1 = X i " ML(z1.i)H(z1,i, z2,i) P 2 12···i M(z1,i) + ML(−z1.i)H(−z1,i, z2,i) P 2 12···i M(−z1,i) + (z1,i ↔z2,i) # (A 7) # (A.7) ( ) where P 2 12···i are propogators, H(a, b) = a(a+1)(b2−1) 2(a2−b2) , and ±z1,i, ±z2,i are the roots of ˆP 2 12···i = 0, the explicit expressions can be found in [38] and [6]. Note that for a given i, L includes 1, · · · , i and R includes i + 1, · · · , n. ( ) where P 2 12···i are propogators, H(a, b) = a(a+1)(b2−1) 2(a2−b2) , and ±z1,i, ±z2,i are the roots of ˆP 2 12···i = 0, the explicit expressions can be found in [38] and [6]. Note that for a given i, L includes 1, · · · , i and R includes i + 1, · · · , n. – 16 – Recall the definition of celestial amplitudes for massless scalars: Mn(∆i, xi) = Y i Z ∞ 0 dωiω∆i−1 i ! Mn(ωi, xi)δ(4) X i pi(xi) ! . (A.8)f (A.8) Since Di,j has no effect on the delta function (it holds the momentum conservation), we have the following recursion relation for celestial amplitudes: Mn = 1 2πi I z=1 D1,nMn z −1 = Z d4P X i H(z1,i, z2,i) P 2 12···i D1,n(z1,i)[AL(P)AR(−P)] + H(−z1,i, z2,i) P 2 12···i D1,n(−z1,i)[AL(P)AR(−P)] + (z1,i ↔z2,i) (A.9) (A.9) JHEP09(2023)193 where A(P) denotes the BG current (multiplied with P 2) with the momentum of the outgoing off-shell leg with momentum P. Note that P can be an off-shell momentum, while D1,nP must be an on-shell complex momentum. This is indeed the BCFW prescription. However, this is not a recursion that constructs a celestial amplitude from lower points celestial amplitudes but lower point BG currents. To restore this, one needs to use some tricks. An example is [23] for 4-pt MHV amplitudes. A 3D BCFW for celestial amplitudes In the general case, since the operator D1,n has no effect on the magnitude ωP of the momentum P we can consider the following formalism: Z ∞ ˆ M(P) = D1,nM(P) = Z ∞ 0 dωP ω∆P −1 P D1,nA(P) →D1,nA(P) = Z c+i∞ c−i∞ d∆P ω−∆P P ˆ M(P) (A.10)i (A.10) Note that we also need to figure out the shifted coordinate xP to obtain ˆ M(P). After substituting this, we will obtain a recursion from lower point celestial amplitudes. Note that we also need to figure out the shifted coordinate xP to obtain ˆ M(P). After substituting this, we will obtain a recursion from lower point celestial amplitudes. B Wrong conformal weights from integrating the celestial BG current An interesting trial is taking the off-shell particle to the celestial sphere. However, this pre- scription must be wrong because such a thing is not gauge-independent. Despite this, We can still make a calculation and see how this wrong thing manifests itself in the conformal behavior of the “celestial amplitude”.f Now we consider the 2-pt gluon BG current case. We regard the off-shell gluon as an on-shell massive spin-1 particle. In order to impose the transverse condition, we choose the Lorenz gauge. Now we define the celestial “amplitude” for this 2-pt BG currents: ˜ M = Z dydwd ¯w y3  y y2 + |z3 −w3|2 ∆3+1 G(1) Jb p2 3A12 · ϵb 3 (B.1) (B.1) G(1) Jb =      √ 2(z−w)2 y −2(z −w) − √ 2y √ 2(z −w) |z−w|2 y √ 2(¯z −¯w) √ 2y 2(¯z −¯w) − √ 2(¯z−¯w)2 y .      (B.2) (B.2) – 17 – We use the tilde to remind us that (B.1) is not the real celestial amplitude but an experi- mental object. Since we have imposed the Lorenz gauge here, the polarization vectors for off-shell gluons are the same as on-shell massive spin-1 particles. ϵ− µ = 1 √ 2(w, 1, i, −w) ϵ0 µ = −1 2y(1 −y2 + w ¯w, w + ¯w, −i(w −¯w), 1 + y2 −w ¯w) ϵ+ µ = 1 √ 2( ¯w, 1, −i, −¯w) (B.3) (B.3) JHEP09(2023)193 JHEP09(2023)193 We take the helicity of both particle 1 and particle 2 “−”. If we take J = −1, then GJbϵb µ = √ 2yϵ+ µ + 2(¯z −¯w)ϵ0 µ − √ 2(¯z−¯w)2 y ϵ− µ . Af l l h fi l l We take the helicity of both particle 1 and particle 2 “−”. If we take J = −1, then GJbϵb µ = √ 2yϵ+ µ + 2(¯z −¯w)ϵ0 µ − √ 2(¯z−¯w)2 y ϵ− µ .i µ µ µ y µ After some calculation, the final result is µ µ µ y µ After some calculation, the final result is ˜ M = 2−∆1−∆2+2m∆1+∆2−3¯z23 |z12|∆1+∆2−∆3−1|z23|−∆1+∆2+∆3+1|z13|∆1−∆2+∆3+1 B ∆1 −∆2 + ∆3 + 1 2 , −∆1 + ∆2 + ∆3 + 1 2  + 2−∆1−∆2+2m∆1+∆2−3z12¯z2 23 |z12|∆1+∆2−∆3+1|z23|−∆1+∆2+∆3+3|z13|∆1−∆2+∆3−1 B ∆1 −∆2 + ∆3 −1 2 , −∆1 + ∆2 + ∆3 + 3 2  . B Wrong conformal weights from integrating the celestial BG current (B.4) (B.4) This is an unphysical result since these two terms have different conformal dimensions. Another example is more interesting. For a 3-pt celestial “amplitude” with 1 massless scalar (particle 1), 1 gluon (particle 2), and 1 off-shell massless scalar (particle 3), we have the result: A3 = m∆1+∆2−5|z23|∆12−∆3+1¯z12 2∆1+∆2−3/2|z12|∆1+∆23+1|z13|∆1−∆23+1 B ∆12 + ∆3 + 1 2 , −∆12 + ∆3 −1 2  (B 5 (B.5) (B.5) ( ) It has wrong weights (∆1+1 2 , ∆1 2 ), (∆2 2 , ∆2−1 2 ), (∆3 2 , ∆3 2 ). However, if we want this “ampli- tude” to behave like a CFT correlator with correct weights, we only need to take the holomorphic collinear limit z1 →z2 so that z13 = z23. In this case, from the momentum conservation, particle 3 is forced to be massless and the gauge invariant is preserved. Open Access. 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Short-term treatment with taurolidine is associated with liver injury

BMC pharmacology & toxicology

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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 DOI 10.1186/s40360-017-0168-z Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 DOI 10.1186/s40360-017-0168-z Open Access * Correspondence: [email protected] 1Department of General, Visceral and Vascular Surgery, University Hospital Jena, 07747 Jena, Germany 2Center for Sepsis Control and Care (CSCC), University Hospital Jena, 07747 Jena, Germany Full list of author information is available at the end of the article Abstract Background: Taurolidine has been used for peritonitis, oncological and catheter-lock treatment because of its anti-inflammatory properties. It has been suggested that taurolidine has no severe side-effects, but after long-term use morphological and functional changes of the liver were reported. The aim of this study was to investigate the effect of short-term use of taurolidine on the liver. Methods: In HepaRG cell cultures and on a novel liver biochip dose-dependent effects of taurolidine treatment on hepatocyte adherence and cell viability was investigated. Furthermore, liver enzymes and interleukin- (IL-) 6 were measured in supernatants. Male rats were treated with low- or high-dose taurolidine, respectively, and compared to controls with physiological saline solution administration regarding blood serum parameters and histology. Results: In HepaRG cell cultures, hepatocyte adherence was significantly decreased, cell death and cleaved caspase-3 were significantly increased after administration of taurolidine in a dose-dependent manner. High-dose application of taurolidine led to elevated liver enzymes and IL-6 secretion in hepatic organoid. After 24 h a significant increase of serum GLDH and ASAT was observed in rats treated with high-dose taurolidine treatment. Conclusions: Our results suggest that taurolidine caused liver injury after short-term use in in vitro and in vivo models probably due to direct toxic effects on hepatocytes. Therefore, the taurolidine dose should be titrated in further investigations regarding liver injury and inflammation. Keywords: Cytokines, Liver injury, Liver biochip, Taurolidine, Liver enzymes Short-term treatment with taurolidine is associated with liver injury René Fahrner1,2*, Anika Möller1, Adrian T. Press2,3, Andreas Kortgen2,3, Michael Kiehntopf2,4, Falk Rauchfuss1, Utz Settmacher1 and Alexander S. Mosig2 René Fahrner1,2*, Anika Möller1, Adrian T. Press2,3, Andreas Kortgen2,3, Michael Kiehntopf2,4, Falk Rauchfuss1, Utz Settmacher1 and Alexander S. Mosig2 Background inflammatory experimental and clinical studies as prophy- laxis against catheter associated infections [14–17]. Add- itionally, taurolidine was investigated in experimental oncology and showed promising results in tumor therapy [18–27]. Several mechanisms were discussed, such as direct apoptosis of the tumor cells [5] or indirectly via inhibition of NF-kappaB and consecutive inhibition of anti-apoptotic regulator leading to cell death [21]. Taurolidine was first introduced decades ago as a broad range antibiotic drug for the treatment of peritonitis, sepsis and pleural empyema [1–4]. Taurolidine is a chemical dimeric molecule of two taurinamide rings [5] which is stable with a half-life time of approximately eight hours [6]. The main anti-inflammatory mechanism results in a reduction of pro-inflammatory cytokine re- lease such as tumor necrosis factor (TNF), interleukin (IL-) 6, and IL-8 [7–13]. In addition, an inactivation of endotoxins was reported [3], as well as anti-microbial, anti-adherent, anti-inflammatory and anti-neoplastic effects [5]. Therefore, taurolidine was used in So far, taurolidine was reported to be a well-tolerated drug without relevant side effects in animal studies and in human local peritoneal and pleural application [11, 14, 15, 17, 28, 29]. However, one recent publica- tion reported that long-term high-dose treatment with taurolidine caused severe hepatic injury in a murine osteosarcoma model [30]. Therefore, the authors sug- gested caution before widespread use of taurolidine as an anti-cancer drug in clinical oncology [31]. Page 2 of 9 Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 containing 10% (v/v) FCS (Life Sciences, Darmstadt, Germany), 2 mM glutamine (GIBCO, Darmstadt, Germany), 5 μg/ml insulin (Sigma-Aldrich, Steinheim, Germany), 50 μM hydrocortisone-hemisuccinate (Sigma-Aldrich, Steinheim, Germany) and 100 U/ml penicillin/100 μg/ml streptomycin mixture (GIBCO). In a humidified cell incubator at 5% CO2 and 37 °C cells were cultured to grow for 14 days with renewal of the medium every three to four days. As described previously, cell differentiation was induced by addition of 2% DMSO for 14 days. Differentiated cells were used for up to four weeks [32]. Endothelial cells (HUVECs) were isolated from human umbilical cord veins as previously described [33]. Donors gave written consent after they were in- formed about the aim of the study. HUVEC cells were cultured in Endothelial Cell Medium (ECM) (Promo- cell, Heidelberg, Germany) up to passage four. Background Periph- eral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient centrifugation as previ- ously described [34] and seeded in X-VIVO 15 medium (Lonza, Cologne, Germany) with 10% (v/v) autologous human serum, 10 ng/ml human granulocyte macrophage colony-stimulating factor (GM-CSF) (PeproTech, Ham- burg, Germany) and penicillin (100 u/ml)/streptomycin (100 μg/ml). After three hours of incubation in a humidi- fied cell incubator at 5% CO2 and 37 °C the cells were washed with X-VIVO 15 medium. For 24 h adherent monocytes were cultivated in X-VIVO 15 medium and seeded into liver biochip. LX-2 stellate cells (kindly pro- vided by Scott L. Friedman, Division of Liver Diseases, Mount Sinai School of Medicine, New York City, NY, USA) were cultured in Dulbecco’s Minimum Essential Medium (DMEM) (Biochrom, Berlin, Germany) with sup- plementation of 10% (v/v) FCS, 1 mM sodium pyruvate (GIBCO) and penicillin/streptomycin. Because of the reported anti-inflammatory properties, taurolidine could be useful as a potential drug for acute liver injury and prevention of hepatic ischemia-reperfusion injury in the future. So far toxic effects of taurolidine on liver function and morphology during short-term treatment have not been investigated. Thus, the aim of this pilot study was to evaluate the effect of taurolidine on hepatocytes in vitro using a conventional cell culture setting as well as a novel human liver biochip model. In addition, the short- term effect in the liver after a systemic use of taurolidine was investigated in an experimental rat model. Immunohistochemical analysis of the liver Fresh liver tissue was fixed overnight in 4% paraformal- dehyde and embedded in paraffin. For hematoxylin/eosin (HE) staining, sections were deparaffinized with xylol and then counterstained with HE. The analysis of all histology slides was performed blinded by the same investigator. Methods All research protocols were carried out in accordance with the National Institutes of Health guidelines for the care and use of experimental animals and approval of the Animal Care Committee of Thuringia, Germany (No. 22-2684-04-02-061/14). Animal preparation and experimental setting p p p g Experiments were performed using male Lewis rats kept at the Central Animal Facility of the Friedrich Schiller University Jena, Germany and were purchased from Charles River, Sulzfeld, Germany. The animals were kept with a 12 h light/dark cycle at 22 °C. All procedures were performed in animals aged 8–10 weeks with a mean weight of 286 ± 28 g. Taurolidine was obtained as a ready-to-use solution with a concentration of 2% (Geistlich Pharma AG, Switzerland). The animals were divided into three groups of six animals each: low dose taurolidine (140 mg/kg body weight), high dose taurolidine (290 mg/kg body weight) and controls with equivalent volume of physiologic saline solution administered in- traperitoneally. The harvesting of blood and tissue was performed 24 h after the treatment under general anesthesia with isoflurane inhalation. During general anesthesia laparotomy was performed and a lethal amount of blood was taken from the inferior vena cava. Immediately after removal of blood the liver was excised and processed. WST-assay WST WST-assay (Roche Diagnostics GmbH) was used to as- sess cell viability and toxic effects of taurolidine 24 h after HepaRG treatment. 3 × 105/cm2 HepaRG cells were cultured in complemented William’s Medium E with or without taurolidine with pre-determined concen- trations for 24 h at 5% CO2 and 37 °C. Subsequently WST-assay dye was added. Briefly, the stable tetrazolium salt WST-1 is cleaved to formazan, a soluble metabolite, by a complex bioreductive mechanism in viable cells. The amount of formazan dye reflects the number of metabolically viable cells. Therefore, the cell medium and WST dye were prepared according to the manufac- turer’s instructions in a 96-well tissue culture plate. Cells were incubated 30 min at 37 °C and 5% CO2 with the WST-reagent. Absorbance was measured at a wave- length of 450 nm with a reference wavelength of 650 nm Biochips MOTiF biochips, a cyclic olefin copolymer (COC), were made from Topas and obtained from the microfluidic ChipShop GmbH (Jena, Germany). The biochips were manufactured as previously described [35]. Briefly, chips were made by injection molding with integration of a 12 μm thick PET membrane with a pore diameter of 8 μm and a density of 2 × 105 pores/cm2 (TRAKETCH Sabeu, Radeberg, Germany). Sealed top and bottom sur- faces of the chips and channel structures were obtained by an application of an extruded 140 μm thick COC film using a low-temperature proprietary bonding method. For perfusion and oxygen equilibration during experimentation gas permeable silicon tubing was used. To avoid unfavor- able flow conditions and trapping of stationary bubbles the ramping structures have been introduced into the chip bulk. Oxygen plasma treatment for hydrophilization of the whole chip surface reduced bubble formation in the chips. Before use, the perfusion medium was stirred and equili- brated overnight under perfusion conditions. After seeding the cells into the upper and lower biochip chamber, tauro- lidine was added for stimulation at different doses. After 24 h, supernatants were collected for further analysis. Taurolidine caused disruption of hepatic cell adherence and cell death in vitro HepaRG cells were treated with increasing doses of taur- olidine in cell cultures. Cell adherence and cell morph- ology were altered by increasing doses of taurolidine (data not shown). Treatment with taurolidine for 24 h affected HepaRG cell viability with a LC50 of 125 μg/ml (Fig. 1a and Additional file 1: Figure S1). Treatment of hepatocytes with taurolidine without previous surface adherence even showed cell death at a concentration of 100 μg/ml (Fig. 1b), reflecting a higher toxicity on cells without adherence and without direct cell-to-cell con- tact. Further, the release of liver enzymes in supernatants of cell cultures such as ASAT and GLDH significantly correlated with increasing taurolidine concentrations (Fig. 1c and d). In addition, levels of cleaved caspase 3, a marker for apoptotic cells, were significantly elevated in hepatocytes after taurolidine application (Fig. 2a-e). Measurements of cytokines y Rat serum samples were analyzed for IL-6, IL-1ß, IL-10 and tumor necrosis factor (TNF) alpha levels by enzyme- linked immunosorbent assays using immunoassay kits (Quantikine, R&D System) according to the manufacturer’s protocol. Biochip supernatants were collected at pre- determined time points and immediately frozen at −80 °C. Cytokines (IL-1ß, IL-6, IL-10 and TNF-α) were measured using Cytometric Bead Array (CBA) assay (BD Biosciences) according to the manufacturer’s protocol using standard CBA flex sets. Cytokine analysis was performed on a BD FACS-Canto II cytometer with FACSDiva software with data analysis using FCAP Array V3 software (Softflow, Pecs, Hungary). Dose-dependent effect of taurolidine treatment in hepatic biochip On the basis of the results of HepaRG cell culture exper- iments with adherent hepatocytes, liver biochips were perfused with a concentration of 100 μg/ml taurolidine, a concentration below the toxic threshold dose in WST assays. Similar to the findings of cell culture experiments, low concentrations of taurolidine caused no significant hepatocyte damage as assessed by measuring liver en- zymes in biochip supernatants. A concentration of 1 mg/ml taurolidine however revealed significantly Statistics All data are expressed as arithmetic means ± standard deviations. For statistical analysis one-way ANOVA test was performed. The statistical analysis was performed with GraphPad Prism Version 5.0 (GraphPad Software, Inc., La Jolla, USA). Statistical differences with p < 0.05 were considered as statistically significant. Cleaved caspase-3 Liver cells were seeded on 13 mm glass coverslips (Menzel, Braunschweig, Germany) in 24 well plates. After washing twice with DPBS, cells were fixed using a 4% paraformalde- hyd solution. After washing with PBS and permeabilisation a 0.1% saponin solution was added for one hour to block the cells. After incubation with the primary antibody against cleaved caspase 3 (Cell Signaling Technology, Leiden, Netherlands) overnight in a humidity chamber in the fridge (at 8 °C), an AF488-flourescence-marked secondary goat anti rabbit antibody (Life Technologies, Karlsruhe, Germany), combined with DAPI (Life Tech- nologies) staining of the cell nucleus was added for 30 min. Microscopy was performed using an Axio Observer Z1 fluorescence microscope with ApoTome.2 equipment (Carl Zeiss AG, Jena, Germany). Finally, the mean fluorescent in- tensity of at least five samples of controls and high-dose taurolidine each were measured using a computer based program (ImageJ®). Cell culture HepaRG hepatocyte cells were obtained from Biopredic International (Rennes, France). Cells were initially seeded at a density of 2.7 × 104 cells/cm2 and cultured in comple- mented William’s Medium E (Biochrom, Berlin, Germany) Page 3 of 9 Page 3 of 9 Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Page 3 of 9 with an ELISA-Reader, reflecting the number of viable cells. Measurement of extracellular liver enzymes in serum and cell culture supernatants Levels of aspartate aminotransferase (ASAT) and glutam- ate dehydrogenase (GLDH) were measured in serum and cell culture supernatants using the Abbott Architect ci8200 Integrated System (Abbott Laboratories, Abbott Park, IL, USA) according to the manufacturer’s protocol. Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Page 4 of 9 Fig. 1 Cell survival of HepaRG cells with taurolidine treatment for 24 h was assessed by WST assay. Cell survival of seeded and adherent HepaRG cells was significantly decreased after treatment with taurolidine concentration of 500 μg/ml (a). HepaRG cells without previous adherence showed cell death at a concentration of 100 μg/ml (b). Liver enzymes in supernatants of cell cultures were analyzed and showed a significant elevation of ASAT and GLDH with increasing taurolidine concentrations (c, d). Representative data of three independent experiments are shown Fig. 1 Cell survival of HepaRG cells with taurolidine treatment for 24 h was assessed by WST assay. Cell survival of seeded and adherent HepaRG cells was significantly decreased after treatment with taurolidine concentration of 500 μg/ml (a). HepaRG cells without previous adherence showed cell death at a concentration of 100 μg/ml (b). Liver enzymes in supernatants of cell cultures were analyzed and showed a significant elevation of ASAT and GLDH with increasing taurolidine concentrations (c, d). Representative data of three independent experiments are shown Fig. 1 Cell survival of HepaRG cells with taurolidine treatment for 24 h was assessed by WST assay. Cell survival of seeded and adherent HepaRG cells was significantly decreased after treatment with taurolidine concentration of 500 μg/ml (a). HepaRG cells without previous adherence showed cell death at a concentration of 100 μg/ml (b). Liver enzymes in supernatants of cell cultures were analyzed and showed a significant elevation of ASAT and GLDH with increasing taurolidine concentrations (c, d). Representative data of three independent experiments are shown group and controls (Fig. 5a and b). Serum IL-6 levels 24 h after treatment showed no significant differences between the groups (Fig. 5c). elevated serum levels of GLDH and ASAT (Fig. 3a and b). IL-6 levels were not elevated after perfusion of the liver biochip with low taurolidine concentration but were sig- nificantly elevated with high taurolidine concentration (Fig. 3c). There were no statistically significant differences regarding TNF-α, IL-1ß or IL-10 serum levels in the con- trols compared to high dose taurolidine group (Fig. 3d). Discussion To the best of our knowledge, this study investigated for the first time the effect of systemic short-term use of taurolidine on liver function and morphology using in vitro models and an in vivo model in rats. In all investi- gated models the treatment with taurolidine caused liver injury at a specific toxic dose. During acute liver injury e.g. after liver resection [36, 37], ischemia-reperfusion injury [38], sepsis [39], and acute in- flammation [40] a pro-inflammatory cytokine release may occur and is associated with an activation of immune cells, such as Kupffer cells, natural killer (NK) cells, NK-T cells and dendritic cells leading to further liver injury [41–43]. In case of severe liver injury, these effects are associated with consecutive liver failure and death due to multiple organ failure [44, 45]. To date there is no specific treat- ment of liver injury to prevent organ failure and poor out- come. Therefore, new treatments and drugs are needed to address this problem and taurolidine might be a potential treatment in the future due to its reported impressive anti- inflammatory effects. Hence, this study was planned to Measurement of extracellular liver enzymes in serum and cell culture supernatants Perfusion of the liver biochip with 1 mg/ml taurolidine showed hepatocellular excretory dysfunction of MRP-2 dependent release of 5(6)-Carboxy-2′,7′-dichlorofluorescein (CDF) secretion assessed by fluorescence microscopy (data not shown). Short-term treatment with taurolidine without histological alterations After 24 h post injection of taurolidine or saline no struc- tural and morphological alterations were seen in rat livers (Fig. 4a and b). Treatment with taurolidine caused neither influx of inflammatory cells nor cell ballooning or necrosis. Alteration of liver function in vivo Serum levels of ASAT and GLDH in rats were significantly elevated after a single systemic taurolidine treatment with high-dose compared to low-dose taurolidine or controls, whereas there were no differences between the low-dose Page 5 of 9 Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Fig. 2 For cell viability investigations previously seeded hepatocytes were treated with taurolidine and stained for cleaved caspase 3. Fluorescence microscopy revealed an increase of cleaved caspase 3 positive cells (green cells) in a dose-dependent manner (a-d). Analysis of mean fluorescent intensity using a computer based program (ImageJ®) showed significantly higher fluorescence after administration of high dose taurolidine (1 mg/ml) (e). Representative data of three independent experiments are shown Fig. 2 For cell viability investigations previously seeded hepatocytes were treated with taurolidine and stained for cleaved caspase 3. Fluorescence microscopy revealed an increase of cleaved caspase 3 positive cells (green cells) in a dose-dependent manner (a-d). Analysis of mean fluorescent intensity using a computer based program (ImageJ®) showed significantly higher fluorescence after administration of high dose taurolidine (1 mg/ml) (e). Representative data of three independent experiments are shown Altogether it is difficult to compare exactly effects of the drug dosages between the different in vitro models and animal studies. So far, there are only few case reports of systemical use of taurolidine in humans available [46–48]. In these reports, patients were treated intravenously with taurolidine with a dose up to 300 mg/kg body weight, for glioblastoma, gastric cancer or metastatic melanoma [46–48]. In these few patients, no severe drug toxicity was seen, but in one patients elevated liver enzymes were reported [48]. In addition, there are several studies in which taurolidine was applied locally to the peritoneal or pleural cavity and for catheter-lock treatment, respectively [1, 4, 15, 16]. The toxic concentra- tion of taurolidine identified in this study, was conse- quently lower than the used concentration in previous clinical case studies, but the toxic concentration in human subjects remains difficult to extrapolate. Finally, this is the first study investigating and comparing the effects of taur- olidine in a liver-on-a-chip model, cell cultures and an animal in vivo model. Therefore, the exact comparison investigate the optimal treatment dose for further liver supportive treatment experiments during e.g. hepatic ischemia-reperfusion injury, toxic liver injury or liver inflammation. Alteration of liver function in vivo Beside the anti-bacterial effect, taurolidine reduced the pro-inflammatory cytokines such as TNF alpha, IL-6 and IL-8 [7–13] and led to endotoxin inactivation [3]. Since the publication of Arlt et al. reporting severe liver tox- icity after long-term oncological treatment of osteosar- coma in rodents with high doses of taurolidine [30], no severe toxic side effects had been reported [28, 29]. But Arlt et al. showed in mice that taurolidine led to hepato- toxicity with histological and morphological changes as well as alterations of serum liver enzymes after 25 days of a daily high-dose intraperitoneal treatment, whereas a low-dose treatment showed no hepatotoxicity [30]. These reported results are similar to our results after short-term treatment with taurolidine in rats and in the in vitro models, whereas the used doses in our in vivo study were about three times lower than in the previous study. Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Page 6 of 9 Fig. 3 Liver biochips were perfused with taurolidine at a concentration of 100 μg/ml and revealed no significant hepatocyte damage assessed by measurement of liver enzymes in biochip supernatants. Whereas a concentration of 1 mg/ml taurolidine revealed significantly increased levels of ASAT and GLDH (a, b). IL-6 levels showed similar results, with a significant increase after high-dose treatment (c). Serum levels of TNF-α, IL-1ß and IL-10 were not significantly different between controls and taurolidine with 1 mg/ml (d). Representative data of three independent experiments are shown Fig. 3 Liver biochips were perfused with taurolidine at a concentration of 100 μg/ml and revealed no significant hepatocyte damage assessed by measurement of liver enzymes in biochip supernatants. Whereas a concentration of 1 mg/ml taurolidine revealed significantly increased levels of ASAT and GLDH (a, b). IL-6 levels showed similar results, with a significant increase after high-dose treatment (c). Serum levels of TNF-α, IL-1ß and IL-10 were not significantly different between controls and taurolidine with 1 mg/ml (d). Representative data of three independent experiments are shown and interpretation of each system will be an issue in fur- ther investigations. more physiologic condition, but beside of macrophages other immune cells are missing in the model. Even though the exact mechanism and responsible ef- fector cells were not investigated, the main negative effect of taurolidine might be a direct toxic effect on hepatocytes, as the treatment even led to hepatic cell death in hepato- cyte monocultures. Alteration of liver function in vivo This effect is emphasized by the in- crease and changes of serum enzymes reflecting hepatic cell death and necrosis. Even though the parameter values were in the maximum only doubled between the treatment In this study we used three different experimental models with similar results reflecting robust and reliable findings. In contrast to monoculture experiments, the use of a microfluidically supported liver biochip compris- ing human cells provides an opportunity to investigate liver processes in a standardized manner [35, 49]. Because of direct cell-to-cell interactions of different hepatic cells within a microfluidic setting, liver biochips represent a Fig. 4 Representative histological figures with H&E staining of rat livers after injection of saline (n = 6) or taurolidine (n = 6). There were no signs of inflammation or necrosis 24 h after treatment with control (a) or high-dose of taurolidine (b) Fig. 4 Representative histological figures with H&E staining of rat livers after injection of saline (n of inflammation or necrosis 24 h after treatment with control (a) or high-dose of taurolidine (b) Fig. 4 Representative histological figures with H&E staining of rat livers after injection of saline (n = 6) or taurolidine (n = 6). There were no signs of inflammation or necrosis 24 h after treatment with control (a) or high-dose of taurolidine (b) Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Page 7 of 9 Fig. 5 Serum levels of ASAT (a), GLDH (b) and IL-6 (c) in rats 24 h after injection of saline or taurolidine, showing a significant elevation of ASAT and GLDH levels in high-dose taurolidine in comparison to controls or low-dose treatment. Animals per group n = 6 Fig. 5 Serum levels of ASAT (a), GLDH (b) and IL-6 (c) in rats 24 h after injection of saline or taurolidine, showing a significant elevation of ASAT and GLDH levels in high-dose taurolidine in comparison to controls or low-dose treatment. Animals per group n = 6 Fig. 5 Serum levels of ASAT (a), GLDH (b) and IL-6 (c) in rats 24 h after injection of saline or taurolidine, showing a significant elevation of ASAT and GLDH levels in high-dose taurolidine in comparison to controls or low-dose treatment. Animals per group n = 6 groups, we are convinced that these changes are still rele- vant in this short-term investigation. Abbreviation ASAT: Aspartate aminotransferase; COC: Cyclic olefin copolymers; GLDH: Glutamate dehydrogenase; GM-CSF: Granulocyte macrophage colony-stimulating factor; H/E: Hematoxylin/eosin; HUVEC: Human umbilical vein endothelial cells; IL: Interleukin; NK: Natural killer; PBMC: Peripheral blood mononuclear cells; TNF alpha: Tumor necrosis factor alpha Conclusions In conclusion, short-term treatment with taurolidine is associated with dose-dependent toxic liver injury with decreased hepatocyte survival in in vitro cell culture ex- periments and in a novel microfluidic hepatic biochip. In addition, liver dysfunction measured by serum levels of liver enzymes in rats showed direct toxic effects on the liver with high-dose treatment. Additional file The lack of histological hepatic alteration in the in vivo model might be further explained by the fact that the ob- servation period of 24 h was quite short. Hepatic necrosis is often seen not before 48 to 72 h after induction of liver injury. Additional file 1: Figure S1. Cell survival and LC50 of HepaRG cells after tauroldine treatment for 24 h was assessed by WST-assay. The LC50 for taurolidine was about 125 μg/ml (n = 6 per concentration, error bars = standard deviation). (TIFF 169 kb) A major experimental limitation should be noted: com- mercially available injectable taurolidine solution used in this study has a concentration of 2%. Therefore, only a restricted volume and concentration of taurolidine could be injected into rats. Thus, it was impossible to reach high hepatotoxic concentrations in rats as those used in in vitro experiments. Alteration of liver function in vivo in liver injury and inflammation, this toxic threshold value should be taken into account to avoid additional direct hepatotoxic drug effects. The increase of IL-6 in the liver biochip might be a re- sponse of Kupffer cells on the induced hepatic injury and inflammation. As other immune cells (NK cells, T- and B-cells) are lacking in the liver biochip model, pro- and anti-inflammatory pathways and processes between these cells are inexistent and might lead to an overwhelming in- flammatory reaction. The effect of increased IL-6 was not seen in rats which is probably due to the lower taurolidine doses. In addition, as taurolidine has an anti-inflammatory effect during acute inflammation in vivo, taurolidine might lead to a protection against hepatoxicity and cytokine release in the in vivo model. Competing interests h h d l h Competing interests 17. Haag GM, Berger AK, Jager D. Treatment of long-term catheter-related bloodstream infections with a taurolidine block: a single cancer center experience. J Vasc Access. 2011;12(3):244–7. p g The authors declare that they have no competing of interest. The authors declare that they have no competing of interest. 18. Hoksch B, Rufer B, Gazdhar A, Bilici M, Beshay M, Gugger M, Schmid RA. Taurolidine in the prevention and therapy of lung metastases. Eur J Cardiothorac Surg. 2009;36(6):1058–63. Acknowledgements We would like to acknowledge the technical assistance of Marko Gröger (Institute of Biochemistry II, Friedrich Schiller University Jena) with performing the in vitro experiments, Kathrin Katenkamp for preparing As the main toxic effect is dose dependent, further in- vestigations of a potential protective effect of taurolidine Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 Fahrner et al. BMC Pharmacology and Toxicology (2017) 18:61 immunohistochemical analyses of the liver samples, and Cora Richert (Department of Clinical Chemistry and Laboratory Diagnostics, University Hospital Jena) for the excellence technical assistance during the measurements of serum samples. We thank Karin Jandt (Department of General, Visceral and Vascular Surgery, University Hospital Jena) for editorial and linguistic revision for this manuscript. immunohistochemical analyses of the liver samples, and Cora Richert (Department of Clinical Chemistry and Laboratory Diagnostics, University Hospital Jena) for the excellence technical assistance during the measurements of serum samples We thank Karin Jandt (Department of General Visceral and immunohistochemical analyses of the liver samples, and Cora Richert (Department of Clinical Chemistry and Laboratory Diagnostics, University Hospital Jena) for the excellence technical assistance during the measurements of serum samples. We thank Karin Jandt (Department of General, Visceral and Vascular Surgery, University Hospital Jena) for editorial and linguistic revision for this manuscript. polymorphonuclear neutrophils in a human in vitro model of peritonitis. Surg Infect. 2002;3(3):235–44. 9. Rosman C, Westerveld GJ, van Oeveren W, Kooi K, Bleichrodt RP. Effect of intraperitoneal antimicrobials on the concentration of bacteria, endotoxin, and tumor necrosis factor in abdominal fluid and plasma in rats. Eur Surg Res. 1996;28(5):351–60. p g of serum samples. We thank Karin Jandt (Department of General, Visceral and Vascular Surgery, University Hospital Jena) for editorial and linguistic revision for this manuscript. 10. Rosman C, Westerveld GJ, Kooi K, Bleichrodt RP. Local treatment of generalised peritonitis in rats; effects on bacteria, endotoxin and mortality. Eur J Surg. 1999;165(11):1072–9. Funding Funding There was no funding of this study. There was no funding of this study. There was no funding of this study. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 19. Aceto N, Bertino P, Barbone D, Tassi G, Manzo L, Porta C, Mutti L, Gaudino G. Taurolidine and oxidative stress: a rationale for local treatment of mesothelioma. Eur Respir J. 2009;34(6):1399–407. Authors’ contribution S d d d Study conception and design: RF, FR, US, AM. Acquisition of data: RF, AM, MK, ATP, AK. Analysis and interpretation of data: RF, AM, FR, US, AM. Drafting Study conception and design: RF, FR, US, AM. Acquisition of data: RF, AM, MK, ATP, AK. Analysis and interpretation of data: RF, AM, FR, US, AM. Drafting of manuscript: RF. Critical revision of manuscript: AM, ATP, AK, MK, FR, US, AM. All authors have read and approved the final version of this manuscript. 11. Watson RW, Redmond HP, Mc Carthy J, Bouchier-Hayes D. Taurolidine, an antilipopolysaccharide agent, has immunoregulatory properties that are mediated by the amino acid taurine. J Leukoc Biol. 1995;58(3):299–306. of manuscript: RF. Critical revision of manuscript: AM, ATP, AK, MK, FR, US, AM. All authors have read and approved the final version of this manuscript. 12. Marcinkiewicz J, Kurnyta M, Biedron R, Bobek M, Kontny E, Maslinski W. Anti-inflammatory effects of taurine derivatives (taurine chloramine, taurine bromamine, and taurolidine) are mediated by different mechanisms. Adv Exp Med Biol. 2006;583:481–92. Availability of data and materials 13. Leithauser ML, Rob PM, Sack K. Pentoxifylline, cyclosporine A and taurolidine inhibit endotoxin-stimulated tumor necrosis factor-alpha production in rat mesangial cell cultures. Exp Nephrol. 1997;5(1):100–4. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 14. Cullis PS, McKee RF. Taurolidine lock - experience from the West of Scotland. Clin Nutr. 2011;30(3):399–400. author reply 401 Received: 11 May 2017 Accepted: 4 August 2017 22. Chromik AM, Daigeler A, Hilgert C, Bulut D, Geisler A, Liu V, Otte JM, Uhl W, Mittelkotter U. Synergistic effects in apoptosis induction by taurolidine and TRAIL in HCT-15 colon carcinoma cells. J Invest Surg. 2007;20(6):339–48. References 1. Linder MM, Ott W, Wesch G: [Antibacterial therapy of purulent peritonitis: a prospective randomized study on the effects of antibiotics and taurolin, a new chemotherapeutic and antiendotoxic agent (author’s transl)]. Chir Forum Exp Klin Forsch. 1980:67–71. 2. Staubach KH. Adjuvant therapy of peritonitis with taurolidine. Modulation of mediator liberation. Langenbecks Arch Chir. 1997;382(4 Suppl 1):S26–30. 3. Reith HB. Therapy of peritonitis today. Surgical management and adjuvant therapy strategies. Langenbecks Arch Chir. 1997;382(4 Suppl 1):S14–7. 4. Conlan AA, Abramor E, Delikaris P, Hurwitz SS. Taurolidine instillation as therapy for empyema thoracis. A prospective study of 50 patient. S Afr Med J. 1983;64(17):653–5. 5. Neary PM, Hallihan P, Wang JH, Pfirrmann RW, Bouchier-Hayes DJ, Redmond HP. The evolving role of taurolidine in cancer therapy. Ann Surg Oncol. 2010;17(4):1135–43. 6. Erb F, Febvay N, Imbenotte M. Structural investigation of a new organic antiseptic: Taurolidine A spectroscopic study of its stability and equilibria in various solvents. Talanta. 1982;29(11 Pt 1):953–8. 7. Traub WH, Leonhard B, Bauer D. Taurolidine: in vitro activity against multiple-antibiotic-resistant, nosocomially significant clinical isolates of Staphylococcus aureus, Enterococcus faecium, and diverse Enterobacteriaceae. Chemotherapy. 1993;39(5):322–30. 8. Sendt W, Mansouri E, Schmitt-Graeff A, Wolff-Vorbeck G, Schoffel U. Influence of antiseptic agents on interleukin-8 release and transmigration of 23. Chromik AM, Daigeler A, Bulut D, Flier A, May C, Harati K, Roschinsky J, Sulberg D, Ritter PR, Mittelkotter U, et al. Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines. J Exp Clin Cancer Res. 2010;29:21. 1. Linder MM, Ott W, Wesch G: [Antibacterial therapy of purulent peritonitis: a prospective randomized study on the effects of antibiotics and taurolin, a new chemotherapeutic and antiendotoxic agent (author’s transl)]. Chir Forum Exp Klin Forsch. 1980:67–71. 24. Darnowski JW, Goulette FA, Cousens LP, Chatterjee D, Calabresi P. Mechanistic and antineoplastic evaluation of taurolidine in the DU145 model of human prostate cancer. Cancer Chemother Pharmacol. 2004;54(3):249–58. 2. Staubach KH. Adjuvant therapy of peritonitis with taurolidine. Modulation of mediator liberation. Langenbecks Arch Chir. 1997;382(4 Suppl 1):S26–30. 2. Staubach KH. Adjuvant therapy of peritonitis with taurolidine. Modulation of mediator liberation. 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Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72

Acta neuropathologica communications

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© 2013 Mann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Cases of Frontotemporal Lobar Degeneration (FTLD) and Motor Neurone Disease (MND) associated with expansions in C9ORF72 gene are characterised pathologically by the presence of TDP-43 negative, but p62 positive, inclusions in granule cells of the cerebellum and in cells of dentate gyrus and area CA4 of the hippocampus. Results: We screened 84 cases of pathologically confirmed FTLD and 23 cases of MND for the presence of p62 positive inclusions in these three brain regions, and identified 13 positive cases of FTLD and 3 of MND. All cases demonstrated expansions in C9ORF72 by Southern blotting where frozen tissues were available. The p62 positive inclusions in both cerebellum and hippocampus were immunostained by antibodies to dipeptide repeat proteins (DPR), poly Gly-Ala (poly-GA), poly Gly-Pro (poly-GP) and poly Gly-Arg (poly-GR), these arising from a putative non-ATG initiated (RAN) sense translation of the GGGGCC expansion. There was also some slight, but variable, immunostaining with poly-AP antibody implying some antisense translation might also occur, though the relative paucity of immunostaining could reflect poor antigen avidity on the part of the antisense antibodies. Of the FTLD cases with DPR, 6 showed TDP-43 type A and 6 had TDP-43 type B histology; one had FTLD-tau with the pathology of corticobasal degeneration. There were no qualitative or quantitative differences in the pattern of immunostaining with antibodies to DPR, or p62, proteins between TDP-43 type A and type B cases. Ratings for frequency of inclusions immunostained by these poly-GA, poly-GP and poly-GR antibodies broadly correlated with those for immunolabelled by p62 antibody in all three regions. Conclusion: We conclude that DPR are a major component of p62 positive inclusions in FTLD and MN Conclusion: We conclude that DPR are a major component of p62 positive inclusions in FTLD and MN Keywords: Frontotemporal lobar degeneration, C9ORF72, p62 inclusions, Dipeptide repeat proteins Keywords: Frontotemporal lobar degeneration, C9ORF72, p62 inclusions, Dipeptide repeat protein : Frontotemporal lobar degeneration, C9ORF72, p62 inclusions, Dipeptide repeat proteins Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72 Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72 David MA Mann1*, Sara Rollinson2, Andrew Robinson1, Janis Bennion Callister2, Jennifer C Thompson1, Julie S Snowden1, Tania Gendron3, Leonard Petrucelli3, Masami Masuda-Suzukake4, Masato Hasegawa4, Yvonne Davidson1 and Stuart Pickering-Brown2 * Correspondence: [email protected] 1Clinical and Cognitive Sciences Research Group, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, Salford Royal Hospital, Salford M6 8HD, UK Full list of author information is available at the end of the article Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Histological methods l ff Serial paraffin sections were cut (at a thickness of 6 μm) from formalin fixed blocks of temporal cortex (with hippo- campus) and cerebellar cortex. Sections within the series were immunostained by routine methods for amyloid β protein (Aβ), tau, TDP-43 and FUS proteins, employing microwaving in 0.1 M citrate buffer, pH 6.0 for antigen retrieval [2]. Pathologically, of the 84 patients with FTLD, 30 had FTLD-tau (9 with FTLD-tau Picks (PiD), 7 with MAPT mutation, 11 with CBD and 3 with PSP), 52 had FTLD-TDP (24 with type A histology, 22 with type B histology, 6 with type C histology), 1 had FTLD-FUS (aFTLD-U) and 1 had FTLD-ni. (see [23] for definitions). Pathologically, most FTLD cases with the expansion [15-20], like many non-mutational cases of FTLD [2,21,22], show inclusion bodies within neurones (NCI) and glial cells of the cerebral cortex and hippocampus that contain the nuclear transcription factor, TDP-43, and are said bear a TDP-43 histological subtype termed FTLD-TDP type B (according to [23]), compatible with a clinical diagnosis of FTD and MND. Others, however, show a TDP-43 histological type characterised by the presence of many short neurites (DN) along with NCI within the outer layers of the cerebral cortex, and termed FTLD-TDP type A [23]. Nonetheless, irrespective of TDP-43 histo- logical type, expansion carriers also show a unique path- ology within the hippocampus [17] and cerebellum [17-19,24] characterised by NCI that are TDP-43 nega- tive, but immunoreactive to p62 protein. Further sections from each case within the series were screened for the presence of p62-immunoreactive NCI by immunostaining with p62-lck ligand (rabbit polyclonal antibody (B D Biosciences, Oxford, UK) 1:100) employing a standard ABC Elite kit (Vector, Burlingame, CA, USA) with DAB as chromagen, and again microwaving in 0.1 M citrate buffer, pH6.0 for antigen retrieval. Positive cases were defined where p62 positive, TDP-43 negative NCI within either the cerebellum (see [18]) or hippocampus (see [17]) could be clearly seen under low power object- ive (×20) and the majority of high power fields (×40) contained at least 2 NCI. Negative cases were either com- pletely devoid of p62 immunostaining, or small amounts of apparently extracellular and ‘extraneous’ p62 positive particulate material was observed in occasional high power fields. P62 is a marker for proteins destined for proteasomal degradation though the precise target protein within such NCI remains uncertain, and the pathogenetic mech- anism underlying the hexanucleotide expansion remains unclear. Methods was well known at that time that a number of large, inde- pendent FTD + MND kindreds demonstrated linkage to chromosome 9 in the region 9p21.2-p13.3 [7-10]. Subse- quently, three GWAS studies for MND [11-13], and one for FTLD [14] identified a susceptibility locus within this linked region, with strongest association coming from a 80 kb haplotype block containing 3 genes, MOBKL2B, IFNK and C9ORF72. It has now been shown that this at least some of this association is due to the presence of a large hexanucleotide (GGGGCC) in C9ORF72 gene in patients with both FTLD and MND [15,16]. The expansion occurs in the first intron or the promoter region of the gene, depending upon the transcript iso- form in question, and can number up to as many as 1500 repeats. The expansion is found in about one in every twelve patients with FTLD, but this varies depend- ing on geographical region with the expansion being rare in Asia [16]. Brain tissues were available in the Manchester Brain Bank from a series of 84 patients with FTLD and 23 with MND. All patients were from the North West of England and North Wales. All FTLD cases fulfilled Lund-Manchester clinical diagnostic criteria for FTLD [28,29], and all those with MND fulfilled El Escorial criteria [30]. All brains had been obtained with full ethical permission following con- sent by the next of kin. Background (semantic dementia) or non-fluent (progressive non-fluent aphasia) nature [1]. All three syndromes can be accompan- ied by Motor Neurone Disease (MND) though the combin- ation of FTD and MND is most common. Histologically, around half of cases have tau-based pathology, half have TDP-43-based pathology, and about 5% have FUS-based pathology [2,3]. Importantly, around 40% of all cases have a strong family history of similar disease, irrespective of clinical or histological subtype [1]. Frontotemporal Lobar Degeneration (FTLD) is a clinical, pathological and genetically heterogeneous condition. The major clinical syndromes principally involve personality and behavioural change (behavioural variant frontotemporal dementia, or bvFTD) or language alterations of a fluent * Correspondence: [email protected] 1Clinical and Cognitive Sciences Research Group, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, Salford Royal Hospital, Salford M6 8HD, UK Full list of author information is available at the end of the article By 2007, causative mutations had been identified in tau (MAPT) [4] and progranulin (GRN) [5,6]. Nonetheless, it Page 2 of 13 Page 2 of 13 Page 2 of 13 Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Histological methods l ff Nonetheless, this is likely to result from one, or a combination, of three possible mechanisms: i) haploinsufficiency leading to loss of C9orf72 protein [15,16], ii) the expansion might form nuclear foci of toxic RNA and sequester other RNA-binding proteins such as Pur Alpha (Pur α) [25], or iii) RAN (repeat asso- ciated non ATG-initiated) translation of the expanded repeat region may lead to the ‘inappropriate’ formation of dipeptide repeat proteins (DPR) which may aggregate and confer neurotoxicity [26,27]. Microscopic analysis Sections of cerebellum and hippocampus immunostained for p62 and each of our own 4 DPR antibodies, and the poly-GA antibody supplied by M Hasegawa were assessed for the presence of DPR immunostained NCI within granule cells of the cerebellum, and dentate gyrus cells and CA4 pyramidal cells of the hippocampus at ×20 magnification. The frequency of DPR-immunoreactive NCI was assessed according to: Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Page 3 of 13 1 = very few immunostained NCI present, in some but not all fields. by Genentech. Antibodies were successfully raised against poly-GP, poly-GR, poly-AP and poly-PR proteins. How- ever, it was not possible to generate antibodies to poly-GA and poly-PG proteins. 2 = a moderate number of immunostained NCI present in each field. Serial sections from those cases showing p62-positive pathology, and those from 11 (non-p62 positive) cases with other histological and genetic forms of FTLD, other neurodegenerative disorders and healthy controls (see Table 1) were immunostained for DPR. Antibodies were employed in standard IHC (as above) though, in this in- stance, antigen unmasking was performed by pressure cooking in citrate buffer (pH 6, 10 mM) for 30 minutes, reaching 120 degrees Celsius and >15 kPa pressure Fol- lowing titration to determine optimal immunostaining, antibodies were employed at dilutions of 1:500 (poly-AP and poly-PR), 1:750 (poly-GR) or 1:2000 (poly-GP). 3 = many immunostained NCI present affecting most cells in each field. 3 = many immunostained NCI present affecting most cells in each field. 4 = very many immunostained NCI present, affecting nearly all cells in every field. Statistical analysis d f Rating data from semiquantitative asssessments was entered into an excel spreadsheet and analysed using Statistical Package for Social Sciences (SPSS) software (version 17.0). Mann–Whitney test was used to compare rating data between pairs of groups. A p-value of less than 0.05 was considered statistically significant. Regression analysis using Intercooled Stata Version 9 (StataCorp) was carried to out to investigate effects between repeat length, age of onset and disease duration. Expansion sizing and analysis Expansion sizing was carried out using ImageQuant TL software (Version 7, GE Healthcare) sizing the repeat num- ber against the DIG labelled lambda Hind III labelled size standard included on each gel (Roche Applied Science). Positive control (gDNA isolated from the B-Lymphocyte cell line ND06769 obtained from the NINDS Repository– Coriell) and negative control were included on each blot, and were required to show a band of the expected size or no signal on hybridisation respectively for each blot to pass quality control. Dipeptide antibody staining d l A non-ATG initiated translation of the expansion would putatively result in DPRs derived from either from for- ward (sense) (poly Gly-Pro, (poly-GP), poly Gly-Ala (poly- GA) and poly Gly-Arg, (poly-GR)) or reverse (antisense) (poly Ala-Pro (poly-AP), poly Pro-Gly (poly-PG) and poly Pro-Arg (poly-PR)) directions. Consequently, we commis- sioned a series of custom made rabbit polyclonal anti- bodies, raised against such putatively translated proteins. Briefly, peptides consisting of 15 repeats with an additional N terminal cysteine were synthesised and N-terminally conjugated to keyhole limpet haemocyanin prior to im- munisation. All steps in the preparation were performed Here, we show that DPR are at least one of the target protein(s) within the TDP-43 negative, p62-positive NCI in cases of FTLD associated with hexanucleotide (GGGGCC) expansions, and that such peptides are not associated with other histological forms, or genetic sub- types, of FTLD. Southern blotting b Frozen brain tissue for Southern blotting was available for most cases employed in the study. Southern blotting was performed using a PCR DIG labelled probe adjacent to the expansion. The PCR probe consisted of 851 bp amplicon using the following primers, forward 5′ CCCACACCTGC TCTTGCTA 3′; reverse 5′ CGTTCTGTGTGATTTTTA GTGATGA 3′. y y y Further sections from the series were immunostained with poly-GA (and poly-GP and poly-GR) antibodies courtesy of Dr M Hasegawa. These antibodies were raised against poly-(GA)8, poly-(GP)8 and poly-(GR)8 peptides with cysteine at N-terminus. The peptides were conju- gated to m-maleimidobenzoyl-N-hydrosuccinimide ester- activated thyroglobulin. The thyroglobulin-peptide complex (200 μg) emulsified in Freund’s complete adjuvant was injected subcutaneously into a New Zealand White rabbit, followed by 4 weekly injections of peptide complex emulsi- fied in Freund’s incomplete adjuvant, starting after 2 weeks after the first immunization. Immunoreactivities of these antisera were characterized by ELISA as follows. The peptide immunogens were coated onto microtiter plates. The plates were blocked with 10% fetal bovine serum (FBS) in PBS, incubated with the rabbit antisera diluted in 10% FBS/PBS at room temperature for 1.5 h, followed by incubation with HRP-goat anti-rabbit IgG (Bio-Rad) at 1:3000 dilution, and reacted with the substrate, 0.4 mg/mL o-phenylenediamine, in citrate phosphate buffer (24 mM citric acid, 51 mM Na2HPO4). The absorbance at 490 nm was measured using Plate Chameleon (HIDEX). All anti- bodies were used for imminohistochemistry at dilution of 1:2000, and pretreated as above. Sections were also immunostained with the anti-dipeptide repeat antibody C9RANT [26] (gift from L Petrucelli) at 1:3000 dilution. Briefly, samples were digested overnight with 20 u of XbaI (New England Biolabs). Samples were electrophoresed in 0.8% agarose 1 xTBE gels run at 1.5 volts/cm for 18 hours. Following standard protocols [31], gDNA was transferred to positively charged nylon membrane. Membranes were fixed using UV light at 365 nm for 3 minutes using a GE Image quant 350. Membranes were hybridized and detected as per the DIG detection Manual (Roche Applied Science). Signals were visualised using the GE Image quant 350 after 1 to 4 hours. Results Screening the 84 FTLD and 23 MND cases with p62 revealed 16 cases, 13 with FTLD (cases #1-13) and 3 0 = no immunostained NCI present in any field. a; MND = Motor Neurone Disease; SD = semantic dementia; FTLD = Frontotemporal Lobar Degeneration; na = data not available/applicable; * = age at death; ** = cases #2 and 14 were brothers. Results ble 1 Selected clinical and pathological details of cases investigated by dipeptide immunostaining e M/F Clinical diagnosis Pathological diagnosis Family history Onset (y) Duration (y) Brain weight (g) M FTD FTLD-TDP type A 2 brothers, 2 sisters FTD 49 9 1050 M FTD FTLD-TDP type A brother MND**, mother and grandmother FTD 60 8 1210 F FTD FTLD-TDP type A none available 59 5 1140 M FTD FTLD-TDP type A father dementia 64 8 1100 M FTD FTLD-TDP type A father similar presentation, paternal grandmother ‘AD’ 63 2 na M FTD FTLD-TDP type A yes 78 4 1200 M FTD + MND FTLD-TDP type B ?paternal aunt said to be ‘strange’ 60 2 na M FTD + MND FTLD-TDP type B mother FTD 57 2 1210 M FTD FTLD-TDP type B mother dementia 54 12 na F FTD FTLD-TDP type B mother and sister FTD 51 19 na F FTD + MND FTLD-TDP type B father ‘AD’, sister MND, paternal nephew MND 63 2 na F FTD + MND FTLD-TDP type B sister MND, brother FTD, mother ‘multiple sclerosis’ 68 5 1363 M FTD Corticobasal degeneration father and 5 sisters had Huntington’s disease 59 70 1271 M MND MND brother FTD**, mother and grandmother FTD 60 5 1350 F MND MND none available 40 5 1330 M MND MND brother MND, sister FTD + MND 53 2 1250 M FTD FTLD-tau Pi none 69 6 na F FTD FTLD-tau MAPT +16 mother: early onset dementia; brother: MND 48 15 1100 M Corticobasal Syndrome FTLD-tau CBD none 65 na 1020 M FTD FTLD-TDP A mother AD, brother AD (but with behavioural problems) 66 7 980 F FTD + MND FTLD-TDP B none available 50 2 1050 F SD FTLD-TDP C none 70 2 1522 F Alzheimer’s disease Alzheimer’s disease none 74 12 1220 M Huntington’s disease Huntington’s disease none available 48 24 na M Huntington’s disease Huntington’s disease none available 56 19 1340 M Normal Normal control none 54* na 1720 F Normal Normal control none 53* na 1220 = Frontotemporal dementia; MND = Motor Neurone Disease; SD = semantic dementia; FTLD = Frontotemporal Lobar Degeneration; na = data not available/applicable; * = age at death; ** = cases #2 and 14 were brothers. al. Acta Neuropathologica Communications 2013, 1:68 Pa ww.actaneurocomms.org/content/1/1/68 Page 4 of 13 Page 5 of 13 Mann et al. Results Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 inclusions were frequent, but none were seen within Golgi neurones, or within Bergmann glia. In some cases, occasional Purkinje cells (Figure 2c) and neurones in the dentate nucleus (Figure 2d) contained small, spicular or granular p62-immunoreactive structures, but these were not immunoreactive with TDP-43 antibodies. In most cases, occasional cells resembling astrocytes were seen to contain p62 immunoreactive intranuclear inclusions, these being located deep in the granule cell layer. All cases also showed abundant, small, rounded NCI within granule cells of the dentate gyrus (Figure 2e), along with spicular or granular inclusions within pyramidal cells of areas CA2/3 and CA4 (Figure 2f), less commonly in CA1 and subiculum (Table 2). with MND (cases #14-16) (Table 1) showing p62 posi- tive, TDP-43 negative NCI within granule cells of the cerebellum and other cerebellar cell types, and in granule cells of the dentate gyrus, and pyramidal cells of CA4, CA3 and CA2 regions of the hippocampus, as described previously [17-19,24]. Twelve of the 13 FTLD cases showed TDP-43 proteinopathy, classifiable [23] as either type A (6 cases) or type B (6 cases): the other case had tauopathy compatible with corticobasal degeneration (CBD) (Table 1). Eight of the FTLD cases had presented with bvFTD clinically, 4 with FTD + MND and one (with CBD pathology) with a combination of FTD and corticobasal syndrome. All MND cases bore typical TDP-43 patho- logical changes in motor neurones of brain stem nuclei and spinal cord (where this was available for analysis). In MND cases, cerebellar granule cell NCI were abun- dant in cases #14 and 16, but were rare in case #15 (Table 2). In all cases, p62 positive NCI were also ob- served within granule cells of the dentate gyrus, and similar (to FTLD cases) small, spicular or granular p62- immunoreactive inclusions were seen within pyramidal cells of areas CA2/3 and CA4, less commonly in CA1 and subiculum. Both of these kinds of changes were occasionally present in cases #14 and 15 but were moder- ately common in case #16 (Table 2). Frozen tissue was available for 12 of the 16 cases show- ing p62 pathological changes in cerebellum and hippo- campus. These included 9 cases with FTLD (cases #1-3, 7, 9-13) and all 3 cases with MND (cases #14-16) (Table 1). All 12 of these cases demonstrated a pathological ex- pansion in C9ORF72 by Southern blotting. p62 immunostaining In FTLD cases, on p62 immunostaining, NCI in granule cells of the cerebellum appeared as small rounded or oat- shaped bodies, though occasionally larger, more rounded and solid NCI were observed (Figure 2a). These were very abundant in case #1, common in cases #4, 7 and 10, moderately plentiful in cases #2 and 5, occasional in cases #3, 8 and 12, and rare in cases #6, 9 and 11 (Table 2). Similar, more granular, NCI were usually present in basket cells (Figure 2b), especially when granule cell Results Expansion size ranged from ~5 kb (~450 repeats) to in excess of 23 kb (over 3600 repeats) (Figure 1). No expansions were detected in cases where no p62 pathological changes were observed. There was no correlation be- tween repeat size and age of disease onset or duration (see Additional file 1: Figure S1). In all FTLD and MND cases variable, but often many, pyramidal neurones, chiefly within the deeper layers of the adjoining cerebral (temporal) cortex also contained NCI similar to those in the hippocampus CA regions (not shown). DPR immunostaining Results of immunostaining for DPR are shown in Table 2. All FTLD and MND cases showed similar patterns of immunostaining though there were quantitative differ- ences between cases with respect to the number of inclu- sions immunostaining. As with p62 immunostaining, immunostaining with both our own custom anti poly-GP and poly GR antibodies and Figure 1 Southern blotting of FTLD and MND cases bearing expansions in C9ORF72. Lane 1: Marker, Lane 2: Negative control brain, Lane 3: ND06769, Lane 4: 01/06 FTD case #3, Lane 5: MND case #15, Lane 6: FTD/MND case #11, Lane 7: FTD case #10, Lane 8: CBD case #13, Lane 9: FTD case #1; Lane 10: control FTD case with tauopathy, Lane 11: FTD case #9, Lane 12: FTD/MND case #12, Lane 13: FTD case #2; Lane 14: MND case #14, Lane 15: FTD/MND case #7, Lane 20: MND case#16. Lanes 16-19 show expansions in other clinically diagnosed cases of MND (positive controls) where no brain tissue was available. Figure 1 Southern blotting of FTLD and MND cases bearing expansions in C9ORF72. Lane 1: Marker, Lane 2: Negative control brain, Lane 3: ND06769, Lane 4: 01/06 FTD case #3, Lane 5: MND case #15, Lane 6: FTD/MND case #11, Lane 7: FTD case #10, Lane 8: CBD case #13, Lane 9: FTD case #1; Lane 10: control FTD case with tauopathy, Lane 11: FTD case #9, Lane 12: FTD/MND case #12, Lane 13: FTD case #2; Lane 14: MND case #14, Lane 15: FTD/MND case #7, Lane 20: MND case#16. Lanes 16-19 show expansions in other clinically diagnosed cases of MND (positive controls) where no brain tissue was available. Mann et al. Acta Neuropathologica Communications 2013, 1:68 Page 6 of 13 http://www.actaneurocomms.org/content/1/1/68 Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Page 6 of 13 a b c d e f Figure 2 Immunostaining of the cerebellum and hippocampus for p62 proteins shows neuronal cytoplasmic inclusions in granular neurones (a), basket cells (b), Purkinje cells (c) and cells of the dentate nucleus (d) of the cerebellum, and in dentate gyrus granule cells (e) and pyramidal cells of CA4 region (f). Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification. DPR immunostaining a b b d c d f Figure 2 Immunostaining of the cerebellum and hippocampus for p62 proteins shows neuronal cytoplasmic inclusions in granular neurones (a), basket cells (b), Purkinje cells (c) and cells of the dentate nucleus (d) of the cerebellum, and in dentate gyrus granule cells (e) and pyramidal cells of CA4 region (f). Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification. those prepared by Hasegawa similarly detected NCI in granule cells of the cerebellum, as did Hasegawa’s poly-GA antibody (Figure 2a). Again, more granular looking NCI were usually present in basket cells (Figure 2b), occasionally in Purkinje cells (Figure 2c) and neurones in the dentate nucleus (Figure 2d), but none were seen within Golgi neurones, or within Bergmann glia. A punctate, or fila- mentous, staining was also seen within the molecular layer of the cerebellum, this probably relating to parallel projection fibres (Figure 3a and b). No nuclear inclu- sions in atrocytes immunostained with p62 appeared to be detected by poly-GA antibody. All FTLD and MND cases also showed abundant, small, rounded NCI within granule cells of the dentate gyrus (Figure 3e), along with spicular or granular inclusions within pyramidal cells of areas CA2/3 and CA4 (Figure 3f), but these were less commonly seen in CA1 and subiculum (Table 2). Our own custom antibody raised against poly-AP showed rare, and weak, immunostaining of NCI within CA4 neurone in occasional cases were (Table 2), similar in appearance to those seen in such cells on p62 immuno- staining, or with poly-GA, poly-GP and poly GR anti- bodies. Cases #1-6 had FTLD TDP-43 type A histology (*), cases #7-12 had FTLD TDP-43 type B histology (**), case #13 had corticobasal degeneration, and cases #14-16 had Motor Neurone Disease. CA4 = pyramidal cells of CA4 region of hippocampus; DG = dentate gyrus granule cells; GCC = granule cells of the cerebellum; na = no data. -6 had FTLD TDP-43 type A histology (*), cases #7-12 had FTLD TDP-43 type B histology (**), case #13 had corticobasal degeneration, yramidal cells of CA4 region of hippocampus; DG = dentate gyrus granule cells; GCC = granule cells of the cerebellum; na = no data. DPR immunostaining Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 a b c d e f Figure 3 Immunostaining of the cerebellum and hippocampus for poly-GA protein shows neuronal cytoplasmic inclusions in granular neurones (a), basket cells (b), Purkinje cells (c) and cells of the dentate nucleus (d) of the cerebellum, and in dentate gyrus granule cells (e) and pyramidal cells of CA4 region (f), similar to those seen on p62 immunostaining. Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification. b d d f f Figure 3 Immunostaining of the cerebellum and hippocampus for poly-GA protein shows neuronal cytoplasmic inclusions in granular neurones (a), basket cells (b), Purkinje cells (c) and cells of the dentate nucleus (d) of the cerebellum, and in dentate gyrus granule cells (e) and pyramidal cells of CA4 region (f), similar to those seen on p62 immunostaining. Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification. especially in Purkinje cells of cerebellum and CA4 neurones of hippocampus in some cases bearing expansions in C9ORF72 (Figure 4). However, not all expansion bearers showed any such immunostaining, nor did any of the control cases. adjoining cerebral (temporal) cortex also contained NCI immunostaining with antibodies to DPR in a similar fash- ion to those in the hippocampus CA regions (not shown). adjoining cerebral (temporal) cortex also contained NCI immunostaining with antibodies to DPR in a similar fash- ion to those in the hippocampus CA regions (not shown). Cases of FTLD of other histological or genetic sub- types showed no immunostaining of TDP-43 positive NCI, DN or NII, or of tau positive structures (Pick bodies, neuro- fibrillary tangles). The non-demented control cases showed no relevant DPR immunostaining, and the NII within the HD cases were also unstained by any DPR antibody. Cases of FTLD of other histological or genetic sub- types showed no immunostaining of TDP-43 positive NCI, DN or NII, or of tau positive structures (Pick bodies, neuro- fibrillary tangles). The non-demented control cases showed no relevant DPR immunostaining, and the NII within the HD cases were also unstained by any DPR antibody. The pattern of immunolabelling of NCI with C9RANT antibody in cerebellum and hippocampus mirrored that reported by others [26], and again appeared to be identi- cal to that seen with both our own poly GP antibody and that prepared by M Hasegawa (not shown). DPR immunostaining The antibody to poly-PR did not stain NCI in any case, but strongly immunostained chromatin granules, Table 2 Ratings for p62 and DPR antibody immunostaining of neuronal cytoplasmic inclusions for all 15 cases with expansions in C9ORF72 Case CA4 DG GCC p62 poly-GA poly-GP poly-GR poly-PR poly-AP p62 poly-GA poly-GP poly-GR poly-PR poly-AP p62 poly-GA poly-GP poly-GR poly-PR poly-AP 1* 2 2 3 3 0.5 0.5 1 2 0 0.5 0.5 0.5 4 4 3 0.5 0 0 2* 3 3 2 3 0.5 1 2 2 0.5 2 0.5 1 2 3 3 0 0 0 3* 3 3 3 2 0.5 0.5 4 3 3 0 0 0 1 4 2 2 0 0 4* 2 2 3 3 0.5 0 3 2 3 0 0.5 0 3 3 3 0 0 0 5* 2 1 3 3 0.5 0.5 3 3 2 0 0 0 2 3 3 1 0 0 6* 1 2 2 1 0 0 1 2 2 0 0 0 1 1 1 0 0 0 7** 3 3 3 3 0 0 3 3 2 0 0 0 4 4 4 0 0 0 8** na na na na na na na na na na na na 1 1 1 0 0 0 9** 3 2 3 3 0.5 0.5 1 2 1 0.5 0 0 0.5 3 1 0 0 0 10** 3 2 2 2 0.5 0.5 4 3 3 1 0.5 0 3 3 2 0 0 0 11** 3 3 3 3 0 0 4 3 2 2 0 1 0.5 3 2 1 0.5 0 12** 3 3 2 3 0 0 4 3 1 1 0 0 1 3 3 1 1 0 13 0 2 2 2 0 0 0 2 2 0 0 0 1 1 1 0 0 0 14 3 2 3 3 0 1 1 2 2 0 0 0 3 4 2 0 0 0 15 1 1 1 1 0 0 2 2 2 2 0 0 1 1 1 0 0 0 16 2 2 2 0 0 0 2 2 2 2 0 0 3 3 3 0 0 0 Cases #1-6 had FTLD TDP-43 type A histology (*) cases #7-12 had FTLD TDP-43 type B histology (**) case #13 had corticobasal degeneration and cases #14-16 had Motor Neurone Disease DG Page 8 of 13 Mann et al. Other observations C ll DP Cells containing DPR did not show obvious cell loss, or any other outward signs of neurodegeneration, whether these were granule cells of dentate gyrus or cerebellum, or pyramidal cells of CA regions or cerebral cortex, or Purkinje neurones of the cerebellum. No cases with C9ORF72 expansion showed excessive tau and Aβ path- ology (i.e. commensurate with a pathological diagnosis of AD) for age. DPR immunostaining In all instances, immunostaining of NCI with anti-DPR antibodies was robust. There were no apparent effects of variable post mortem delay or prolonged fixation upon In all expansion carriers, variable numbers of, but often many, pyramidal neurones within the deeper layers of the Mann et al. Acta Neuropathologica Communications 2013, 1:68 Page 9 of 13 http://www.actaneurocomms.org/content/1/1/68 Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Page 9 of 13 a b Figure 4 Immunostaining of Pukinje cells of the cerebellum (a) and pyramidal cells of CA4 region of hippocampus (b) for poly-PR protein shows strong immunoreactivity of chromatin. Note lack of staining of NCI in either cell type. Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification. b a Figure 4 Immunostaining of Pukinje cells of the cerebellum (a) and pyramidal cells of CA4 region of hippocampus (b) for poly-PR protein shows strong immunoreactivity of chromatin. Note lack of staining of NCI in either cell type. Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification. poly-AP and poly-PR. Hence, ratings for poly-GA cor- related significantly with those for p62 in cerebellum (p = 0.032), dentate gyrus (p = 0.000) and CA4 (p = 0.006), with poly-GR in CA4 region (p = 0.044) and with poly-GP in cerebellum (p = 0.003). In CA4, ratings for poly-GP correlated with those for poly-GR (p = 0.007). In the cerebellum, ratings for poly-GA correlated with those for poly-GP (p = 0.008), as did ones for poly-GR with poly-PR (p = 0.027). Only in CA4 did poly-AP correlate with poly-PR ratings (p = 0.014). the quality or quantity of NCI immunostaining, with cases that had been stored in formalin for up to 20 years before new blocks had been taken for processing into wax sections still showing robust immunostaining for DPR, as did cases where post mortem delay periods had exceeded 4 days. Comparisons and correlations between p62 and DPR immunostaining The numbers of NCI immunostained by each DPR anti- body did not always correspond either when compared to p62 immunostaining, or when compared among them- selves. Those in CA2, CA3 and CA4 of hippocampus, and those in Purkinje cells and cells of the dentate nucleus appeared numerically similar in p62 and DPR immuno- staining (except for poly-AP), though it appeared from microscopic inspection that p62-positive NCI in granule cells of the cerebellum were not all, or always, immuno- stained by both poly-GR and poly-GP antibodies, and none were stained by poly-AP antibody (Table 2). In the hippocampus only a minority of the p62-positive NCI in the dentate gyrus were immunostained by poly-GR and poly-GP antibodies, and again none were stained by poly AP antibody (Table 2). Comparison of p62 and DPR immunostaining between FTLD-TDP subtypes There were no statistically significant differences between ratings for p62 and DPR immunostaining in either FTLD- TDP type A or type B cases for NCI in granule cells of the cerebellum, or for those of the dentate gyrus, and CA4 neurones, of hippocampus. Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 All of the 12 p62 positive cases where frozen brain tis- sues were available for analysis showed an expansion in C9ORF72 by Southern blotting, and are therefore con- sistent with the suggestion [17,18] that this type of p62 pathology is pathognomic for cases of FTLD and/or MND associated with C9ORF72 mutation. Thus, it can be presumed that an expansion was also present in the other four FTLD cases with relevant p62 pathology where no southern blotting (or repeat primed PCR) was possible due to lack of fresh frozen brain tissue. The lack of associ- ation between expansion length and age of disease onset or duration presumably reflects that a minimum size of expansion is required for disease. Present data suggests this might be around 500 repeats, and expansions be- yond this are not additionally detrimental. The exact target protein within the p62 immunoreactive NCI remains uncertain. Here, we performed immunohis- tochemistry employing antibodies to DPR putatively pro- duced in the brain through non-ATG initiated (RAN) translation of the expansion itself. Although double im- munofluorescence labelling was not performed in the present study, microscopic observations on consecutive serial sections stained with each DPR antibody suggested that most, if not all, of the p62-positive NCI within Purkinje and dentate nucleus cells of the cerebellum, and neurones of CA2, CA3 and CA4 of the hippocam- pus are similarly immunoreactive to poly-GA, poly-GP and poly-GR antibodies (and to a much lesser extent, poly-AP antibody), whereas only a (variable) subset of p62-positive NCI within granule cells of the cerebellum and dentate gyrus appeared immunoreactive to these antibodies. These microscopic impressions were supported by statistical analysis showing good correlations between ratings of inclusion body frequencies as assessed using poly-GA, poly-GP and poly-GR antibodies. Other studies [26,27] where double immunofluorescence labelling was indeed performed substantiate the present observations. Nevertheless, it appears that DPR may not be the only proteins present within these structures since the p62 positive inclusions have also been shown to contain hnRNPA3 [33], and others reported the inclusions in granule cells of the dentate gyrus and cerebellum (at least) to be immunoreactive to ubiquilin-2 [34]. There is little or no DPR immunostaining of TDP-43 in hippocampus or cerebral cortex in either expansion bearers, or patients with other histological forms of FTLD [29], consistent with those observations of NCI based on p62 immuno- staining [17-19,24]. Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Page 10 of 13 in some C9ORF72 expansion bearers. Although this kind of immunostaining was not seen in any of the control subjects in such cells, it was not consistently present in all expansion bearers, and so the relevance of this (to pathogenesis) remains uncertain. None of the DPR anti- bodies were immunoreactive to any other kinds of inclu- sions (TDP or tau) associated with other histological or genetic forms of FTLD, or the NII containing CAG re- peats seen in HD. These findings are consistent with those recently reported by Mori and colleagues [27]. The pattern of DPR immunolabelling in the cerebellum and hippo- campus in C9ORF72 expansion bearers mirrored that reported by Ash et al. [26], and this appeared identical to that shown by our own poly GP antibody, and that prepared by M Hasegawa. C9RANT antibody was raised to a panel of dipeptide repeat immunogens [26], but judging from the similarities between its pattern of immunolabelling and that of our own poly-GP antibody, it might be consid- ered that its specificity, or at least, it avidity is greatest for the poly-GP component of the immunogen mix. The finding of strong and consistent staining with poly-GA, poly-GP and poly-GR antibodies suggests that most of the aberrant translation relates to sense transcripts, though the slight, but variable immunostaining with poly-AP antibody implies some antisense translation might also occur. However, it remains possible that the relative paucity of immunostaining seen here with antibodies to antisense transcripts reflects poor antigen avidity on the part of the antibody rather than indicating an absence of the relevant polypeptide with DPR inclusions per se. shown), and on Southern blot (see lane #8 in Figure 1). Although the case showed no TDP-43 pathology, DPR were present in cells of the cerebellum and hippocam- pus (see Table 2), though curiously those NCI in granule cells of the cerebellum were p62-immunopositive, while those in hippocampus dentate gyrus and CA4 regions were not positive for p62. None of the other cases with other histological or genetic forms of FTLD showed p62 positive, TDP-43 negative NCI within either cerebellar granule cells or within the hippocampus. The pathological findings de- scribed here are therefore broadly consistent with those previously reported by others in unselected series of cases of FTLD and/or MND [17-19,24]. Discussion In the present study we have observed p62 positive, TDP-43 negative NCI within cerebellar granule cells, and granule cells of the dentate gyrus and pyramidal cells of the hippocampus (see [17-19,24]) in 13 of 84 (15%) cases of FTLD and in 3 of 23 (13%) cases of MND. Interest- ingly, all cases with expansion showed a FTD, FTD + MND or MND clinical phenotype, and 15/16 bore appropriate TDP-43 protein pathological changes. However, one ex- pansion carrier with clinical FTD (case #13 in Table 1) was of FTLD-tau with CBD pathology. This case was described by us previously [20] and is important as it is only the second case of CBD to be described with expan- sion in C9ORF72 (see [32] for details of the other case), though there was no post-mortem confirmation of CBD in this latter instance. The present case demonstrated an expansion in C9ORF72 both on repeat-primed PCR (not Ratings for DPR immunostaining of NCI were corre- lated with those for each other, and those for p62 immu- nostaining, in granule cells of cerebellum, and dentate gyrus and CA4 neurones of hippocampus. In general, there were significant correlations between p62, poly-GA, poly GP and poly-GR ratings, but not with those involving Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Page 10 of 13 Baker M, Mackenzie IRA, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, Snowden J, Adamson J, Sadovnick AD, Rollinson S, Cannon A, Dwosh E, Neary D, Melquist S, Richardson A, Dickson D, Eriksen J, Robinson T, Zehr C, Dickey CA, Crook R, McGowan E, Mann D, Boeve B, Feldman H, Hutton M: Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 2006, 442:916–919. Author details 1Clinical and Cognitive Sciences Research Group, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, Salford Royal Hospital, Salford M6 8HD, UK. 2Clinical and Cognitive Sciences Research Group, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, A V Hill Building, Oxford Rd, Manchester M13 9PL, UK. 3Mayo Clinic, Jacksonville FLFL32224, USA. 4Department of Neuropathology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 areas CA2/3/4 in hippocampus), although it is acknowl- edged that cells in the dentate gyrus of the hippocampus may contain one or other, but rarely both, types of inclu- sion [19,27]. Those cells containing DPR did not show any other outward signs of neurodegeneration, whether these being small granule cells of dentate gyrus or cerebel- lum, or larger pyramidal cells of hippocampal CA regions, cerebral cortex or Purkinje neurones of the cerebellum. Likewise, there appeared to be no obvious loss (thinning out) of granule cells in either region or depletion of cells from hippocampal pyramidal or cerebellar Purkinje cell layers. Moreover, such changes in the cerebellum appear to be without functional (clinical) consequence, although there have been reports of cerebellar atrophy in expansion bearers [35,36]. whether DPR mediated changes are anything more than a pathological ‘curiosity’, but nonetheless they appear to provide a specific diagnostic tissue marker for the pres- ence of the genetic expansion. Acknowledgements We acknowledge the support of Alzheimers Research UK and Alzheimer’s Society through their funding of the Manchester Brain Bank under the Brains for Dementia Research (BDR) initiative. DMAM and SPB also receive funding from MRC and Wellcome Trust which supported this study in part. Authors’ contributions bl f DMAM was responsible for study design, microscopic assessments, data analysis and paper writing. SR, JBC and SPB performed all genetic analyses and Southern blotting, and assisted with preparation of the manuscript. AR provided technical assistance. JCT performed statistical analysis. JSS assisted with case diagnosis and classification. TG and LP provided C9RANT antibody and details of specificity. MMS and MH provided antibodies and details of specificity, and assisted with manuscript preparation. YD performed all immunohistochemical staining. All authors read and approved the final manuscript. Interestingly, there appeared to be no qualitative or quantitative differences between either the number, or the pattern, of DPR stained NCI between FTLD-TDP type A and type B cases, in either hippocampus or cerebellum. This observation begs the question as to the role of C9ORF72 expansions in determining histological pheno- type and their relationship to TDP-43 pathology. It is possible that variability in repeat size may have a role in this, but we were unable to show any obvious differences in repeat size between Type A and type B cases by Southern blot. Alternatively, it is possible that the expansion con- fers an ‘additive’ effect, through induction of a process marked by p62 pathology, which is superimposed upon a ‘background TDP-43 proteinopathy derived through a similar mechanism to that seen in non-expansion bearers sharing the same TDP-43 histological phenotype. If that were so, then it might be presumed that patients bearing the mutation suffer a ‘double whammy’, and that the ex- pansion plays no real part in determining the TDP-43 proteinopathy and the basic underlying disorder. How- ever, if this ‘chance scenario’ were true, then it might be anticipated that C9ORF72 repeat expansions might com- monly occur in association with other disorders, though there is little, and conflicting, evidence for this in, for ex- ample, Alzheimer’s disease [37-43] or Parkinson’s disease and other parkinsonian syndromes such as Corticobasal syndrome [44-47]. Indeed, it is possible that in at least some of these instances where an expansion has been reported in patients with conditions other than FTLD or MND, the underlying condition may still be FTLD, though presenting in an atypical way [39]. References 1 N D 1. Neary D, Snowden JS, Mann DMA: Frontotemporal lobar degeneration: clinical and pathological relationships. Acta Neuropathol 2007, 114:31–38. 2. Baborie A, Griffiths TD, Jaros E, Richardson A, Ferrari R, Moreno J, Momeni P, McKeith IG, Burn DJ, Duplessis D, Pal P, Rollinson S, Pickering-Brown SM, Thompson JC, Neary D, Snowden JS, Perry R, Mann DMA: Pathological correlates of frontotemporal lobar degeneration in the elderly. Acta Neuropathol 2011, 121:365–373. 3. Shi J, Shaw CL, Richardson AMT, Bailey K, Tian J, Varma AR, Neary D, Snowden JS, Mann DMA: Histopathological changes underlying frontotemporal lobar degeneration with clinicopathological correlation. Acta Neuropathol 2005, 110:501–512. 4. Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, Houlden M, Pickering-Brown SM, Chakraverty S, Isaacs A, Grover A, Hackett J, Adamson J, Lincoln S, Dickson D, Davies P, Petersen RC, Stevens M, De Graaf E, Wauters E, Van Baren J, Hillebrand M, Joosse M, Kwon JM, Nowotny P, Che LK, Norton J, Morris JC, Reed LA, Trojanowski JQ, Basun H, et al: Association of missense and 5′–splice-site mutation in tau with inherited dementia FTDP-17. Nature 1998, 393:702–705. 4. Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, Houlden M, Pickering-Brown SM, Chakraverty S, Isaacs A, Grover A, Hackett J, Adamson J, Lincoln S, Dickson D, Davies P, Petersen RC, Stevens M, De Graaf E, Wauters E, Van Baren J, Hillebrand M, Joosse M, Kwon JM, Nowotny P, Che LK, Norton J, Morris JC, Reed LA, Trojanowski JQ, Basun H, et al: Association of missense and 5′–splice-site mutation in tau with inherited dementia FTDP-17. Nature 1998, 393:702–705. Additional file Additional file 1: Figure S1. Relationship between minimum and maximum size of repeat and age at onset/disease duration. Competing interests The authors declare that they have no competing interests. Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Furthermore there was no immuno- staining of NII containing expanded poly-Q stretches in patients with HD (see also [27]). Collectively, these findings reinforce the suggestion that p62-, DPR-positive NCI are pathognomic for FTLD (and MND) associated with C9ORF72 expansions. It remains uncertain as to how these NCI containing DPR may relate to disease pathogenesis and other fun- damental aspects of disease pathology such as TDP-43 positive NCI and neurites. While NCI containing DPR are located in clinically and pathologically relevant regions, such as the hippocampus and adjacent temporal cortex, they are actually present in cells distant from the major TDP-43 changes (layers V and VI in cerebral cortex and None of the NCI was immunoreactive to poly-PR anti- body, though this antibody did immunolabel chromatin granules, especially in Purkinje cells and CA4 neurones, Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Page 11 of 13 Mann et al. 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Ash PE, Bieniek KF, Gendron TF, Caulfield T, Lin WL, Dejesus-Hernandez M, Van Blitterswijk MM, Jansen-West K, Paul JW III, Rademakers R, Boylan KB, Dickson DW, Petrucelli L: Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 2013, 77:639–646. 14. Van Deerlin VM, Sleiman PMA, Martinez-Lage M, Chen-Plotkin A, Wang LS, Graff Radford NR, Dickson DW, Rademakers R, Boeve BF, Grossman M, Arnold SE, Mann DMA, Pickering-Brown SM, Seelaar H, Heutink P, Van Swieten JC, Murrell JR, Ghetti B, Spina S, Grafman J, Hodges J, Spillantini MG, Gilman S, Lieberman AP, Kaye JA, Woltjer RL, Bigio EH, Mesulam M, Al-Sarraj S, Troakes C, et al: Common variants at 7p21 are associated with frontotemporal lobar degeneration with TDP-43 inclusions. Nat Genet 2010, 42:234–243. 27. Mori K, Weng SM, Arzberger T, May S, Rentzsch K, Kremmer E, Schmid B, Kretzschmar HA, Cruts M, Van Broeckhoven C, Haass C, Edbauer D: The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 2013, 339:1335–1338. 28. Brun A, Englund E, Gustafson L, Passant U, Mann DMA, Neary D, Snowden JS: Clinical, neuropsychological and neuropathological criteria of fronto- temporal dementia. J Neurol Neurosurg Psychiatry 1994, 57:416–418. 15. Conclusion Although it is clear that production of DPR from un- conventional translation of the expansion is a feature of C9ORF72 associated diseases, it is far from certain as to whether such changes drive cell damage and loss, and how they might relate to changes in TDP-43 function and contribute clinical disability. It remains an open question Page 12 of 13 Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Page 13 of 13 46. Lesage S, Le Ber I, Condroyer C, Broussolle E, Gabelle A, Thobois S, Pasquier F, Mondon K, Dion PA, Rochefort D, Rouleau GA, Dürr A, Brice A, French Parkinson’s Disease Genetics Study Group: C9orf72 repeat expansions are a rare genetic cause of Parkinsonism. Brain 2013, 136:385–391. 47. Majounie E, Abramzon Y, Renton AE, Keller MF, Traynor BJ, Singleton AB: Large C9orf72 repeat expansions are not a common cause of Parkinson’s disease. Neurobiol Aging 2013, 33(2527):e1–e2. doi:10.1186/2051-5960-1-68 Cite this article as: Mann et al.: Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathologica Communications 2013 1:68. 33. Mori K, Lammich S, Mackenzie IR, Forné I, Zilow S, Kretzschmar H, Edbauer D, Janssens J, Kleinberger G, Cruts M, Herms J, Neumann M, Van Broeckhoven C, Arzberger T, Haass C: hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations. Acta Neuropathol 2013, 125:413–423. 47. Majounie E, Abramzon Y, Renton AE, Keller MF, Traynor BJ, Singleton AB: Large C9orf72 repeat expansions are not a common cause of Parkinson’s disease. Neurobiol Aging 2013, 33(2527):e1–e2. 34. Brettschneider J, Van Deerlin VM, Robinson JL, Kwong L, Lee EB, Ali YO, Safren N, Monteiro MJ, Toledo JB, Elman L, McCluskey L, Irwin DJ, Grossman M, Molina-Porcel L, Lee VMY, Trojanowski JQ: Pattern of ubiquilin pathology in ALS and FTLD indicates presence of C9ORF72 hexanucleotide expansion. Acta Neuropathol 2012, 123:825–839. doi:10.1186/2051-5960-1-68 Cite this article as: Mann et al.: Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathologica Communications 2013 1:68. 35. Mahoney CJ, Beck J, Rohrer JD, Lashley T, Mok K, Shakespeare T, Yeatman T, Warrington EK, Schott JM, Fox NC, Rossor MN, Hardy J, Collinge J, Revesz T, Mead S, Warren JD: Frontotemporal dementia with the C9ORF72 hexanucleotide repeat expansion: clinical, neuroanatomical and neuropathological features. Brain 2012, 135:736–750. 36. Whitwell JL, Weigand SD, Boeve BF, Senjem ML, Gunter JL, DeJesus-Hernandez M, Rutherford NJ, Baker M, Knopman DS, Wszolek ZK, Parisi JE, Dickson DW, Petersen RC, Rademakers R, Jack CR Jr, Josephs KA: Neuroimaging signatures of frontotemporal dementia genetics: C9ORF72, tau, progranulin and sporadics. Brain 2012, 135:794–806. 37. Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Rollinson S, Halliwell N, Young K, Bennion Callister J, Toulson G, Gibbons L, Davidson Y, Robinson A, Gerhard A, Richardson A, Neary D, Snowden J, Mann D, Pickering Brown SM: Analysis of the hexanucleotide repeat in C9ORF72 in Alzheimer’s disease. Neurobiol Ageing 1846, 2012(33):e5–e6. 38. Beck J, Poulter M, Hensman D, Rohrer JD, Mahoney CJ, Adamson G, Campbell T, Uphill J, Borg A, Fratta P, Orrell RW, Malaspina A, Rowe J, Brown J, Hodges J, Sidle K, Polke JM, Houlden H, Schott JM, Fox NC, Rossor MN, Tabrizi SJ, Isaacs AM, Hardy J, Warren JD, Collinge J, Mead S: Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population. Am J Hum Genet 2013, 92:345–353. 39. Cacace R, Van Cauwenberghe C, Bettens K, Gijselinck I, van der Zee J, Engelborghs S, Vandenbulcke M, Van Dongen J, Bäumer V, Dillen L, Mattheijssens M, Peeters K, Cruts M, Vandenberghe R, De Deyn PP, Van Broeckhoven C, Sleegers K: C9orf72 G4C2 repeat expansions in Alzheimer’s disease and mild cognitive impairment. Neurobiol Aging 2013, 34(1712):e1–e7. 40. Davidson YS, Robinson AC, Snowden JS, Mann DMA: Pathological assessments for the presence of hexanucleotide repeat expansions in C9ORF72 in Alzheimer’s disease. Acta Neuropathol Comm 2013, 1:50. 41. Harms M, Benitez BA, Cairns N, Cooper B, Cooper P, Mayo K, Carrell D, Faber K, Williamson J, Bird T, Diaz-Arrastia R, Foroud TM, Boeve BF, Graff-Radford NR, Mayeux R, Chakraverty S, Goate AM, Cruchaga C, NIA-LOAD/NCRAD Family Study Consortium: C9orf72 hexanucleotide repeat expansions in clinical Alzheimer disease. JAMA Neurol 2013, 70:736–741. 42. Harms MB, Neumann D, Benitez BA, Cooper B, Carrell D, Racette BA, Perlmutter JS, Goate A, Cruchaga C: Parkinson disease is not associated with C9ORF72 repeat expansions. Neurobiol Aging 2013, 34(1519):e1–e2. 43. Kohli MA, John-Williams K, Rajbhandary R, Naj A, Whitehead P, Hamilton K, Carney RM, Wright C, Crocco E, Gwirtzman HE, Lang R, Beecham G, Martin ER, Gilbert J, Benatar M, Small GW, Mash D, Byrd G, Haines JL, Pericak-Vance MA, Züchner S: Repeat expansions in the C9ORF72 gene contribute to Alzheimer’s disease in Caucasians. Neurobiol Aging 2013, 34(1519):e5–e12. 44. Majounie E, Abramzon Y, Renton AE, Perry R, Bassett SS, Pletnikova O, Troncoso JC, Hardy J, Singleton AB, Traynor BJ: Repeat expansion in C9ORF72 in Alzheimer’s disease. New Engl J Med 2013, 366:283–284. 45. Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J, Kouri N, Wojtas A, Sengdy P, Hsiung GY, Karydas A, Seeley WW, Josephs KA, Coppola G, Geschwind DH, Wszolek ZK, Feldman H, Knopman DS, Petersen RC, Miller BL, Dickson DW, Boylan KB, Graff-Radford NR, Rademakers R, et al: Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 2011, 72:245–256. 29. Neary D, Snowden JS, Gustafson L, Passant U, Stuss D, Black S, Freedman M, Kertesz A, Robert PH, Albert M, Boone K, Miller BL, Cummings J, Benson DF: Frontotemporal lobar degeneration: a consensus on clinical diagnostic criteria. Neurology 1998, 51:1546–1554. 30. Brooks BR: El escorial world federation of neurology criteria for the diagnosis of amyotrophic lateral sclerosis: subcommittee on motor neuron diseases/amyotrophic lateral sclerosis of the world federation of neurology research group on neuromuscular diseases and the el escorial “Clinical limits of amyotrophic lateral sclerosis” workshop contributors. J Neurol Sci 2004, 124(suppl):96–107. 16. Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, Laaksovirta H, Schymick JC, Van Swieten J, Myllykangas L, Kalimo H, Paetau A, Abramzon Y, Remes AM, Kaganovich A, Scholz SW, Duckworth J, Ding J, Harmer DW, Hernandez DG, Johnson JO, Mok K, Ryten M, Trabzuni D, Guerreiro RJ, Orrell RW, Neal J, Murray A, Pearson J, Jansen IE, et al: A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked amyotrophic lateral sclerosis-frontotemporal dementia. Neuron 2011, 72:257–268. J Neurol Sci 2004, 124(suppl):96–107. 31. Sambrook J: Molecular cloning: a laboratory manual. 3rd edition. 2000. 32. Lindquist SG, Duno M, Batbayli M, Puscmann A, Braendgaard H, Mardosiene S, Svenstrup K, Pinborg LH, Vestergaard K, Hjermind LE, Stokholm J, Andersen BB, Johanssen P, Nielsen JE: Corticobasal and ataxia syndromes widen the spectrum of C9ORF72 hexanucleotide expansion disease. Clin Genet 2013, 83:279–283. 17. Al-Sarraj S, King A, Troakes C, Smith B, Maekawa S, Bodi I, Rogelj B, Al-Chalabi A, Hortobagyi T, Shaw CE: p62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the 46. Lesage S, Le Ber I, Condroyer C, Broussolle E, Gabelle A, Thobois S, Pasquier F, Mondon K, Dion PA, Rochefort D, Rouleau GA, Dürr A, Brice A, French Parkinson’s Disease Genetics Study Group: C9orf72 repeat expansions are a rare genetic cause of Parkinsonism. Brain 2013, 136:385–391. 47. Majounie E, Abramzon Y, Renton AE, Keller MF, Traynor BJ, Singleton AB: Large C9orf72 repeat expansions are not a common cause of Parkinson’s disease. Neurobiol Aging 2013, 33(2527):e1–e2. doi:10.1186/2051-5960-1-68 Cite this article as: Mann et al.: Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathologica Communications 2013 1:68. Mann et al. Acta Neuropathologica Communications 2013, 1:68 http://www.actaneurocomms.org/content/1/1/68 Daoud H, Noreau A, Rochefort D, Paquin-Lanthier G, Gauthier MT, Provencher P, Pourcher E, Dupré N, Chouinard S, Jodoin N, Soland V, Fon EA, Dion PA, Rouleau GA: Investigation of C9orf72 repeat expansions in Parkinson’s disease. Neurobiol Aging 2013, 34(1710):e7–e9. 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Cost effectiveness of using surgery versus skeletal traction in management of femoral shaft fractures at Thika level 5 hospital, Kenya

˜The œPan African medical journal

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Abstract Introduction: A prospective quasi experimental study was undertaken at the Thika level 5 hospital. The study aimed to compare the costs of managing femoral shaft fracture by surgery as compared to skeletal traction. Methods: sixty nine (46.6%) patients were enrolled in group A and managed surgically by intramedullary nailing while 79 (53.4%) patients were enrolled in group B and managed by skeletal traction. Exclusion criteria included patients with pathological fractures and previous femoral fractures. Data was collected by evaluation of patients in patient bills using a standardized questionnaire. The questionnaire included cost of haematological and radiological tests, bed fees, theatre fees and physiotherapy costs. The data was compiled and analyzed using SPSS version 16. Person's chi square and odds ratios were used to measure associations and risk analysis respectively. Results: A higher proportion of patients (88.4%) in group A were hospitalized for less than one month compared to 20 patients (30.4%) in group B (p, 0.001).Total cost of treatment in group A was significantly lower than in group B. Nineteen (27.9%) patients who underwent surgery paid a total bill of Ksh 5000-7500 compared to 7(10.4%) who were treated by traction. The financial cost benefit of surgery was further complimented by better functional outcomes. Conclusion: The data indicates a cost advantage of managing femoral shaft fracture by surgery compared to traction. Furthermore the longer hospital stay in the traction group is associated with more malunion, limb deformity and shortening. Open Access Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Everisto Opondo1,&, Peter Wanzala2, Ansellimo Makokha3 1Jomo Kenyatta University of Agriculture and Technology, College of Health Sciences, Nairobi, Kenya, 2Kenya Medical Research Institute, Nairobi, Kenya, 3Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya &Corresponding author: Opondo Everisto, Jomo Kenyatta University of Agriculture and Technology, College of Health Sciences,, Box 105 002 02 Nairobi, Kenya Key words: Fracture shaft femur, cost effectiveness, functional outcome Key words: Fracture shaft femur, cost effectiveness, functional outcome Received: 11/02/2013 - Accepted: 16/05/2013 - Published: 06/06/2013 Received: 11/02/2013 - Accepted: 16/05/2013 - Published: 06/06/2013 Introduction Globally there is a growing incidence of traumatic injury to long bones especially involving the lower limbs .The femur is the largest bone of the body and principal weight bearing bone of the lower extremity that is commonly injured following lower extremity trauma. Femoral fractures are usually associated with considerable mortality and morbidity especially if they follow road traffic accidents. The economic burden of these injuries is felt most in the low resource setting countries with low Gross Domestic Product (GDP) and per capita incomes [1-3]. The allocation into each treatment group was done after the patients were given sufficient information on the treatment options and allowed to make a choice. At casualty detailed history about injury, co morbidity and clinical findings were recorded in a questionnaire. Patients diagnosed to have pathological fractures based on history and radiological findings were excluded from the study. After basic data was collected and investigations done the patients in group A underwent surgery while those in group B were managed by skeletal traction (Perkins method). For the patients in group A, intramedullary fixation of the fracture was done on the next elective surgical list. The surgery was done through a lateral approach and the fracture fixed after reduction with an interlocking nail without fluoroscopy. For the patients enrolled into group B skeletal traction was applied within 24 hour from admission with a Steinman's traction pin inserted in the proximal tibia. While in the ward and after discharge all the patients were followed up regularly every two weeks and evaluated for clinical and radiological union and complications. Over 3,000 Kenyans die as a result of trauma related injuries on Kenyan roads every year, most of them being aged between the ages of 15 and 44 years [3]. The cost to the economy from these accidents has been estimated to exceed US$ 50 million exclusive of the actual loss of life [4]. Unfortunately, road safety trends in Kenya are worsening with more than 75% of road traffic casualties being economically productive young adults in urban areas. Pedestrians and passengers are the most vulnerable and they account for 80% of the deaths from injuries sustained following road traffic accidents. Pedestrians are more likely to be killed in urban areas, whereas passengers are the majority killed on intercity highways that transverse rural setting. Introduction Traction has been the traditional method of treatment of femoral fractures but this method has proved to have a high rate of malunion and knee stiffness. There has been a gradual shift towards surgery as the ideal treatment but very few studies have evaluated the cost effectiveness of the newer interventions in comparison to traction [1]. Review after discharge was done every two weeks for first two months then monthly thereafter. The review in the outpatient clinics included evaluation for fracture healing, local complications and limb mobility. The items considered in treatment cost analysis included the costs of ward bed, drugs, radiographs, laboratory investigations, physiotherapy and theatre fees. The fixed costs such as infrastructure and depreciation value of the initial equipment costs were not considered. The goal of treatment is usually restoration of limb anatomy and early mobilization without long term complications [4,5]. Traditionally in third world countries femoral shaft fractures are managed conservatively by traction and to a small extent by surgery using various nailing and plating techniques. In poor resource setting lack of human capital and material resources may preclude internal fixation of long bone fractures. In early 2000s, the Surgical Implant Generation Network (SIGN) nailing system was introduced in selected district and regional hospitals in Kenya including Thika Level 5 Hospital. It is a solid nail designed for insertion without image intensification, fracture table or use of power reamer and thus specifically adapted for resource poor environments. The frequencies and crosstabs procedure were used to create two way and multiway tables. Statistics and graphical displays were used for describing variables, charts and graphs. After tabulation p values were determined using persons' chi-squares. P value of less than 0.05 was considered significant. All the statistical methods were carried out through the SPSS for windows version 17.0. The results were analyzed for risk of occurrence and associations using the odds ratio and logistic regression. Economic cost evaluations (CEA) are widely and commonly used tools in the developed countries for comparison of different interventions and strategies. The purpose of CEA is to assess the relative cost of one intervention over another for a given condition. One intervention is more cost effective if: (1) it is less costly with equal or better outcome or (2) it is more costly with better outcomes, and the added benefit is worth the added cost [3,4]. Socio demographics of study population A total of 148 cases of femoral shaft fractures were seen in the study. 102(75.7%) of the patients were aged between 18 and 50 years with a mean age of 42 years. Males (115) had a significantly higher frequency compared to females (33) (Table 1) with a ratio of 3.5:1. The right side (81.1%) was affected more commonly than the left side (18.9%) (Table 1). This study was carried out to compare the cost effectiveness of managing femoral shaft fractures surgically versus traction at a regional referral hospital in Kenya. The study findings indicate a better cost advantage of treating femoral shaft fractures by intramedullary nailing as compared to using skeletal traction. Severe trauma due to road traffic accidents was the most common injury mode seen in 83.1% of the patients and mild trauma due to falls was seen in 16.9% of the study patients. Pan African Medical Journal. 2013; 15:42. doi:10.11604/pamj.2013.15.42.2451 This article is available online at: http://www.panafrican-med-journal.com/content/article/15/42/full/ © Everisto Opondo et al. The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) allocated to either of the treatment groups based on treatment modality. Sixty nine (46.6%) patients were enrolled in group A and managed surgically by intramedullary nailing while 79 (53.4%) patients were enrolled in group B and managed by skeletal traction. Among the 148 patients 115(77.7%) were males and 33(22.3%) were females. Age range was 19 to 96 years with a mean age of 42 years. Right femoral fracture was seen in 120(81.1%) patients and left femur fracture in 28(18.9%). Functional outcomes Functional outcomes in terms of limb mobility, malunion and limb length discrepancies were analyzed after 12 weeks post trauma. Majority of the patients (55.1 %) who underwent surgery attained normal mobility without any support compared to 29.1% in the group managed by traction (OR 3.80 and p 0.004). More patients (43.1%) in the conservative group developed malunion compared to the surgery group with 30.4% having angular malunions greater than 10 degrees radiologically. Overall 58 (84.1%) patients in the surgery group archived union without malunion compared to 45(57%) patients in the conservative group (p=0.001). Discussion With the considerable advances having been achieved in the development of orthopaedic implants it's imperative that newer modes of treatment are analysed against the routine conservative options that are commonly used in developing countries. The evidence derived from such studies should then form the informed basis for changes in patient care especially in resource constrained setting like ours. Patients choice of treatment modalities should based on available evidence of advantage of one method over another. Authors’ contributions All the authors contributed to the study design, data collection and analysis and final manuscript preparation. Conclusion It is safe to conclude that in this setting better clinical outcome was attained at a lower cost with surgery making it more cost effective than Perkins traction in management of adult femoral shaft fractures. The results also confirm the safety of intramedullary nailing at a lower level referral Hospital with minimal equipment. Tables Throughout the world, there are many sites that have reported reduction in variable costs after use of surgery as compared to traction in treatment of femoral shaft fractures [4-10]. The reduction in costs can be attributed to reduced hospital stay, cost of physiotherapy and X-rays. Gosselin and Lovary in a study on a cohort of 53 patients with similar demographic (mean age of 34.2 and 81% male) to ours treated by Perkins traction in Sierra Leone, a West African country reported malunion rates of 9% and shortening of more than 2cm of 6% [12]. Table 1: Distribution of age in years, duration of hospital stay in months and total bill paid in Kenya shillings among the study patients by total and by treatment option Table 2: Fracture patterns among the study patients by treatment method Table 3: Local and systemic complications among the study patients by treatment method Table 3: Local and systemic complications among the study patients by treatment method In a study evaluating the impact of introduction of interlocking nail at Mulago hospital in Uganda Sekimpe et, al reported a malunion rate of 8% that compares with our malunion rate of 8.7% for the patients who underwent surgery in this study. The limitation of that study though was the lack of a comparison group. Injury patterns Fracture was seen most commonly in the middle third of the diaphysis. The most common fracture pattern was comminuted and oblique (Table 2). 62.8% of the patients had fracture femur plus one associated systemic injury while 22.3% had more than two injuries systemic injuries and 14.9% had more than three systemic injuries. The most common associated injury was head injury in 27(18.2%) patients followed by other musculoskeletal injuries in 20(13.5%). There was no significant difference in local and systemic complications seen in both groups (Table 3). Methods The average duration of hospital stay in the surgery groups was thirty days and sixty days in the conservative group. 61(88.4%) of the patients who underwent surgery were discharged within one This prospective quasi-experimental study was carried out at Thika level 5 Hospital located in central province of Kenya from June 2010 to June 2011. Patients were consecutively recruited at casualty and Page number not for citation purposes 2 month of admission while 24(30.4%) in the traction group were discharged in the same duration. month of admission while 24(30.4%) in the traction group were discharged in the same duration. patients who were treated by traction being discharged in the same duration. The reduced hospital stay in the surgery group lead to reduced bed charges, physiotherapy and radiology costs. The average cost of treatment for patients who underwent surgery was Ksh 9761 compared to those managed conservatively Ksh 13594. The average cost of treatment for patients who underwent surgery was Ksh 9761 compared to those managed conservatively Ksh 13594. Thirteen patients had their bills waived due to financial and social reasons. A similar retrospective study in Cambodia at provincial trauma hospital reported the cost of treating one patient in traction as corresponding to treatment of three patient's surgically. That study also reported 57% of patients treated by nailing as having full mobility at discharge and only 22% in the traction group [5]. These results compare favourably with our study where 51% of patients in the surgery group had excellent functional outcome and 29% in the traction group. Acknowledgments Special thanks to the Medical superintendent and ethical committee at the Thika Level 5 Hospital for granting permission to carry out the study. The authors would like to thank Mr Moses Mwangi of Centre for Public Health Research, KEMRI for statistical input. Competing interests The authors declare no conflicting interests in this study. References 1. Whittle AP and Wood GW II. Fractures of the lower extremity. In: Campbell's Operative Orthopedics, 10th ed, Vol 3, Canale, ST (Ed). 2010, Mosby, St. Louis p.2825. This study found an average duration of hospital stay in the surgery group to be thirty days and sixty days in the conservative group. A higher proportion of patients (88.4%) who underwent surgery were discharged within one month of admission compared to 30.4% of Page number not for citation purposes 3 2. Beveridge M, Howard A. The burden of orthopaedic disease in developing countries. J Bone Joint Surg Am. 2004 Aug;86- A(8):1819-22. PubMed | Google Scholar 3. Oyaya CO, Rifkin SB. Health sector reforms in Kenya: an examination of district level planning. Health Policy. 2003 Apr;64(1):113-27. PubMed | Google Scholar 4. Gekonge NN. Traffic accident statistics and their economic implications in Kenya. Medicus. 1990; 9(12): 3-19.2. PubMed | Google Scholar 5. Robert JB et al. Intramedullary nailing of femoral shaft fractures. J Bone Joint Surg. 1998; 70-A: 1453-62. PubMed | Google Scholar 6. Fadero PE, Alabi S, Adebule GT, Odunubi OO et al. Locked intramedullary nailing for the treatment of femoral shaft fractures: experience and result in 19 cases. Niger J Med. 2008 Apr-Jun;17(2):168-72. PubMed | Google Scholar 7. Bozic KJ, Rosenberg AG, Huckman RS, Herndon JH. Economic evaluation in orthopaedics. J Bone Joint Surg Am. 2003 Jan;85- A(1):129-42. PubMed | Google Scholar 8. Gosselin RA, Heitto M, Zirkle L. Cost-effectiveness of replacing skeletal traction by interlocked intramedullary nailing for femoral shaft fractures in a provincial trauma hospital in Cambodia. Int Orthop. 2009 Oct;33(5):1445-8. PubMed | Google Scholar 2. Beveridge M, Howard A. The burden of orthopaedic disease in developing countries. J Bone Joint Surg Am. 2004 Aug;86- A(8):1819-22. PubMed | Google Scholar 3. Oyaya CO, Rifkin SB. Health sector reforms in Kenya: an examination of district level planning. Health Policy. 2003 Apr;64(1):113-27. PubMed | Google Scholar 9. Sekimpi P, Okike K, Zirkle L, Jawa A. Femoral fracture fixation in developing countries: an evaluation of the Surgical Implant Generation Network (SIGN) intramedullary nail. J Bone Joint Surg Am. 2011 Oct 5;93(19):1811-8. PubMed | Google Scholar 4. Gekonge NN. Traffic accident statistics and their economic implications in Kenya. Medicus. 1990; 9(12): 3-19.2. PubMed | Google Scholar 10. Downing ND, Griffin DR, Davis TR. A comparison of the relative costs of cast treatment and intramedullary nailing for tibial diaphyseal fractures in the UK. Injury. References 1997 Jun-Jul;28(5- 6):373-5. PubMed | Google Scholar 5. Robert JB et al. Intramedullary nailing of femoral shaft fractures. J Bone Joint Surg. 1998; 70-A: 1453-62. PubMed | Google Scholar 6. Fadero PE, Alabi S, Adebule GT, Odunubi OO et al. Locked intramedullary nailing for the treatment of femoral shaft fractures: experience and result in 19 cases. Niger J Med. 2008 Apr-Jun;17(2):168-72. PubMed | Google Scholar 11. Reeves RB, Ballard RI, Hughes JL. Internal fixation versus traction and casting of adolescent femoral shaft fractures. J Pediatr Orthop. 1990 Sep-Oct;10(5):592-5. PubMed | Google Scholar 11. Reeves RB, Ballard RI, Hughes JL. Internal fixation versus traction and casting of adolescent femoral shaft fractures. J Pediatr Orthop. 1990 Sep-Oct;10(5):592-5. PubMed | Google Scholar 7. Bozic KJ, Rosenberg AG, Huckman RS, Herndon JH. Economic evaluation in orthopaedics. J Bone Joint Surg Am. 2003 Jan;85- A(1):129-42. PubMed | Google Scholar 12. Gosselin R, Lavaly D. Perkins traction for adult femoral shaft fractures: a report on 53 patients in Sierra Leone. Int Orthop. 2007 Oct;31(5):697-702. Page number not for citation purposes References PubMed | Google Scholar Table 1: Distribution of age in years, duration of hospital stay in months and total bill paid in Kenya shillings among the study patients by total and by treatment option All patients Variables N Mean SD Minimum Maximum Age in years 148 42 19 18 96 Duration of hospital stay in months 148 2 1 1 4 Total bill paid 135 11663 6111 400 40000 Patients treated with surgery option Variables N Mean SD Minimum Maximum Age in years 69 35 13 18 73 Duration of hospital stay in days 69 30 30 30 90 Total bill paid 68 9761 4565 4750 27000 Patients treated with conservative option Variables N Mean SD Minimum Maximum Age in years 79 47 22 18 96 Duration of hospital stay in days 79 60 30 30 120 Total bill paid 67 13594 6867 400 40000 Table 1: Distribution of age in years, duration of hospital stay in months and total bill paid in Kenya shillings among the study patients by total and by treatment option Page number not for citation purposes 4 Table 2: Fracture patterns among the study patients by treatment method Table 2: Fracture patterns among the study patients by treatment method Variables Total (n=148) Surgery (n=69) Conservative (n=79) N % N % N % Plain femur X-ray Right 120 81.1 53 76.8 67 84.8 Left 28 18.9 16 23.2 12 15.2 Fracture pattern Transverse 34 23.0 14 20.3 20 25.3 Spiral 20 13.5 13 18.8 7 8.9 Oblique 57 38.5 22 31.9 35 44.3 Comminuted 37 25.0 20 29.0 17 Table 3: Local and systemic complications among the study patients by treatment method Variables Total (n=148) Surgery (n=69) Conservative (n=79) OR 95% CI p value N % n % N % Lower Upper Local complications Yes 72 48.6 33 47.8 39 49.4 Reference No 76 51.4 36 52.2 40 50.6 1.06 0.56 2.03 0.852 Systemic complications Yes 45 30.4 26 37.7 19 24.1 Reference No 103 69.6 43 62.3 60 75.9 0.52 0.26 1.06 0.072 ocal and systemic complications among the study patients by treatment method Page number not for citation purposes 5

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English

Critical elliptic systems involving multiple strongly–coupled Hardy–type terms

Advances in nonlinear analysis

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Adv. Nonlinear Anal. 2020; 9: 866–881 Dongsheng Kang*, Mengru Liu, and Liangshun Xu Critical elliptic systems involving multiple strongly–coupled Hardy–type terms Dongsheng Kang*, Mengru Liu, and Liangshun Xu https://doi.org/10.1515/anona-2020-0029 Received November 25, 2018; accepted March 29, 2019. https://doi.org/10.1515/anona-2020-0029 Received November 25, 2018; accepted March 29, 2019. https://doi.org/10.1515/anona-2020-0029 Received November 25, 2018; accepted March 29, 2019. Abstract: In this paper, we study the radially–symmetric and strictly–decreasing solutions to a system of critical elliptic equations in RN, which involves multiple critical nonlinearities and strongly–coupled Hardy– type terms. By the ODEs analysis methods, the asymptotic behaviors at the origin and infnity of solutions are proved. It is found that the singularities of u and v in the solution (u, v) are at the same level. Finally, an explicit form of least energy solutions is found under certain assumptions, which has all of the mentioned properties for the radial decreasing solutions. Keywords: critical elliptic system; radial decreasing solution; asymptotic property; Hardy term Keywords: critical elliptic system; radial decreasing solution; asymptotic property; Hardy term MSC: 35J47, 35J50 Keywords: critical elliptic system; radial decreasing solution; asymptotic property; Hardy term Open Access. © 2019 D. Kang et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution alone 4.0 License. *Corresponding Author: Dongsheng Kang, School of Mathematics and Statistics, South–Central University for Nationalities, Wuhan 430074, China, E-mail: [email protected] 1 Introduction In this paper, we study the following critical elliptic system involving multiple coupled Hardy–type terms:             −∆u −µ1u + λv |x|2 = u2*−1 + ηα 2* uα−1vβ, x ∈RN \ {0}, −∆v −λu + µ2v |x|2 = v2*−1 + ηβ 2* uαvβ−1, x ∈RN \ {0}, (1.1) (1.1)    (u, v) ∈(D1,2(RN))2, u, v > 0, x ∈RN \ {0}, where D1,2(RN) is the completion of C∞ 0 (RN) with respect to (R RN |∇· |2dx)1/2 and the parameters satisfy the assumption: where D1,2(RN) is the completion of C∞ 0 (RN) with respect to (R RN |∇· |2dx)1/2 and the parameters satisfy the assumption: (H1) N ≥5, 1 < α, β < 2, α + β = 2*, η > 0, ¯µ > µ1 ≥µ2 > 0, λ > 0, λ2 < minµ1µ2, (¯µ −µ1)(¯µ −µ2) . (H1) N ≥5, 1 < α, β < 2, α + β = 2*, η > 0, ¯µ > µ1 ≥µ2 > 0, λ > 0, λ2 < minµ1µ2, (¯µ −µ1)(¯µ −µ2) . 2* := 2N N−2 is the critical Sobolev exponent and ¯µ := N−2 2
2 is the best constant in the Hardy inequality ([1]): 2* := 2N N−2 is the critical Sobolev exponent and ¯µ := N−2 2
2 is the best constant in the Hardy inequality ([1]): ritical Sobolev exponent and ¯µ := N−2 2
2 is the best constant in the Hardy inequality ([1]): 2 Z RN u2 |x|2 dx ≤1 ¯µ Z RN |∇u|2dx, ∀u ∈D1,2(RN). Z RN u2 |x|2 dx ≤1 ¯µ Z RN |∇u|2dx, ∀u ∈D1,2(RN). Mengru Liu, School of Mathematics and Statistics, South–Central University for Nationalities, Wuhan 430074, China Liangshun Xu, School of Mathematics and Statistics, Central China Normal University, Wuhan 430079, China 1 Introduction In particular, Terracini proved that S(µ) has the unique positive extremals ([5]): Vε µ(x) := ε 2−N 2 Uµ(ε−1x) , ∀ε > 0, µ ∈[0, ¯µ), (1.3) Vε µ(x) := ε 2−N 2 Uµ(ε−1x) , ∀ε > 0, µ ∈[0, ¯µ), (1.3) Uµ(x) := 2N (¯µ −µ) p ¯µ  √¯µ 2  |x| √¯µ −√¯µ −µ √¯µ + |x| √¯µ + √¯µ −µ √¯µ −√¯µ , by which many elliptic problems involving the Hardy inequality have been studied. Furthermore, elliptic sys- tems corresponding to the case λ = 0 in (1.1) have been studied (e.g. [11] and [3] for the existence of solutions, [4], [13] and [14] for the asymptotic properties of solutions, [15] and [16] for the case without Hardy terms). by which many elliptic problems involving the Hardy inequality have been studied. Furthermore, elliptic sys- tems corresponding to the case λ = 0 in (1.1) have been studied (e.g. [11] and [3] for the existence of solutions, [4], [13] and [14] for the asymptotic properties of solutions, [15] and [16] for the case without Hardy terms). In this paper, we study the asymptotic behaviors at the origin and infnity of the radially–symmetric and strictly–decreasing solution (u, v) to (1.1) by the ODEs analysis methods. The existence of this kind of solutions to (1.1) can be proved by the arguments similar to those of [3]. Elliptic regularity argument shows that the solution (u, v) to (1.1) satisfes u, v ∈C2(RN \ {0}). The following constants and functions are all well defned under (H1): The following constants and functions are all well defned under (H1): A = A(µ1, µ2, λ) := 2λ p (µ1 −µ2)2 + 4λ2 + (µ1 −µ2) , l(µ1, µ2, λ) := s ¯µ −µ1 + µ2 + p (µ1 −µ2)2 + 4λ2 2 , a(l) := p ¯µ −l(µ1, µ2, λ), b(l) := p ¯µ + l(µ1, µ2, λ), (1.4) (1.4) f(τ) := ηβ 2* + τα −τ2−β −ηα 2* τ2, τ ≥0, g(τ) := λτ2 + (µ1 −µ2)τ −λ, τ ≥0. (1.5) f(τ) := ηβ 2* + τα −τ2−β −ηα 2* τ2, τ ≥0, (1.5) (1.5) g(τ) := λτ2 + (µ1 −µ2)τ −λ, τ ≥0. g(τ) := λτ2 + (µ1 −µ2)τ −λ, τ ≥0. g(τ) := λτ2 + (µ1 −µ2)τ −λ, τ ≥0. 1 Introduction Under the assumption (H1), the matrix E :=  µ1 λ λ µ2  is positive defnite and for all (u, v) ∈(D1,2(RN))2 there holds that 0 < γ1 < γ2 < ¯µ, and furthermore, γ1(u2 + v2) ≤µ1u2 + 2λuv + µ2v2 ≤γ2(u2 + v2), γ1(u2 + v2) ≤µ1u2 + 2λuv + µ2v2 ≤γ2(u2 + v2), γ1(u2 + v2) ≤µ1u2 + 2λuv + µ2v2 ≤γ2(u2 + v2), *Corresponding Author: Dongsheng Kang, School of Mathematics and Statistics, South–Central University for Nationalities, Wuhan 430074, China, E-mail: [email protected] *Corresponding Author: Dongsheng Kang, School of Mathematics and Statistics, South–Central University for Nationalities, Wuhan 430074, China, E-mail: [email protected] Mengru Liu, School of Mathematics and Statistics, South–Central University for Nationalities, Wuhan 430074, China Mengru Liu, School of Mathematics and Statistics, South–Central University for Nationalities, Wuhan 430074, China Liangshun Xu, School of Mathematics and Statistics, Central China Normal University, Wuhan 430079, China Open Access. © 2019 D. Kang et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution alone 4.0 License. D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 867 D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 867 867 where γ1, γ2, are eigenvalues of the matrix E. According to the Hardy, Sobolev and Young inequalities, the following best constants are well defned ([2–5]): S(µ) := inf u∈D1,2(RN)\{0} Z RN  |∇u|2 −µ u2 |x|2  dx  Z RN |u|2*dx  2 2* , µ < ¯µ, ( ) (1.2) R S(µ1, µ2, λ) := inf (u,v)∈D Z RN  |∇u|2 +|∇v|2 −µ1u2 + 2λuv +µ2v2 |x|2  dx  Z RN |u|2* + |v|2* + η|u|α|v|β
dx  2 2* , (1.2) where D := (D1,2(RN))2 \ {(0, 0)}. Critical elliptic equations and systems involving the Hardy inequality have been studied by many authors (e.g. [2, 5–10] for the problems of single equation, [3], [4], [11–14] for related elliptic systems). and none of above infmums and supremum can be achieved. and none of above infmums and supremum can be achieved. Let (u, v) be a radial decreasing solution to (1.1) and set r = |x| = et, x ∈RN \ {0}, t ∈R. By (H1), (1.4) and above Theorem 1.1, we have that λA < ¯µ −µ1 and the following constant and function are well defned: T0 := inf  T > 0  ¯µ −µ1 −λ v(et) u(et) > 0, ∀t ∈(−∞, −T) ∪(T, ∞)  , ω(t) := s ¯µ −µ1 −λ v(et) u(et) , t ∈(−∞, −T0) ∪(T0, ∞). T0 := inf  T > 0  ¯µ −µ1 −λ v(et) u(et) > 0, ∀t ∈(−∞, −T) ∪(T, ∞)  , ω(t) := s ¯µ −µ1 −λ v(et) u(et) , t ∈(−∞, −T0) ∪(T0, ∞). Theorem 1.2. Suppose that (H1) holds with A < Λ0 and (u, v) is a radially–symmetric and strictly–decreasing solution to the problem (1.1). Set r = |x| = et, x ∈RN \ {0}. Then there exist the constants C1, C2 > 0, such that Theorem 1.2. Suppose that (H1) holds with A < Λ0 and (u, v) is a radially–symmetric and strictly–decreasing solution to the problem (1.1). Set r = |x| = et, x ∈RN \ {0}. Then there exist the constants C1, C2 > 0, such that lim t→+∞e √¯µt+ R t T0 ω(s)dsu(et) = C1, lim t→+∞e(√¯µ+1)t+ R t T0 ω(s)ds|u′(et)| = C1b(l), lim t→−∞e √¯µt+ R −T0 t ω(s)dsu(et) = C2, lim t→−∞e(√¯µ+1)t+ R −T0 t ω(s)ds|u′(et)| = C2a(l), lim t→+∞e √¯µt+ R t T0 ω(s)dsv(et) = C1A, lim t→+∞e(√¯µ+1)t+ R t T0 ω(s)ds|v′(et)| = C1Ab(l), lim t→−∞e √¯µt+ R −T0 t ω(s)dsv(et) = C2A, lim t→−∞e(√¯µ+1)t+ R −T0 t ω(s)ds|v′(et)| = C2Aa(l). lim t→+∞e √¯µt+ R t T0 ω(s)dsu(et) = C1, lim t→+∞e(√¯µ+1)t+ R t T0 ω(s)ds|u′(et)| = C1b(l), lim t→−∞e √¯µt+ R −T0 t ω(s)dsu(et) = C2, lim t→−∞e(√¯µ+1)t+ R −T0 t ω(s)ds|u′(et)| = C2a(l), lim t→+∞e(√¯µ+1)t+ R t T0 ω(s)ds|v′(et)| = C1Ab(l), lim t→−∞e(√¯µ+1)t+ R −T0 t ω(s)ds|v′(et)| = C2Aa(l). lim t→+∞e √¯µt+ R t T0 ω(s)dsv(et) = C1A, lim t→+∞e(√¯µ+1)t+ R t T0 ω(s)ds|v′(et)| = C1Ab(l), lim t→−∞e √¯µt+ R −T0 t ω(s)dsv(et) = C2A, lim t→−∞e(√¯µ+1)t+ R −T0 t ω(s)ds|v′(et)| = C2Aa(l). 1 Introduction Under the assumption (H1), since f(0) = ηβ 2* > 0, f(+∞) = −∞, for all η ∈(0, ∞), then there exists naturally the smallest positive zero Λ0 of f (e.g. [16]). Furthermore, f(τ) > 0 in [0, Λ0) and a Under the assumption (H1), since f(0) = ηβ 2* > 0, f(+∞) = −∞, for all η ∈(0, ∞), Under the assumption (H1), since f(0) = ηβ 2* > 0, f(+∞) = −∞, for all η ∈(0, ∞), 2 then there exists naturally the smallest positive zero Λ0 of f (e.g. [16]). Furthermore, f(τ) > 0 in [0, Λ0) and a direct calculation shows that g(A) = 0 and g(τ) < 0 for all τ ∈[0, A). To study the explicit form minimizers to S(µ1, µ2, λ), we also defne 2 F(τ) := 1 + τ2 (1 + ητβ + τ2*) 2 2* , τ ≥0, F(τmin) := min 0 F(τ), (1.6) F(τ) := 1 + τ2 (1 + ητβ + τ2*) 2 2* , τ ≥0, (1.6) F(τ) := 1 + τ2 (1 + ητβ + τ2*) 2 2* , τ ≥0, (1.6) F(τmin) := min τ≥0 F(τ), 868 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 868 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms µ* := µ1 + 2λτmin + µ2(τmin)2 1 + (τmin)2 , µ* := µ1 + µ2 + p (µ1 −µ2)2 + 4λ2 2 , s* :=  1 + ηα 2* Aβ 1 2−2* , (1.7) µ* : 1 + (τmin)2 , µ* := µ1 + µ2 + p (µ1 −µ2)2 + 4λ2 2 , s* :=  1 + ηα * Aβ 1 2−2* , (1.7) 1 (τmin) µ* := µ1 + µ2 + p (µ1 −µ2)2 + 4λ2 2 , *  ηα β 1 * (1.7) (1.7) s* :=  1 + ηα 2* Aβ 2−2* , t of F(τ). Direct calculation shows that n ≥0 is a minimal point of F(τ). Direct calculation shows that where τmin ≥0 is a minimal point of F(τ). Direct calculation shows that 2λ ≤Aλ + λ A , µ1 + Aλ = µ2 + λ A = µ*, µ* ∈(0, ¯µ). (1.8) (1.8) Note that Λ0, τmin and µ* depend on η but are independent of λ. However, A and µ* depend on λ but are independent of η. 1 Introduction Note that Λ0, τmin and µ* depend on η but are independent of λ. However, A and µ* depend on λ but are independent of η. The main results of this paper are summarized in the following theorems and they are new to the best of our knowledge. It can be checked that the intervals for the parameters are not empty. Theorem 1.1. Suppose that (H1) holds and (u, v) is a radially–symmetric and strictly–decreasing solution to the problem (1.1). Set r = |x|, x ∈RN \ {0}. p ( (i) Then lim r→0+ v(r) u(r) = A, lim r→0+ r u′(r) u(r) = lim r→0+ r v′(r) v(r) = −a(l), lim r→+∞ v(r) u(r) = A, lim r→+∞r u′(r) u(r) = lim r→+∞r v′(r) v(r) = −b(l). (ii) Assume furthermore that A < Λ0, then (ii) Assume furthermore that A < Λ0, then inf r>0 v(r) u(r) = A, inf r>0 r u′(r) u(r) = −b(l), sup r>0 r u′(r) u(r) = −a(l), and none of above infmums and supremum can be achieved. and none of above infmums and supremum can be achieved. lim t→+∞e √¯µt+ R t T0 ω(s)dsv(et) = C1A, lim t→−∞e √¯µt+ R −T0 t ω(s)dsv(et) = C2A, In the following theorem, we fnd the explicit radially–symmetric and strictly–decreasing minimizers to S(µ1, µ2, λ) under certain assumptions, among which there exists an explicit form of least energy solutions to (1.1) satisfying all of the properties mentioned in Theorems 1.1 and 1.2. In the following theorem, we fnd the explicit radially–symmetric and strictly–decreasing minimizers to S(µ1, µ2, λ) under certain assumptions, among which there exists an explicit form of least energy solutions to (1.1) satisfying all of the properties mentioned in Theorems 1.1 and 1.2. D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 869 Theorem 1.3. Suppose that (H1) holds and Vεµ(x) is the minimizer of S(µ) defned as in (1.3). Assume further- more that τmin = A. Then Theorem 1.3. Suppose that (H1) holds and Vεµ(x) is the minimizer of S(µ) defned as in (1.3). Assume further- more that τmin = A. Then µ* = µ*, S(µ1, µ2, λ) = F(A)S(µ*) = F(A)S(µ*), µ* = µ*, S(µ1, µ2, λ) = F(A)S(µ*) = F(A)S(µ*), and S(µ1, µ2, λ) has the radially–symmetric and strictly–decreasing minimizers of the form C(Vε µ*, AVε µ*), C, ε > 0 , among which the problem (1.1) has the explicit form of least energy solutions ns*Vε µ*(x), s*AVε µ*(x)
, ε > 0 o . Corollary 1.4. Suppose that (H1) holds with β < α, η ≥ N N−2. Then there exist µ1, µ2, λ ∈(0, ¯µ) such that τmin = A, S(µ1, µ2, λ) = F(A)S(µ*), and thus S(µ1, µ2, λ) has the minimizers of the form C(Vε µ*(x), AVε µ*(x)), C, ε > 0 . Remark 1.5. Suppose that (H1) holds. Then the existence of radial decreasing ground state solutions to (1.1) can be proved by the arguments similar to those of [3], where the case λ = 0 was studied and by which S(µ1, µ2, λ) is achieved. Taking in (1.1) µ1 > µ2, η = 2, α = β = 2* 2 , then a direct calculation shows that 0 < A < 1, Λ0 = 1, and thus A < Λ0 holds naturally. Remark 1.6. Suppose that (H1) holds with A < Λ0. and none of above infmums and supremum can be achieved. Taking T > max{|t1|, |t2|} and by Lemma 3.3 of this paper, the radial decreasing solution (u, v) to (1.1) satisfes lim t→±∞ω(t) = l(µ1, µ2, λ) > 0, ω(t) < l(µ1, µ2, λ), ∀t ∈R, lim t→±∞ω(t) = l(µ1, µ2, λ) > 0, ω(t) < l(µ1, µ2, λ), ∀t ∈R, lim t→+∞ p ¯µt + R t T0 ω(s)ds b(l) t = 1, lim t→−∞ p ¯µt + R −T0 t ω(s)ds a(l) t = 1, which together with Theorem 1.2 reveals that u and v have the singularities of same level at the origin and infnity. When λ = 0 and the other parameters satisfy (H1), then there exists a critical surface Π for the positive solution (u, v) to (1.1) (e.g. [14]): which together with Theorem 1.2 reveals that u and v have the singularities of same level at the origin and infnity. When λ = 0 and the other parameters satisfy (H1), then there exists a critical surface Π for the positive solution (u, v) to (1.1) (e.g. [14]): Π : α p ¯µ −µ1 = (2 −β) p ¯µ −µ2, Π : α p ¯µ −µ1 = (2 −β) p ¯µ −µ2, such that the singularities of v are diferent above and below Π, which implies that the strongly–coupled critical terms in (1.1) plays a key role for the asymptotic properties of u, v. However, when (H1) holds with λ > 0, then the asymptotic properties of u, v, depend only on the Hardy terms in (1.1). Hence, the methods and conclusions of this paper have crucial diferences with those of [14]. such that the singularities of v are diferent above and below Π, which implies that the strongly–coupled critical terms in (1.1) plays a key role for the asymptotic properties of u, v. However, when (H1) holds with λ > 0, then the asymptotic properties of u, v, depend only on the Hardy terms in (1.1). Hence, the methods and conclusions of this paper have crucial diferences with those of [14]. Remark 1.7. Suppose that (H1) holds. Then Theorem 1.3 shows that the minimizers of S(µ1, µ2, λ) has an explicit relation with those of S(µ*) if τmin = A. In our following work, we will study singularities of solutions to (1.1) when τmin ≠ A. Then combining Remark 1.7. Suppose that (H1) holds. and none of above infmums and supremum can be achieved. Then Theorem 1.3 shows that the minimizers of S(µ1, µ2, λ) has an explicit relation with those of S(µ*) if τmin = A. In our following work, we will study singularities of solutions to (1.1) when τmin ≠ A. Then combining with the conclusions of this paper, the asymptotic properties of the radial decreasing minimizer (U, V) to S(µ1, µ2, λ) will be clear and further studies on (1.1) and related problems can be done, even without the explicit forms of (U, V). with the conclusions of this paper, the asymptotic properties of the radial decreasing minimizer (U, V) to S(µ1, µ2, λ) will be clear and further studies on (1.1) and related problems can be done, even without the explicit forms of (U, V). This paper is organized as follows. Some preliminary results are established in Section 2, Theorems 1.1 and 1.2 are proved in Section 3, and Theorem 1.3 is verifed in Section 4. For convenience, we always denote positive constants as C and omit dx in integrals if no confusion is caused. 870 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 870 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 870 870 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 2 Preliminary results Assume that (H1) holds. Let r = |x| and (u(r), v(r)) be a radially–symmetric and strictly–decreasing solution to (1.1). Then from (1.1) it follows that      rN−1u′(r)
′ = −rN−1 µ1 u r2 + λ v r2 + u2*−1 + ηα 2* uα−1vβ , rN−1v′(r)
′ = −rN−1 λ u r2 + µ2 v r2 + v2*−1 + ηβ 2* uαvβ−1 , (2.1) (2.1) which implies that rN−1u′(r) and rN−1v′(r) are strictly decreasing in (0, +∞). Since u′(r), v′(r) ≤0, then we obtain that u′(r), v′(r) < 0 in (0, +∞). Set which implies that rN−1u′(r) and rN−1v′(r) are strictly decreasing in (0, +∞). Since u′(r), v′(r) ≤0, then we obtain that u′(r), v′(r) < 0 in (0, +∞). Set t = ln r, r = et, r ∈(0, +∞), t ∈R, y1(t) = r √¯µu(r), z1(t) = r √¯µ+1u′(r), y2(t) = r √¯µv(r), z2(t) = r √¯µ+1v′(r). (2.2) (2.2) Since u(r), v(r) ∈C2(0, +∞), y1(t), y2(t) ∈C2(R), by (2.1) and (2.2) we have that Since u(r), v(r) ∈C2(0, +∞), y1(t), y2(t) ∈C2(R), by (2.1) and (2.2) we have that                y′ 1 = p ¯µy1 + z1, z′ 1 = − p ¯µz1 −µ1y1 −λy2−  y2*−1 1 + ηα 2* yα−1 1 yβ 2  , y′ 2 = p ¯µy2 + z2, z′ 2 = − p ¯µz2 −λy1 −µ2y2−  y2*−1 2 + ηβ 2* yα 1yβ−1 2  , (2.3) (2.3) which implies that y1 and y2 satisfy the following ODEs system: which implies that y1 and y2 satisfy the following ODEs system: which implies that y1 and y2 satisfy the following ODEs system: which implies that y1 and y2 satisfy the following ODEs system:      y ′′ 1 = (¯µ −µ1)y1 −λy2 −y2*−1 1 −ηα 2* yα−1 1 yβ 2, y ′′ 2 = (¯µ −µ2)y2 −λy1 −y2*−1 2 −ηβ 2* yα 1yβ−1 2 . 2 Preliminary results Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 871 871 3 Asymptotic properties hat (H1) holds and the functions y1(t), y2(t), are defned as in (2.2). For all l ∈R, i = 1, 2, a direct Assume that (H1) holds and the functions y1(t), y2(t), are defned as in (2.2). For all l ∈R, i = 1, 2, a direct calculation shows that R t y′ i (s)
Assume that (H1) holds and the functions y1(t), y2(t), are defned as in (2.2). For all l ∈R, i = 1, 2, a direct calculation shows that eltyi(t) = yi(0)e R t 0 l+ y′ i (s) yi(s)
ds, t ∈(0, +∞), eltyi(t) = yi(0)e R t 0 l+ y′ i (s) yi(s)
ds, t ∈(0, +∞), e−ltyi(t) = yi(0)e R 0 t l− y′ i (s) yi(s)
ds, t ∈(−∞, 0). (3.1) eltyi(t)
′ = eltyi(t)  l + y′ i(t) yi(t)  , t ∈(0, +∞), e−ltyi(t)
′ = e−ltyi(t)  −l + y′ i(t) yi(t)  , t ∈(−∞, 0). eltyi(t) = yi(0)e R t 0 l+ y′ i (s) yi(s)
ds, t ∈(0, +∞), e−ltyi(t) = yi(0)e R 0 t l− y′ i (s) yi(s)
ds, t ∈(−∞, 0). (3.1) e yi(t) = yi(0)e yi( )
, t ∈(0, +∞), e−ltyi(t) = yi(0)e R 0 t l− y′ i (s) yi(s)
ds, t ∈(−∞, 0). (3.1) (3.1) Furthermore, eltyi(t)
′ = eltyi(t)  l + y′ i(t) yi(t)  , t ∈(0, +∞), e−ltyi(t)
′ = e−ltyi(t)  −l + y′ i(t) yi(t)  , t ∈(−∞, 0). For i = 1, 2, defne the functions gi(l) := +∞ Z 0  l + y′ i(s) yi(s)  ds, l ∈R. gi(l) := +∞ Z 0  l + y′ i(s) yi(s)  ds, l ∈R. gi(l) := +∞ Z 0  l + y′ i(s) yi(s)  ds, l ∈R. From Lemma 2.1 it follows that From Lemma 2.1 it follows that − p ¯µ −µi + y′ i(s) yi(s) < 0 < p ¯µ −µi + y′ i(s) yi(s) , s ∈R. (3.2) − p ¯µ −µi + y′ i(s) yi(s) < 0 < p ¯µ −µi + y′ i(s) yi(s) , s ∈R. (3.2) (3.2) For any ε > 0, from (3.2) it follows that For any ε > 0, from (3.2) it follows that gi( p ¯µ −µi + ε) = +∞, gi(0) = −∞. 2 Preliminary results (2.4) (2.4) The complete integral of (2.4) is given by The complete integral of (2.4) is given by The complete integral of (2.4) is given by The complete integral of (2.4) is given by V(y1, y2, y′ 1, y′ 2) := 1 2 |y′ 1|2 + |y′ 2|2
−1 2 ¯µ −µ1
y2 1 −1 2 ¯µ −µ2
y2 2 + λy1y2 + 1 2* y2* 1 + y2* 2 + ηyα 1yβ 2
. V(y1, y2, y′ 1, y′ 2) := 1 2 |y′ 1|2 + |y′ 2|2
−1 2 ¯µ −µ1
y2 1 −1 2 ¯µ −µ2
y2 2 + λy1y2 + 1 2* y2* 1 + y2* 2 + ηyα 1yβ 2
. Set V(t) = V(y1(t), y2(t), y′ 1(t), y′ 2(t)). From (2.4) it follows that V′(t) = 0. Then V(t) is a constant an Set V(t) = V(y1(t), y2(t), y′ 1(t), y′ 2(t)). From (2.4) it follows that V′(t) = 0. Then V(t) is a constant and we set V(t) ≡K0, ∀t ∈R. Set V(t) = V(y1(t), y2(t), y′ 1(t), y′ 2(t)). From (2.4) it follows that V′(t) = 0. Then V(t) is a constant and we set V(t) ≡K0, ∀t ∈R. Set V(t) = V(y1(t), y2(t), y′ 1(t), y′ 2(t)). From (2.4) it follows that V′(t) = 0. Then V(t) is a constant and we set V(t) ≡K0, ∀t ∈R. V(t) ≡K0, ∀t ∈R. V(t) ≡K0, ∀t ∈R. V(t) ≡K0, ∀t ∈R. V(t) ≡K0, ∀t ∈R. Lemma 2.1. Suppose that (H1) holds. Then K0 = 0. Furthermore, y′ i(t) yi(t)  < p ¯µ −µi, ∀t ∈R, i = 1, 2, lim t→±∞yi(t) = lim t→±∞y′ i(t) = lim t→±∞y′′ i (t) = 0, i = 1, 2. (2.5) (2.5) Proof. The arguments are similar to those of Lemmas 2.1 and 2.2 in [14], where the case λ = 0 of (1.1) was studied. The details are omitted for simplicity. Proof. The arguments are similar to those of Lemmas 2.1 and 2.2 in [14], where the case λ = 0 of (1.1) was studied. The details are omitted for simplicity. Proof. The arguments are similar to those of Lemmas 2.1 and 2.2 in [14], where the case λ = 0 of (1.1) was studied. The details are omitted for simplicity. D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 871 D. 3 Asymptotic properties Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 872 Furthermore, Furthermore, Furthermore, 0 ≤l1, l3 ≤ p ¯µ −µ1, 0 ≤l2, l4 ≤ p ¯µ −µ2. Therefore, we can assume that l ≥0 when studying li, 1 ≤i ≤4. Therefore, we can assume that l ≥0 when studying li, 1 ≤i ≤4. Lemma 3.2. Suppose that (H1) holds. Then l1 = l2, l3 = l4. Lemma 3.2. Suppose that (H1) holds. Then l1 = l2, l3 = l4. Proof. For all ε > 0, from the defnitions of li it follows that Proof. For all ε > 0, from the defnitions of li it follows that t Z 0  li ± ε + y′ i(s) yi(s)  ds →±∞as t →+∞, ε > 0, i = 1, 2, which together with (3.1) implies that which together with (3.1) implies that e(li−ε)tyi(t) = yi(0)e R t 0 li−ε+ y′ i (s) yi(s)
ds →0 as t →+∞, i = 1, 2, e(li+ε)tyi(t) = yi(0)e R t 0 li+ε+ y′ i (s) yi(s)
ds →+∞as t →+∞, i = 1, 2. Consequently, 0 < e(li−ε)tyi(t) < 1 < e(li+ε)tyi(t) as t →+∞, i = 1, 2, (3.5) (3.5) which implies that which implies that e(l1−l2−2ε)t < y2 y1 < e(l1−l2+2ε)t as t →+∞, ε > 0, e(l1−l2−2ε)t < y2 y1 < e(l1−l2+2ε)t as t →+∞, ε > 0, If l1 > l2, then by taking 2ε < l1 −l2 we have that lim t→+∞ y2 y1 = +∞. From (2.4) and the L’Hospital rule it follows that If l1 > l2, then by taking 2ε < l1 −l2 we have that lim t→+∞ y2 y1 = +∞. From (2.4) and the L’Hospital rule it follows that 2 lim t→+∞ |y′ 1|2 y2 1 = lim t→+∞ y′′ 1 y1 = lim t→+∞  ¯µ −µ1 −λ y2 y1 −ηα 2* yα−2 1 yβ 2  = −∞, a contradiction. If l1 < l2, then lim t→+∞ y1 y2 = +∞and therefore lim t→+∞ |y′ 2|2 y2 2 = lim t→+∞ y′′ 2 y2 = lim t→+∞  ¯µ −µ2 −λ y1 y2 −ηβ 2* yα 1yβ−2 2  = −∞, a contradiction. a contradiction. Therefore l1 = l2 must hold and similarly l3 = l4 also holds. Therefore l1 = l2 must hold and similarly l3 = l4 also holds. 3 Asymptotic properties (3.3) gi( p ¯µ −µi + ε) = +∞, gi(0) = −∞. (3.3) gi( p ¯µ −µi + ε) = +∞, gi(0) = −∞. (3.3) Therefore the following constants are well defned: Therefore the following constants are well defned: l* i := supl | gi(l) < +∞ , l** i := infl | gi(l) > −∞ . Since ε > 0 is arbitrary, from (3.3) it follows that Since ε > 0 is arbitrary, from (3.3) it follows that 0 ≤l** i ≤l* i ≤ p ¯µ −µi, i = 1, 2. (3.4) 0 ≤l** i ≤l* i ≤ p ¯µ −µi, i = 1, 2. 0 ≤l** i ≤l* i ≤ p ¯µ −µi, i = 1, 2. (3.4) Lemma 3.1. Suppose that (H1) holds. Then l* i = l** i ≥0, i = 1, 2. Lemma 3.1. Suppose that (H1) holds. Then l* i = l** i ≥0, i = 1, 2. Proof. Assume that l** i < l* i . Then there exist a, b ∈R such that l** i ≤b < a ≤l* i and −∞< gi(b) < gi(a) < +∞. Furthermore, gi(a) −gi(b) = +∞ Z 0 (a −b)ds = +∞, gi(a) −gi(b) = +∞ Z 0 (a −b)ds = +∞, a contradiction. From (3.4) it follows that l* i = l** i ≥0. a contradiction. From (3.4) it follows that l* i = l** i ≥0. According to Lemma 3.1, the following constants are well defned: li := sup l | gi(l) < +∞ = inf l | gi(l) > −∞ , i = 1, 2. According to Lemma 3.1, the following constants are well defned: 3.1, the following constants are well defned: li := sup l | gi(l) < +∞ = inf l | gi(l) > −∞ , i = 1, 2. Similarly, we defne the functions Similarly, we defne the functions gi+2(l) := 0 Z −∞  l −y′ i(s) yi(s)  ds, i = 1, 2. gi+2(l) := 0 Z −∞  l −y′ i(s) yi(s)  ds, i = 1, 2. Arguing as above, the following constants are well defned Arguing as above, the following constants are well defned li := sup l | gi(l) < +∞ = inf l | gi(l) > −∞ , i = 3, 4. 872 | D. Lemma 3.3. Suppose that (H1) holds. Let A and l(µ1, µ2, λ) be defned as in (1.4). Then Lemma 3.3. Suppose that (H1) holds. Let A and l(µ1, µ2, λ) be defned as in (1.4). Then lim t→+∞ y2(t) y1(t) = lim t→−∞ y2(t) y1(t) = A, lim t→+∞ y2(t) y1(t) = lim t→−∞ y2(t) y1(t) = A, lim t→+∞ y′ i(t) yi(t) = −lim t→−∞ y′ i(t) yi(t) = −l(µ1, µ2, λ), i = 1, 2. Proof. From (3.5) and Lemma 3.2 it follows that Proof. From (3.5) and Lemma 3.2 it follows that 3 Asymptotic properties Therefore l1 = l2 must hold and similarly l3 = l4 also holds. Therefore l1 = l2 must hold and similarly l3 = l4 also holds. Lemma 3.3. Suppose that (H1) holds. Let A and l(µ1, µ2, λ) be defned as in (1.4). Then Proof. From (3.5) and Lemma 3.2 it follows that ( ) ( ) y y (ii) We claim that if y2(t) y1(t) is bounded, then there exists the limit lim t→+∞ y2(t) y1(t) = A. ( ) ( ) (ii) We claim that if y2(t) y1(t) is bounded, then there exists the limit lim t→+∞ y2(t) y1(t) = A. In fact, if lim t→+∞ y2(t) y1(t) doesn’t exist, then the continuity implies that y2(t) y1(t) must have sequences of local minimum points {¯σn} ⊂(0, +∞) and of local maximum points {¯τn} ⊂(0, +∞), such that (ii) We claim that if y1(t) is bounded, then there exists the limit lim t→+∞y1(t) = A. In fact, if lim t→+∞ y2(t) y1(t) doesn’t exist, then the continuity implies that y2(t) y1(t) must have sequences of local minimum points {¯σn} ⊂(0, +∞) and of local maximum points {¯τn} ⊂(0, +∞), such that ( ) y1(t) , t→+∞y1(t) In fact, if lim t→+∞ y2(t) y1(t) doesn’t exist, then the continuity implies that y2(t) y1(t) must have sequences of local minimum points {¯σn} ⊂(0, +∞) and of local maximum points {¯τn} ⊂(0, +∞), such that lim n→∞¯σn = ∞,  y2(t) y1(t) ′ t=¯σn = 0,  y2(t) y1(t) ′′ t=¯σn ≥0, lim n→∞¯τn = ∞,  y2(t) y1(t) ′ t=¯τn = 0,  y2(t) y1(t) ′′ t=¯τn ≤0. lim n→∞¯τn = ∞,  y2(t) y1(t) ′ t=¯τn = 0,  y2(t) y1(t) ′′ t=¯τn ≤0. Then for all n ∈N, a direct calculation shows that Then for all n ∈N, a direct calculation shows that y′ 2(¯σn) y′ 1(¯σn) = y2(¯σn) y1(¯σn) , y′ 2(¯τn) y′ 1(¯τn) = y2(¯τn) y1(¯τn) ,  y′′ 2(t) y2(t) −y′′ 1(t) y1(t)  t=¯σn ≥0,  y′′ 2(t) y2(t) −y′′ 1(t) y1(t)  t=¯τn ≤0. Proof. From (3.5) and Lemma 3.2 it follows that For any local minimum points {¯σn} ⊂(0, +∞) of y2(t) y1(t), since ¯σn is a local maximum point of y1 y2 −y2 y1 , from (3.6) and (3.7) it follows that For any local minimum points {¯σn} ⊂(0, +∞) of y2(t) y1(t), since ¯σn is a local maximum point of y1 y2 −y2 y1 , from (3.6) and (3.7) it follows that For any local minimum points {¯σn} ⊂(0, +∞) of y2(t) y1(t), since ¯σn is a local maximum point of y1 y2 −y2 y1 , from (3.6) and (3.7) it follows that µ1 −µ2 + o(1) ≥λ  y1(¯σn) y2(¯σn) −y2(¯σn) y1(¯σn)  , as n →∞, µ1 −µ2 + o(1) ≥λ  y1(¯σn) y2(¯σn) −y2(¯σn) y1(¯σn)  , as n →∞, which implies that which implies that µ1 −µ2 + o(1) ≥λ  y1(t) y2(t) −y2(t) y1(t)  , as t →+∞. µ1 −µ2 + o(1) ≥λ  y1(t) y2(t) −y2(t) y1(t)  , as t →+∞. Similarly, for any local maximum points {¯τn} ⊂(0, +∞) of y2(t) y1(t), since ¯τn is a local minimum point of y1 y2 −y2 y1 such that Similarly, for any local maximum points {¯τn} ⊂(0, +∞) of y2(t) y1(t), since ¯τn is a local minimum point of y1 y2 −y2 y1 such that ( ) ( ) Similarly, for any local maximum points {¯τn} ⊂(0, +∞) of y2(t) y1(t), since ¯τn is a local minimum point of y1 y2 −y2 y1 such that  (¯τ ) (¯τ ) µ1 −µ2 + o(1) ≤λ  y1(¯τn) y2(¯τn) −y2(¯τn) y1(¯τn)  , as n →∞, µ1 −µ2 + o(1) ≤λ  y1(¯τn) y2(¯τn) −y2(¯τn) y1(¯τn)  , as n →∞, at µ1 −µ2 + o(1) ≤λ  y1(¯τn) y2(¯τn) −y2(¯τn) y1(¯τn)  , as n →∞, and therefore and therefore µ1 −µ2 + o(1) ≤λ  y1(t) y2(t) −y2(t) y1(t)  , as t →+∞. µ1 −µ2 + o(1) ≤λ  y1(t) y2(t) −y2(t) y1(t)  , as t →+∞. µ1 −µ2 + o(1) ≤λ  y1(t) y2(t) −y2(t) y1(t)  , as t →+∞. µ1 −µ2 + o(1) ≤λ  y1(t) y2(t) −y2(t) y1(t)  , as t →+∞. Consequently, lim t→+∞  y1(t) y2(t) −y2(t) y1(t)  = µ1 −µ2 λ , (3.8) which implies that lim t→+∞  y1(t) y2(t) + y2(t) y1(t)  = r µ1 −µ2 λ 2 + 4 . Proof. From (3.5) and Lemma 3.2 it follows that lim t→±∞yα−2 1 yβ 2 = 0, lim t→±∞yα 1yβ−2 2 = 0. (3.6) (3.6) (i) We claim that either y2(t) y1(t) or y1(t) y2(t) is bounded in (0, +∞). ( ) ( ) ( ) (i) We claim that either y2(t) y1(t) or y1(t) y2(t) is bounded in (0, +∞). that either y2(t) y1(t) or y1(t) y2(t) is bounded in (0, +∞). (i) We claim that either y2(t) y1(t) or y1(t) y2(t) is bounded in (0, +∞). y y In fact, if neither y2(t) y1(t) nor y1(t) y2(t) is bounded in (0, +∞), then the continuity implies that y2(t) y1(t) must have sequences of local minimum points {σn} ⊂(0, +∞) such that In fact, if neither y2(t) y1(t) nor y1(t) y2(t) is bounded in (0, +∞), then the continuity implies that y2(t) y1(t) must have sequences of local minimum points {σn} ⊂(0, +∞) such that In fact, if neither y1(t) nor y2(t) is bounded in (0, + ), then sequences of local minimum points {σn} ⊂(0, +∞) such that y1(t) y2(t) sequences of local minimum points {σn} ⊂(0, +∞) such that lim n→∞σn = +∞, lim n→∞ y2(σn) y1(σn) = 0,  y2(t) y1(t) ′ t=σn = 0,  y2(t) y1(t) ′′ t=σn ≥0. D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 873 Then for all n ∈N, a direct calculation shows that Then for all n ∈N, a direct calculation shows that y′ 2(σn) y′ 1(σn) = y2(σn) y1(σn) ,  y′′ 2(t) y2(t) −y′′ 1(t) y1(t)  t=σn ≥0, y′ 2(σn) y′ 1(σn) = y2(σn) y1(σn) ,  y′′ 2(t) y2(t) −y′′ 1(t) y1(t)  t=σn ≥0, which together with (2.4) imply that which together with (2.4) imply that which together with (2.4) imply that  ¯µ −µ2 −λ y1 y2 −y2*−2 2 −ηβ 2* yα 1yβ−2 2  t=σn ≥  ¯µ −µ1 −λ y2 y1 −y2*−2 1 −ηα 2* yα−2 1 yβ 2  t=σn . (3.7) (3.7) Since Since Since lim n→∞y1(σn) = lim n→∞y2(σn) = 0, lim n→∞ y1(σn) y2(σn) = +∞, Since lim n→∞y1(σn) = lim n→∞y2(σn) = 0, lim n→∞ y1(σn) y2(σn) = +∞, by (3.6) and (3.7) we get a contradiction, which implies that either y2(t) y1(t) or y1(t) y2(t) is bounded in by (3.6) and (3.7) we get a contradiction, which implies that either y2(t) y1(t) or y1(t) y2(t) is bounded in (0, +∞). Proof. From (3.5) and Lemma 3.2 it follows that (3.9) Consequently, lim t→+∞  y1(t) y2(t) −y2(t) y1(t)  = µ1 −µ2 λ , (3.8) (3.8) lim t→+∞  y1(t) y2(t) + y2(t) y1(t)  = r µ1 −µ2 λ 2 + 4 . (3.9) (3.9) 874 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 874 From (3.8) and (3.9) it follows that lim t→+∞ y2(t) y1(t) exists, a contradiction. From (3.8) and (3.9) it follows that lim t→+∞ y2(t) y1(t) exists, a contradiction. ( ) From (3.8) and (3.9) it follows that lim t→+∞ y2(t) y1(t) exists, a contradiction. ( ) Therefore, there must exist the limit B := lim t→+∞ y2(t) y1(t) . Since l1 = l2, from (2.4) and the L’Hospital rule it follows that Therefore, there must exist the limit B := lim t→+∞ y2(t) y1(t) . Since l1 = l2, from (2.4) and the L’Hospital rule it follows that ( ) ′ ( ) ′′( ) ( )B λ B = lim t→+∞ y2(t) y1(t) = lim t→+∞ y′ 2(t) y′ 1(t) = lim t→+∞ y′′ 2(t) y′′ 1(t) = (¯µ −µ2)B −λ ¯µ −µ1 −λB , which implies that which implies that B = lim t→+∞ y2(t) y1(t) = A = 2λ p (µ1 −µ2)2 + 4λ2 + (µ1 −µ2) > 0. (3.10) (3.10) (iii) Similarly, if y1(t) y2(t) is bounded, then there exists the limit lim t→+∞ y1(t) y2(t) = 1 A. From (i)–(iii) it follows that (3 10) holds under (H1) Arguing as above under (H1) we also have that (iii) Similarly, if y1(t) y2(t) is bounded, then there exists the limit lim t→+∞ y1(t) y2(t) = 1 A. From (i)–(iii) it follows that (3.10) holds under (H1). Arguing as above, under (H1) we also have that (iii) Similarly, if y1(t) y2(t) is bounded, then there exists the limit lim t→+∞ y1(t) y2(t) = 1 A. From (i)–(iii) it follows that (3.10) holds under (H1). Arguing as above, under (H1) we also have that y2(t) t→+∞y2(t) A From (i)–(iii) it follows that (3.10) holds under (H1). Arguing as above, under (H1) we also have that lim t→−∞ y2(t) y1(t) = A. (3.11) (3.11) Let l(µ1, µ2, λ) be defned as in (1.4). Then a direct calculation shows that 0 < l(µ1, µ2, λ) ≤ p ¯µ −µi, i = 1, 3, l(µ1, µ2, λ) = p ¯µ −µ1 −λA = r ¯µ −µ2 −λ A . Proof. From (3.5) and Lemma 3.2 it follows that (3.12) (3.12) From (2.4), (3.6), (3.11), (3.12) and the L’Hospital rule it follows that From (2.4), (3.6), (3.11), (3.12) and the L’Hospital rule it follows that lim t→±∞ |y′ 1|2 y2 1 = lim t→±∞ y′′ 1 y1 = lim t→±∞  ¯µ −µ1 −λ y2 y1  = l(µ1, µ2, λ)2, lim t→±∞ |y′ 2|2 y2 2 = lim t→±∞ y′′ 2 y2 = lim t→±∞  ¯µ −µ2 −λ y1 y2  = l(µ1, µ2, λ)2. (3.13) (3.13) y1 y1 y1 lim t→±∞ |y′ 2|2 y2 2 = lim t→±∞ y′′ 2 y2 = lim t→±∞  ¯µ −µ2 −λ y1 y2  = l(µ1, µ2, λ)2. (3.13) By (3.13) we deduce that y′′ 1, y′′ 2 > 0 as |t| large enough, which together with (2.5) implies that y′ 1, y′ 2 < 0 as t →+∞and y′ 1, y′ 2 > 0 as t →−∞. Consequently, lim t→+∞ y′ i(t) yi(t) = −lim t→−∞ y′ i(t) yi(t) = −l(µ1, µ2, λ), i = 1, 2. (3.14) (3.14) The proof is complete. The proof is complete. The proof is complete. The proof is complete. Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then inf t∈R y2(t) y1(t) = A, sup t∈R y′ 1(t) y1(t) = l(µ1, µ2, λ), inf t∈R y′ 1(t) y1(t) = −l(µ1, µ2, λ). Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then i f y2(t) y′ 1(t) l( λ) i f y′ 1(t) l( λ) Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then pose that (H1) holds and A < Λ0. Then inf t∈R y2(t) y1(t) = A, sup t∈R y′ 1(t) y1(t) = l(µ1, µ2, λ), inf t∈R y′ 1(t) y1(t) = −l(µ1, µ2, λ). Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then inf t∈R y2(t) y1(t) = A, sup t∈R y′ 1(t) y1(t) = l(µ1, µ2, λ), inf t∈R y′ 1(t) y1(t) = −l(µ1, µ2, λ). Proof. If inf t∈R y2(t) y1(t) can’t be achieved at any fnite point, then from Lemma 3.3 it follows that inf t∈R y2(t) y1(t) = A. If the infmum is achieved at a fnite point t3 ∈R, then Proof. If inf t∈R y2(t) y1(t) can’t be achieved at any fnite point, then from Lemma 3.3 it follows that inf t∈R y2(t) y1(t) = A. If the infmum is achieved at a fnite point t3 ∈R, then 0 < inf t∈R y2(t) y1(t) = y2(t3) y1(t3) ≤A,  y2 y1 ′ t=t3 = h y2 y1  y′ 2 y2 −y′ 1 y1 i t=t3 = 0,  y2 y1 ′′ t=t3 = h y2 y1  y′′ 2 y2 −y′′ 1 y1 i t=t3 ≥0. (3.15) (3.15) D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 875 875 Let the functions f(τ) and g(τ) be defned as in (1.5) and Λ0 be the smallest positive zero of f(τ). Then f(τ) > 0 for all τ ∈[0, Λ0), g(A) = 0 and g(τ) < 0 for all τ ∈[0, A). Since A < Λ0, from (2.4) and (3.15) it follows that 0 ≥g  y2(t3) y1(t3)  ≥f  y2(t3) y1(t3)  y2(t3) y1(t3) β−1 y1(t3)2*−2 > 0, a contradiction, which together with (3.10) implies that inf t∈R y2(t) y1(t) can’t be achieved at any fnite point and furthermore, a contradiction, which together with (3.10) implies that inf t∈R y2(t) y1(t) can’t be achieved at any fnite point and furthermore, ( ) ( ) lim t→±∞ y2(t) y1(t) = inf t∈R y2(t) y1(t) = A. (3.16) (3.16) Consider the supremum of  y′ 1(t) y1(t)  in R. If the supremum can’t be achieved at any fnite point, then (3.16) Consider the supremum of  y′ 1(t) y1(t)  in R. If the supremum can’t be achieved at any fnite point, then (3.16) ′ ( ) implies that sup t∈R  y′ 1(t) y1(t)  = l(µ1, µ2, λ). If the supremum is achieved at fnite point t4 ∈R, then y′ 1(t4) y1(t4)  = sup t∈R  y′ 1(t) y1(t)  ≥l(µ1, µ2, λ). Furthermore,  y′ 1(t) y1(t) ′ t=t4 =  y′′ 1(t) y1(t) −|y′ 1(t)|2 |y1(t)|2  t=t4 = 0. Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then (3.17)  y′ 1(t) y1(t) ′ t=t4 =  y′′ 1(t) y1(t) −|y′ 1(t)|2 |y1(t)|2  t=t4 = 0. (3.17) (3.17) Since inf t∈R y2(t) y1(t) can’t be achieved at any fnite point, from (2.4), (3.16) and (3.17) it follows that Since inf t∈R y2(t) y1(t) can’t be achieved at any fnite point, from (2.4), (3.16) and (3.17) it follows that |y′ 1|2 y2 1  t=t4 = h (¯µ −µ1) −λ y2 y1 −y2*−2 1 −ηα 2* yα−2 1 yβ 2 i t=t4 ≤¯µ −µ1 −λ y2(t4) y1(t4) < ¯µ −µ1 −λA = l(µ1, µ2, λ)2, ≤¯µ −µ1 −λ y2(t4) y1(t4) < ¯µ −µ1 −λA = l(µ1, µ2, λ)2, a contradiction, which implies that sup t∈R  y′ 1(t) y1(t)  can’t be achieved at any fnite points. Then from (3.14) and (3.16) it follows that a contradiction, which implies that sup t∈R  y′ 1(t) y1(t)  can’t be achieved at any fnite points. Then from (3.14) and (3.16) it follows that a contradiction, which implies that sup t∈R  y′ 1(t) y1(t)  can’t be achieved at any fnite points. Then from (3.14) and (3.16) it follows that lim t→+∞ y′ 1(t) y1(t) = inf t∈R y′ 1(t) y1(t) = − p ¯µ −µ1 −λA = −l(µ1, µ2, λ), lim t→−∞ y′ 1(t) y1(t) = sup t∈R y′ 1(t) y1(t) = p ¯µ −µ1 −λA = l(µ1, µ2, λ). (3.18) (3.18) The proof is complete. The proof is complete. Lemma 3.5. Suppose that (H1) holds and A < Λ0. Then li = l(µ1, µ2, λ), 1 ≤i ≤4. Lemma 3.5. Suppose that (H1) holds and A < Λ0. Then li = l(µ1, µ2, λ), 1 ≤i ≤4. Lemma 3.5. Suppose that (H1) holds and A < Λ0. Then li = l(µ1, µ2, λ), 1 ≤i ≤4. Proof. For any ε > 0, by the defnition of l1 we have that Proof. For any ε > 0, by the defnition of l1 we have that Proof. For any ε > 0, by the defnition of l1 we have that +∞ Z 0 l1 + ε −l(µ1, µ2, λ)
+ l(µ1, µ2, λ) + y′ 1 y1  ds = +∞ Z 0  l1 + ε + y′ 1 y1  ds = +∞. Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then +∞ Z 0 l1 + ε −l(µ1, µ2, λ)
+ l(µ1, µ2, λ) + y′ 1 y1  ds = +∞ Z 0  l1 + ε + y′ 1 y1  ds = +∞. From Lemma 3.4 it follows that l1 + ε −l(µ1, µ2, λ) ≥0, ∀ε > 0, 876 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 876 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 876 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 876 that is, l1 −l(µ1, µ2, λ) ≥0. For any ε > 0, from Lemma 3.4 it follows that µ1, µ2, λ) ≥0. For any ε > 0, from Lemma 3.4 it follows that g1(l(µ1, µ2, λ) + ε) = +∞. g1(l(µ1, µ2, λ) + ε) = +∞. Then l1 ≤l(µ1, µ2, λ). Therefore, l1 = l(µ1, µ2, λ), which together with Lemma 3.2 implies that l1 = l2 = l(µ1, µ2, λ). µ µ Under the assumption (H1), arguing similarly as above we also have that Under the assumption (H1), arguing similarly as above we also have that lim t→−∞ y′ 1 y1 = lim t→−∞ y′ 2 y2 = l(µ1, µ2, λ), l3 = l4 = l(µ1, µ2, λ). te. The proof is complete. The proof is complete. Lemma 3.6. Suppose that (H1) holds with A < Λ0 and let t1, t2, be defned as in Theorem 1.1. Then there exist the positive constants C1, C2 > 0, such that r r Lemma 3.6. Suppose that (H1) holds with A < Λ0 and let t1, t2, be defned as in Theorem 1.1. Then there exist the positive constants C1, C2 > 0, such that lim t→+∞e R t T0 r ¯µ−µ1−λ y2(s) y1(s) dsy1(t) = C1, lim t→−∞e R −T0 t r ¯µ−µ1−λ y2(s) y1(s) dsy1(t) = C2, lim t→+∞e R t T0 r ¯µ−µ1−λ y2(s) y1(s) dsy2(t) = C1A, lim t→−∞e R −T0 t r ¯µ−µ1−λ y2(s) y1(s) dsy2(t) = C2A. Proof. We only prove the frst equality. The second one can be verifed similarly and the last two can be con- cluded by the frst two equalities and (3.10). ′ ( ) Proof. We only prove the frst equality. The second one can be verifed similarly and the last two can be con- cluded by the frst two equalities and (3.10). Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 877 877 We claim that the integral I := R +∞ T0 q ¯µ −µ1 −λ y2(s) y1(s) + H1(s)
ds converges. I f f (3 18) d (3 19) i f ll h We claim that the integral I := R +∞ T0 q ¯µ −µ1 −λ y2(s) y1(s) + H1(s)
ds converges. In fact, from (3.18) and (3.19) it follows that We claim that the integral I := R +∞ T0 q ¯µ −µ1 −λ y2(s) y1(s) + H1(s)
ds converges. I f f (3 8) d (3 9) i f ll h We claim that the integral I := R +∞ T0 q ¯µ −µ1 −λ y2(s) y1(s) + H1(s)
ds converges. We claim that the integral I := R +∞ T0 q ¯µ −µ1 −λ y2(s) y1(s) + H1(s)
ds converges. In fact, from (3.18) and (3.19) it follows that g R T0 q µ µ1 y1(s) 1( )
g In fact, from (3.18) and (3.19) it follows that lim t→+∞ s ¯µ −µ1 −λ y2(s) y1(s) = l(µ1, µ2, λ), lim t→+∞H1(s) = −l(µ1, µ2, λ). (3.21) (3.21) Since A < Λ0, arguing as in the proof of Lemma 3.4 we have that y2(s) y1(s) and H1(s) are strictly decreasing as s →+∞. Taking ¯τ > T0 large enough we have that H′ 1(s) < 0 for all s > ¯τ and Since A < Λ0, arguing as in the proof of Lemma 3.4 we have that y2(s) y1(s) and H1(s) are strictly decreasing as s →+∞. Taking ¯τ > T0 large enough we have that H′ 1(s) < 0 for all s > ¯τ and +∞ Z ¯τ H′ 1(s) q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds  =  +∞ Z ¯τ H′ 1(s) l(µ1, µ2, λ) −H1(s) l(µ1, µ2, λ) −H1(s) q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds  ≤C  +∞ Z ¯τ H′ 1(s) l(µ1, µ2, λ) −H1(s) ds  = C ln 2l(µ1, µ2, λ) l(µ1, µ2, λ) −H1(¯τ)  < +∞. (3.22) +∞ Z ¯τ H′ 1(s) q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds =  +∞ Z ¯τ H′ 1(s) l(µ1, µ2, λ) −H1(s) l(µ1, µ2, λ) −H1(s) q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds  (3.22) (3.22) ≤C  +∞ Z ¯τ H′ 1(s) l(µ1, µ2, λ) −H1(s) ds  ( ) = C ln 2l(µ1, µ2, λ) l(µ1, µ2, λ) −H1(¯τ)  < +∞. Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then ′ ( ) (s) := y′ 1(s) y1(s). From (2.4) and Lemma 3.4 it follows that l(µ1, µ2, λ) ± H1(s) > 0, ∀s ∈R. l(µ1, µ2, λ) ± H1(s) > 0, ∀s ∈R. l(µ1, µ2, λ) ± H1(s) > 0, ∀s ∈R. Note that A < Λ0 and Note that A < Λ0 and lim t→+∞  ¯µ −µ1 −λ y2(t) y1(t)  = l(µ1, µ2, λ)2 > 0. (3.19) lim t→+∞  ¯µ −µ1 −λ y2(t) y1(t)  = l(µ1, µ2, λ)2 > 0. (3.19) Arguing as in the proof of Lemma 3.4, we have that Arguing as in the proof of Lemma 3.4, we have that ¯µ −µ1 −λ y2(t) y1(t) > 0, ∀t ∈(T0, +∞). ¯µ −µ1 −λ y2(t) y1(t) > 0, ∀t ∈(T0, +∞). According to (2.4) and by direct calculation we have that According to (2.4) and by direct calculation we have that According to (2.4) and by direct calculation we have that H′ 1(s) = ¯µ −µ1 −λ y2(s) y1(s) −H2 1(s) −y2*−2 1 −ηα 2* yα−2 1 yβ 2, H′ 1(s) = ¯µ −µ1 −λ y2(s) y1(s) −H2 1(s) −y2*−2 1 −ηα 2* yα−2 1 yβ 2, which implies that which implies that s ¯µ −µ1 −λ y2(s) y1(s) + H1(s) = H′ 1(s) + y2*−2 1 + ηα 2* yα−2 1 yβ 2 q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) , ∀t > T0. (3.20) Defne I := +∞ Z T0 s ¯µ −µ1 −λ y2(s) y1(s) + H1(s)  ds, I1 := +∞ Z T0 H′ 1(s) q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds, I2 := +∞ Z T0 y2*−2 1 + ηα 2* yα−2 1 yβ 2 q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds. s ¯µ −µ1 −λ y2(s) y1(s) + H1(s) = H′ 1(s) + y2*−2 1 + ηα 2* yα−2 1 yβ 2 q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) , ∀t > T0. (3.20) (3.20) Defne I := +∞ Z T0 s ¯µ −µ1 −λ y2(s) y1(s) + H1(s)  ds, I1 := +∞ Z T0 H′ 1(s) q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds, I2 := +∞ Z T0 y2*−2 1 + ηα 2* yα−2 1 yβ 2 q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds. D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 877 D. Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then Then the integral I1 converges. Furthermore, (3.21) implies that there exists R > 0 such that s ¯µ −µ1 −λ y2(s) y1(s) −H1(s) > l(µ1, µ2, λ), ∀s > R. s ¯µ −µ1 −λ y2(s) y1(s) −H1(s) > l(µ1, µ2, λ), ∀s > R. Therefore, 0 < I2 := +∞ Z R 2*y2*−2 1 + ηαyα−2 1 yβ 2 q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds < 1 l(µ1, µ2, λ) +∞ Z R 2*y2*−2 1 + ηαyα−2 1 yβ 2
ds. (3.23) 0 < I2 := +∞ Z R 2*y2*−2 1 + ηαyα−2 1 yβ 2 q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds (3 23) 0 < I2 := +∞ Z R 2*y2*−2 1 + ηαyα−2 1 yβ 2 q ¯µ −µ1 −λ y2(s) y1(s) −H1(s) ds (3.23) < 1 l(µ1, µ2, λ) +∞ Z R 2*y2*−2 1 + ηαyα−2 1 yβ 2
ds. (3.23) < 1 l(µ1, µ2, λ) +∞ Z R 2*y2*−2 1 + ηαyα−2 1 yβ 2
ds. For any ε > 0, t > 0, from (3.5) and Lemma 3.5 it follows that For any ε > 0, t > 0, from (3.5) and Lemma 3.5 it follows that +∞ Z R  y2*−2 1 + yα−2 1 yβ 2  ds < C(ε) +∞ Z R e−((2*−2)l(µ1,λ,µ2)−(2*+2)ε)sds. By taking ε →0+ we have that By taking ε →0+ we have that −((2* −2)l(µ1, µ2, λ) −(2* + 2)ε) < 0. −((2* −2)l(µ1, µ2, λ) −(2* + 2)ε) < 0. Then the integral R +∞ T0 2*y2*−2 1 + ηαyα−2 1 yβ 2
ds converges, which together with (3.23) implies that the integral I2 converges. Then the integral R +∞ T0 2*y2*−2 1 + ηαyα−2 1 yβ 2
ds converges, which together with (3.23) implies that the integral I2 converges. Then the integral R +∞ T0 2*y2*−2 1 + ηαyα−2 1 yβ 2
ds converges, which together with (3.23) implies that the integral I2 converges. By (3.22) and (3.23) we have that the integral I = I1 + I2 converges. Furthermore, By (3.22) and (3.23) we have that the integral I = I1 + I2 converges. Proof of Theorem 1.1. The results follow directly from (2.2), (2.3), Lemma 3.3 and Lemma 3.4. Proof of Theorem 1.1. The results follow directly from (2.2), (2.3), Lemma 3.3 and Lemma 3.4. Proof of Theorem 1.2. The asymptotic properties of u(r) and v(r) at the origin and infnity follow from (2.2) and Lemma 3.6, and the asymptotic properties of u′(r) and v′(r) follow from (2.2), (2.3), (3.14) and Lemma 3.6. Lemma 3.4. Suppose that (H1) holds and A < Λ0. Then Furthermore, lim t→+∞e R t T0 r ¯µ−µ1−λ y2(s) y1(s) dsy1(t) = y1(T0) lim t→+∞e R t T0 r ¯µ−µ1−λ y2(s) y1(s) +H1(s)
ds = y1(T0)e R +∞ T0 r ¯µ−µ1−λ y2(s) y1(s) +H1(s)
ds = C1, where C1 := y1(T0)e R +∞ T0 r ¯µ−µ1−λ y2(s) y1(s) +H1(s)
ds > 0. 878 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 878 Proof of Theorem 1.1. The results follow directly from (2.2), (2.3), Lemma 3.3 and Lemma 3.4. 4 Explicit form solutions In this section, we study the explicit form of radially–symmetric and strictly–decreasing minimizers to S(µ1, µ2, λ), among which there exists an explicit form of least energy solutions to (1.1), satisfying all of the properties in Theorems 1.1 and 1.2. For convenience we set k(τ) := −f(τ), τ > 0, where f(τ) is defned as in (1.5). Proof of Theorem 1.3. Suppose that (H1) holds with τmin = A. We frst investigate the functions F(τ) and k(τ). A direct calculation shows that F′(τ) = 2τβ−1k(τ) (1 + ητβ + τ2*) 2 2* +1 , τ > 0. (4.1) (4.1) Note that Note that Note that Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 879 879 Then from (1.2), (1.6), (1.8) and (4.4) it follows that Then from (1.2), (1.6), (1.8) and (4.4) it follows that Then from (1.2), (1.6), (1.8) and (4.4) it follows that ≥ Z RN  |∇un|2 −µ* u2n |x|2   (1 + ηs−β n + s−(2*) n ) Z RN |un|2* 2 2* + s−2 n Z RN  |∇zn|2 −µ* z2n |x|2   (1 + ηs−β n + s−(2*) n ) Z RN |zn|2* 2 2* ≥F(s−1 n )S(µ*) ≥F(τmin)S(µ*). Taking n →∞we have that * S(µ1, µ2, λ) ≥F(τmin)S(µ*). (4.5) hat τmin = A ⇐=⇒µ* = µ*. (4.6) f ll h S(µ1, µ2, λ) ≥F(τmin)S(µ*). (4.5) S(µ1, µ2, λ) ≥F(τmin)S(µ*). (4.5) Note that Note that lim τ→0+ F(τ) = lim τ→+∞F(τ) = 1, k(τ) < 0 as τ →0+, k(τ) > 0 as τ →+∞. lim τ→0+ F(τ) = lim τ→+∞F(τ) = 1, lim τ→0+ F(τ) = lim τ→+∞F(τ) = 1, lim τ→0+ F(τ) = lim τ→+∞F(τ) = 1, k(τ) < 0 as τ →0+, k(τ) > 0 as τ →+∞. k(τ) < 0 as τ →0+, k(τ) > 0 as τ →+∞. k(τ) < 0 as τ →0+, k(τ) > 0 as τ →+∞. k(τ) < 0 as τ →0+, k(τ) > 0 as τ →+∞. Then min τ≥0 F(τ) must be achieved at fnite τmin > 0 and from (4.1) it follows that F′(τmin) = 0, k(τmin) = 0, 0 < F(τmin) < 1. F′(τmin) = 0, k(τmin) = 0, 0 < F(τmin) < 1. For all w ∈D1, 2(RN) \ {0}, testing the second Rayleigh quotient in (1.2) by (w, τminw), we have th S(µ1, µ2, λ) ≤F(τmin) Z RN  |∇w|2 −µ* w2 |x|2   Z RN |w|2* 2 2* , which together with (1.2) implies that which together with (1.2) implies that S(µ1, µ2, λ) ≤F(τmin)S(µ*). S(µ1, µ2, λ) ≤F(τmin)S(µ*). S(µ1, µ2, λ) ≤F(τmin)S(µ*). (4.2) (4.2) Let {(un, vn)} ⊂D be a minimizing sequence of S(µ1, µ2, λ) and defne zn = snvn, where Let {(un, vn)} ⊂D be a minimizing sequence of S(µ1, µ2, λ) and defne zn = snvn, where sn =  Z RN |vn|2*−1 Z RN |un|2* 1 2* , Z RN |zn|2* = Z RN |un|2*. (4.3) sn =  Z RN |vn|2*−1 Z RN |un|2* 1 2* , which implies that Z RN |zn|2* = Z RN |un|2*. (4.3) which implies that Z RN |zn|2* = Z RN |un|2*. (4.3) (4.3) R By the Young inequality and (4.3) we have that that By the Young inequality and (4.3) we have that that Z RN |un|α|zn|β ≤α 2* Z RN |un|2* + β 2* Z RN |zn|2* = Z RN |un|2* = Z RN |zn|2*. (4.4) (4.4) D. ≥ Z RN  |∇un|2 −µ* u2n |x|2   (1 + ηs−β n + s−(2*) n ) Z RN |un|2* 2 2* + s−2 n Z RN  |∇zn|2 −µ* z2n |x|2   (1 + ηs−β n + s−(2*) n ) Z RN |zn|2* 2 2* A direct calculation shows that Since τmin depends on α, β, η, and A depends on µ1, µ2, λ, then τmin is indepen- dent of A. Obviously, by (4.1) a sufcient condition to ensure τmin = A is k(A) = 0, k(τ) < 0 in (0, A), k(τ) > 0 in (A, +∞). In the following discussion, we only consider the case k(τ) > 0 in [1, +∞). Suppose that 1 < β < α < 2, η ≥ N N−2 . Since In the following discussion, we only consider the case k(τ) > 0 in [1, +∞). Suppose that 1 < β < α < 2, η ≥ N N−2 . Since k′(τ) = τ1−β 2 −β + 2ηα 2* τβ −ατ2*−2 , k(1) = η 2* (α −β) > 0, 2 −β > 0, β > 2* −2, 2ηα 2* ≥α, k′(τ) > 0, k(τ) > k(1) > 0, ∀τ ∈(1, +∞). (4.7) k′(τ) = τ1−β 2 −β + 2ηα 2* τβ −ατ2*−2 , then we have that then we have that k(1) = η 2* (α −β) > 0, 2 −β > 0, β > 2* −2, 2ηα 2* ≥α, (4.7) (4.7) k′(τ) > 0, k(τ) > k(1) > 0, ∀τ ∈(1, +∞). k′(τ) > 0, k(τ) > k(1) > 0, ∀τ ∈(1, +∞). Since k(τ) < 0 as τ →0+, from (4.1) and (4.7) it follows that min τ≥0 F(τ) must be achieved at fnite τmin ∈(0, 1). Noting that 0 < A ≤1, A →0 as λ →0 and A = 1 as µ1 = µ2, and A(µ1, µ2, λ) is a continuous function, there must exist certain µ1, µ2, λ ∈(0, ¯µ), such that A(µ1, µ2, λ) = τmin. Then the desired result follows directly from Theorem 1.3. Acknowledgement The authors acknowledge the anonymous referee for carefully reading this paper and making many important comments. This work is supported by the Fundamental Research Funds for the Central Universities of China, South– Central University for Nationalities (No. CZT18008). This work is supported by the Fundamental Research Funds for the Central Universities of China, South– Central University for Nationalities (No. CZT18008). A direct calculation shows that τmin = A ⇐=⇒µ* = µ*. (4.6) (4.6) τmin = A ⇐=⇒µ* = µ*. Then from (4.2), (4.5) and (4.6) it follows that Then from (4.2), (4.5) and (4.6) it follows that S(µ1, µ2, λ) = F(A)S(µ*) = F(A)S(µ*), S(µ1, µ2, λ) = F(A)S(µ*) = F(A)S(µ*), S(µ1, µ2, λ) = F(A)S(µ*) = F(A)S(µ*), which implies that S(µ1, µ2, λ) has the minimizers of the form: which implies that S(µ1, µ2, λ) has the minimizers of the form: which implies that S(µ1, µ2, λ) has the minimizers of the form: n CVε µ*(x), AVε µ*(x)
, C, ε > 0 o . Since k(τmin) = k(A) = 0, a direct calculation shows that the problem (1.1) has the explicit form of least energy solutions ) = 0, a direct calculation shows that the problem (1.1) has the explicit form of least energy ns*Vε µ*(x), s*AVε µ*(x)
, ε > 0 o , that is, (uε, vε) :=s*Vε µ*(x), s*AVε µ*(x)
is a solution to (1.1) such that that is, (uε, vε) :=s*Vε µ*(x), s*AVε µ*(x)
is a solution to (1.1) such that Z RN  |∇uε|2 +|∇vε|2 −µ1u2ε + 2λuεvε +µ2v2ε |x|2  = Z RN u2* ε + v2* ε + ηuα ε vβ ε
= Z RN u2* ε + v2* ε + ηuα ε vβ ε
880 | D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms 880 =F(A)S(µ*)
N 2 = S(µ1, µ2, λ) N 2 , where s* is defned as in (1.7). By (3.18) we have that l(µ1, µ2, λ) = p ¯µ −µ1 −λA and therefore the solution s*Vε µ*(x), s*AVε µ*(x)
satisfes all of the properties mentioned in Theorems 1.1 and 1.2 with T0 = 0. where s* is defned as in (1.7). By (3.18) we have that l(µ1, µ2, λ) = p ¯µ −µ1 −λA and therefore the solution s*Vε µ*(x), s*AVε µ*(x)
satisfes all of the properties mentioned in Theorems 1.1 and 1.2 with T0 = 0. The proof is complete. Proof of Corollary 1.4.. Since τmin depends on α, β, η, and A depends on µ1, µ2, λ, then τmin is indepen- dent of A. Obviously, by (4.1) a sufcient condition to ensure τmin = A is Proof of Corollary 1.4.. References [1] G. Hardy, J. Littlewood, G. Polya, Inequalities, Cambridge University Press, Cambridge, 1952. [1] G. Hardy, J. Littlewood, G. Polya, Inequalities, Cambridge University Press, Cambridge, 1952 [2] B. Abdellaoui, V. Felli, I. Peral, Existence and nonexistence for quasilinear equations involving the p-laplacian, Unione Mat. Ital. B 9, (2006), 445–484. [3] Z. Chen and W. Zou, Existence and symmetry of positive ground states for a doubly critical Schrödinger system, Trans. Amer. Math. Soc. 367, (2015), 3599–3646. [3] Z. Chen and W. Zou, Existence and symmetry of positive ground states for a doubly critical Schrödinger system, Trans. Amer. Math. Soc. 367, (2015), 3599–3646. [4] D. Kang, Systems of elliptic equations involving multiple critical nonlinearities and diferent Hardy–type terms in RN, J. Math. Anal. Appl. 420, (2014), 930–941. [4] D. Kang, Systems of elliptic equations involving multiple critical nonlinearities and diferent Hardy–type terms in RN, J. Math. Anal. Appl. 420, (2014), 930–941. [5] S. Terracini, On positive entire solutions to a class of equations with a singular coefcient and critical exponent, Adv. Diferential Equations 1, (1996), 241–264. [5] S. Terracini, On positive entire solutions to a class of equations with a singular coefcient and critical exponent, Adv. Diferential Equations 1, (1996), 241–264. [6] D. Cao and P. Han, Solutions to critical elliptic equations with multi–singular inverse square potentials, J. Diferential Equations 224(2006) 332–372. [6] D. Cao and P. Han, Solutions to critical elliptic equations with multi–singular inverse square potentials, J. Diferential Equations 224(2006) 332–372. [8] F. Catrina and Z. Wang, On the Cafarelli–Kohn–Nirenberg inequalities: sharp constants, existence(and nonexis and symmetry of extremal functions, Comm. Pure Appl. Math. 54, (2001), 229–257. D. Kang et al., Critical elliptic systems involving multiple strongly–coupled Hardy–type terms | 88 881 [9] L. Dupaigen, A nonlinear elliptic PDE with the inverse square potential, J. Anal. Math. 86, (2002), 359–398. [10] V. Felli and S. Terracini, Elliptic equations with multi–singular inverse–square potentials and critical nonlinearity, Comm. Partial Diferential Equations 31, (2006), 469–495. [11] B. Abdellaoui, V. Felli, I. Peral, Some remarks on systems of elliptic equations doubly critical in the whole RN, Calc. Var. Partial Diferential Equations 34, (2009), 97–137. [12] Z. Chen and W. Zou, A remark on doubly critical elliptic systems, Calc. Var. Partial Diferential Equations 50, (20 965. [13] D. Kang and X. Liu, Singularities of solutions to elliptic systems involving diferent Hardy–type terms, J. Math. Anal. Appl. 468, (2018), 757–765. [14] D. Kang and L. References Xu, A critical surface for the solutions to singular elliptic systems, J. Math. Anal. Appl. 472, (2019), 2017– 2033. [15] Z. Chen and C.–S. Lin, Removable singularity of positive solutions for a critical elliptic system with isolated singularity, Math. Ann. 363, (2015), 501–523. [16] S. Peng, Y. Peng, Z.–Q. Wang, On elliptic systems with Sobolev critical growth, Calc. Var. Partial Diferential Equations 55:142, (2016), https://doi.org/10.1007/s00526-016-1091-7.

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The Rise and Fading Away of Charisma. Leadership Transition and Managerial Ethics in the Post-Soviet Media Holdings

Journal of business ethics

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Journal of Business Ethics (2021) 174:847–860 https://doi.org/10.1007/s10551-021-04923-z Journal of Business Ethics (2021) 174:847–860 https://doi.org/10.1007/s10551-021-04923-z ORIGINAL PAPER Abstract This paper examines post-communist managerial ethics during the emergence and transition of charismatic leadership in two privately owned media holdings in Russia and Kyrgyzstan. These media holdings were bootstrapped in the 1990s and 2000s by people without management experience and connections. This paper argues that Weberian charismatic leadership was a necessary leadership style to start a private business for people without links to elite networks. However, once firms establish themselves on the market, charisma fades and yields itself to a legal-rational leadership style. In particular, the paper compares and contrasts the managerial ethics issues arising from the loyalty-based leader–follower relations in the charismatic leadership phase and the legal-rational phase of a firm’s development and maturation. While the legal-rational phase brings positive changes to workload management and employees’ rights for vacation and p/maternity leave, task delegation remains an unsolved issue. Ambiguous career advancement criteria of the legal-rational phase replace rapid career progression of junior and middle managers during the charismatic phase. By examining the dynamics of managerial ethics transformation, this study adds to the literature on post-communist leadership, management and governance. Recommendations are provided for privately owned firms on how to advance managerial ethics to attract and retain qualified talent. Keywords  Post-communist · Managerial ethics · Charismatic leadership · Weber · Legal-rational leadership · Russia · Kyrgyzstan · Media holding · Emerging markets The Rise and Fading Away of Charisma. Leadership Transition and Managerial Ethics in the Post‑Soviet Media Holdings Dinara Tokbaeva1 Received: 5 November 2019 / Accepted: 20 August 2021 / Published online: 1 October 2021 © The Author(s) 2021 * Dinara Tokbaeva [email protected] 1 Media, Management and Transformation Centre (MMTC), Jönköping International Business School (JIBS), Jönköping University, Gjuterigatan 5, Lokal B6061A, Box 1026, 551 11 Jönköping, Sweden Charisma and Leadership leadership and managerial ethics in the companies that are run by post-communist businesspeople. This investigation emphasises charismatic leadership and charismatic author- ity in Weberian understanding of it. The study explores the transition of charisma to a legal-rational authority (Weber, 1922/1947) on the example of post-communist leaders of media holdings. The challenge is to find out whether there is a change of managerial ethical issues once leadership style changes, and how is this change manifested in the workload management, task delegation, career progression of junior and middle managers, and employees’ rights for vacation and paternity and maternity leave. With this goal in mind, this paper observes two examples of leadership transition of self-made post-communist businesspeople, one from Russia and one from Kyrgyzstan, both of whom have created and been running a media company after the collapse of the Soviet Union. In this light, the paper con- siders the following research questions: leadership and managerial ethics in the companies that are run by post-communist businesspeople. This investigation emphasises charismatic leadership and charismatic author- ity in Weberian understanding of it. The study explores the transition of charisma to a legal-rational authority (Weber, 1922/1947) on the example of post-communist leaders of media holdings. The challenge is to find out whether there is a change of managerial ethical issues once leadership style changes, and how is this change manifested in the workload management, task delegation, career progression of junior and middle managers, and employees’ rights for vacation and paternity and maternity leave. With this goal in mind, this paper observes two examples of leadership transition of self-made post-communist businesspeople, one from Russia and one from Kyrgyzstan, both of whom have created and been running a media company after the collapse of the Soviet Union. In this light, the paper con- siders the following research questions: Although charismatic authority as a concept of leadership is a well-known sociological concept, the manifestation and the impact of charisma in a business setting is still not well understood. This paper defines charisma as a personal charm or a ‘gift of grace’ which helps one to influence, inspire and lead others. The German sociologist Weber (1922/1947) defined charisma as “a certain quality of an individual personality by virtue of which s/he is set apart from ordinary men and treated as endowed with super- natural, superhuman, or at least specifically exceptional powers or qualities” (p. 358). Charisma and Leadership This paper defines charis- matic leadership as a leadership type whenever an owner, a founder or a large department head (the charismatic leader) inspires the employees to follow his/her vision (Bryman, 1992; Conger & Kanungo, 1987; Kotter, 1990; Küng, 2008). Unlike visionary or transformational leader- ship, charismatic leadership allows creating strong emo- tional ties between a leader and followers in such a way that they develop a mutual attachment (Weber, 1922/1947; Küng, 2008). Weber (1922/1947) distinguished between traditional, charismatic and legal-rational authority. 1. Does managerial ethics in a company change when a leadership style transitions from charismatic to a legal- rational one within post-communist media holdings in Russia and Kyrgyzstan? 2. In the case of post-communist privately owned busi- nesses, how does leadership transition impact manage- rial ethics, namely the issues of workload management, task delegation, career progression, and employees’ rights for vacation and p/maternity leave? Heredity-based traditional authority is of limited rel- evance for the post-soviet small business context. Due to the change of ownership structures and the rise of new entrepreneurship initiatives, inherited family businesses were not typical during the first 20 years after the fall of the Soviet Union. However, research on elite networks (Ivlevs et al., 2020) argued that some post-soviet entrepre- neurs benefited from previous communist party links. One may, for example, view the current post-soviet oligarchic structures as an example of Weberian traditional author- ity. Yet, this paper focuses on small businesses started by people without prior communist party connections, who lived outside of centres of power (e.g. Moscow), and were in their 20 s or early 30 s when the Soviet Union fell. Charismatic authority emanates from a specific leader who inspires others. This leader is perceived to possess the extraordinary individual characteristics of a divine or supernatural origin (Weber, 1922/1947). On the other hand, legal-rational authority is based on the rule of law, rather than on any specific leader (Weber, 1922/1947). The principles of law empower those who hold this authority type and allow them to influence their subordinates. This paper speaks of charismatic authority and legal-rational authority as two distinct leadership styles—a charismatic one and a legal-rational one. These questions will be addressed through a comparative case study based on in-depth semi-structured interviews with company owners, senior, middle and junior management, and former employees. Charisma and Leadership The case study aims at covering the period of company development from its origin—in the early 1990s in case of the Russian firm and early 2000s in case of the Kyrgyz firm—until 2016. A journey of two firms from a basement to a media holding, it’s an investigation into differ- ent phases of leadership across time, the transition of lead- ership and the issues of managerial ethics associated with it. It’s the first study of managerial ethics in media firms in Russia and Kyrgyzstan that are owned and run by the first generation of post-soviet businesspeople. Introduction entire post-Soviet space was full of commercial and risk- taking initiatives. The first generation of post-communist businesspeople enjoyed a carte-blanche or freedom to act as they wish. Needless to say, that only a few of those companies managed to survive on the market until now and eventually become self-sustaining businesses. It is what is known so far. But the interest of this paper is that in the course of bootstrapping and growing a business, the self-made post-communist managers developed and nurtured their understanding of managerial ethics. Their knowledge and values of managerial ethics laid the basis for the emerging institution of post-communist manage- ment. By performing a comparative case study of two phases of leadership in two media holdings, this paper aims at understanding the connection between leader- ship, authority and managerial ethics, and how it has been changing throughout two decades after the collapse of the Soviet Union. The post-communist setting is characterised by large-scale transformations, the ongoing political, eco- nomic and social turbulence, and the weakness of formal institutions. It is a suitable setting to observe changes in How has managerial ethics changed in the post-Soviet space 30 years after the collapse of the Soviet Union? How different and how similar are leadership styles and work ethics in different parts of the former USSR? These are the questions of investigation of this paper, which looks at two sample cases of the first generation of self-made post-communist businesspeople from Russia and Kyr- gyzstan. Russia was the largest Soviet economy, while Kyrgyzstan’s economy was one of the smallest in the for- mer Soviet Union. Perestroika and glasnost paved the way for a whole new generation of self-made post-communist businesspeople. Throughout the 1990s and the 2000s, the (0123 1 3456789) 3 3456789) 3 848 D. Tokbaeva Theoretical Framework Theory-wise, this paper is focused on the concepts of cha- risma and authority in relation to managerial ethics in the post-communist context of media firms. The two subsec- tions, one on charisma, and another one on managerial eth- ics, aim at presenting the previous research which laid the basis for this paper. A crisis is necessary for the occurrence of charismatic leadership in organisations (De Cremer & Van Dijke, 2010; Shamir & Howell, 1999). Followers are eager to support the charismatic leader because they develop 3 3 The Rise and Fading Away of Charisma. Leadership Transition and Managerial Ethics in the… 849 mutual attachment and dependency to each other as a result of their close interaction (Bryman, 1992; Conger & Kanungo, 1987; Kets de Vries, 1988) including the one in media firms (Küng, 2008). Anyhow, it is inevita- ble that charisma over time becomes routinised (Weber, 1922/1947) and fades away. Conger and Kanungo (1987) found evidence that the routinisation of charisma turns charismatic leaders into administrators (caretakers) or managers (nudging followers), and as soon as it hap- pens, their charisma wanes. Following this logic, char- ismatic leaders lose their charisma, the more immersed they become into the structures which emerge with rou- tinisation. Some prefer to work with or for a charismatic leader regardless of charisma’s fading, is because they are comfortable as task-doers as much as the others are com- fortable as task-creators. Inter-family relations in one’s early childhood influence the way a personality is formed (Freud, 1901/1975). Later, alongside other factors, that impacts one’s career choices and behaviour. Zaleznik (2004) adds that appointed managers (or those in senior and middle management) seek stability and control and instinctively try to quickly resolve problems, sometimes before they fully understand what a problem is. Therefore, a combination of internal predispositions and external aspects shapes both leaders and followers. to stay at the company, without changing. With this, we move on to discussing business ethics and, in particular, ethical dimensions of management decision-making. Managerial Ethics in the Post‑communist Businesses Previous research identified that workplace flourishing, career progression and compensation within an organisa- tion depend as much on emotional intelligence of the parties (Hui-Hua & Schutte, 2015) as on the informal ties between them, which is referred to as cronyism (Oxelheim & Clark- son, 2014). Thirty years after the fall of the Berlin Wall, doing business in post-communist countries remains largely persona-based and dependent on informal practices. Poór et al. (2020) point at the inconsistency between HR policies and HR practices in the newly emerged post-socialist market economies. The informal HR practices are persistent and influential even in MNC subsidiaries operating in Central and Eastern Europe, Russia and Central Asia because local HR departments often experience autonomy in decision- making and implementation (Poór et al., 2020). These infor- mal practices, may, for instance, affect the hiring process, workload management arrangements, and workers’ rights for a vacation that may lead to favouritism. These param- eters vary—in case of weak legal and institutional frame- works in the post-communist space they are country- and industry-specific. Edwards and Lawrence (2000) compared managers in Hungary, Poland, Russia, Czech Republic and Slovakia, Romania, Bulgaria, Estonia, former Yugoslavia and concluded that “Russian managers’ experience of the collapse of the communist system in the early 1990s has been particularly acute” (p. 43). The formal process of de-communisation in Russia began later than in Central Europe. The Central Asian states—to which Kyrgyzstan belongs to—became independent as a result of the Soviet Union’s dissolution in 1991. It arguably took them longer to adapt their institutions to the market economy. In Cen- tral Asia, responsible leadership, personal integrity, human and worker rights are the emerging major ethical issues as well as relatively unexplored concepts both by management and employees (Rossouw, 2011). Furthermore, industry- specific aspect in the post-soviet space is under-researched, and therefore, there are various combinations of leadership styles and managerial ethics.i p ( ) The research findings on post-communist business ethics suggest that companies in the Former Soviet Union represent the mixture of leadership norms, for instance, an autocratic one and a participatory one (Rees & Miazhevich, 2009). Educated post-communist people prefer secured full-time employment over fixed-term contracts (Rees & Miazhevich, 2009; Smirnykh & Wörgotter, 2019), and they are content with being active followers and adhering to permissive lead- ership styles (Sotiroska Ivanoska et al., 2019). Business Ethics, Managerial Ethics and HRM Practices Tokbaeva the lack of planning and opportunistic approach to the man- agement of the first-generation of Russian businesspeople. “[…] a person in such uncertain conditions [when rules of the game change without notice] has to be very flexible. Most of them do not dream of building the best company or one that will last forever. They focus on making the most of the opportunities they see around them” (Shekshnia & Kets de Vries, 2008, p. 292). Besides, during the 1990s and early 2000s, many founders and owners of businesses in Russia served as CEOs themselves. Their role included inspiring and energising the staffers regularly. However, the market economy cannot run without the ethos of bureaucratic office (Weber, 1922/1947, as cited in Du Gay, 2009). The research on post-communist business ethics confirms that. During the mid-2000s, the roles of owner and manager in Russia became increasingly separated, and the relationship between leaders and followers is now built on more rational grounds (Serdukov, 2012; Shirokova et al., 2015). For example, it is more common that share and option schemes are used to motivate the personnel (Shekshnia & Kets de Vries, 2008). The research findings on post-communist business ethics suggest that companies in the Former Soviet Union represent the mixture of leadership norms, for instance, an autocratic one and a participatory one (Rees & Miazhevich, 2009). Educated post-communist people prefer secured full-time employment over fixed-term contracts (Rees & Miazhevich, 2009; Smirnykh & Wörgotter, 2019), and they are content with being active followers and adhering to permissive lead- ership styles (Sotiroska Ivanoska et al., 2019). Perceptions of various business practices such as falsifying time/quan- tity/quality reports, the acceptance of gifts and favours in exchange of preferential treatment and other differ among Russian male and female managers (Deshpande et al., 2000). Younger CEOs in Russia “show equal or less need for change in management style” (Shirokova et al., 2015, p. 90) than older CEOs; therefore, age is not a change-maker. These studies confirm previous findings by Donaldson (1996) who found that in multi-national corporations, the understanding of managerial ethics, for example, the tolerance for a bribe, is culture-specific. Therefore, these arguments suggest that there are multiple dimensions to consider when analysing the development of managerial ethics in post-communist firms. Therefore, a variety of qualitative and quantitative studies are needed to fill in this gap. Managerial Ethics in the Post‑communist Businesses Perceptions of various business practices such as falsifying time/quan- tity/quality reports, the acceptance of gifts and favours in exchange of preferential treatment and other differ among Russian male and female managers (Deshpande et al., 2000). Younger CEOs in Russia “show equal or less need for change in management style” (Shirokova et al., 2015, p. 90) than older CEOs; therefore, age is not a change-maker. These studies confirm previous findings by Donaldson (1996) who found that in multi-national corporations, the understanding of managerial ethics, for example, the tolerance for a bribe, is culture-specific. Therefore, these arguments suggest that there are multiple dimensions to consider when analysing the development of managerial ethics in post-communist firms. Therefore, a variety of qualitative and quantitative studies are needed to fill in this gap. Business Ethics, Managerial Ethics and HRM Practices the lack of planning and opportunistic approach to the man- agement of the first-generation of Russian businesspeople. “[…] a person in such uncertain conditions [when rules of the game change without notice] has to be very flexible. Most of them do not dream of building the best company or one that will last forever. They focus on making the most of the opportunities they see around them” (Shekshnia & Kets de Vries, 2008, p. 292). Besides, during the 1990s and early 2000s, many founders and owners of businesses in Russia served as CEOs themselves. Their role included inspiring and energising the staffers regularly. However, the market economy cannot run without the ethos of bureaucratic office (Weber, 1922/1947, as cited in Du Gay, 2009). The research on post-communist business ethics confirms that. During the mid-2000s, the roles of owner and manager in Russia became increasingly separated, and the relationship between leaders and followers is now built on more rational grounds (Serdukov, 2012; Shirokova et al., 2015). For example, it is more common that share and option schemes are used to motivate the personnel (Shekshnia & Kets de Vries, 2008).i Regardless of the region of the world, business people tend to overestimate how ethical they are (Baumhart, 1961), and post-communist managers are not an exception. The essential aspects of business ethics can be identified, plac- ing them in a relevant economic and social perspective. With this, we move on to discussing managerial ethics, focusing on the examples from post-communist countries. Business Ethics, Managerial Ethics and HRM Practices Business ethics can be discussed from various perspectives, including one of the employee, the company and society as a whole. The ownership and the legitimacy of managerial authority (McMahon, 1989), organisational values as per- ceived by employees (Jin et al., 2007), and employees’ pro- fessional value systems (von Weltzien Hoivik, 2002) shape the development of managerial ethics. This paper focuses on managerial ethics, which relates to the leader–follower relationship and ethical decision-making of executives. In the literature, this field overlaps with workplace ethics or the ethics of human resource management (HRM). It covers ethical issues arising around an employer-employee rela- tionship from the employees’ perspective (Sennett, 1998), such as the rights and duties owed between employer and employee. The HRM practices include career development and opportunities for advancement, training opportunities, job influence and challenge, involvement and communica- tion, performance management and appraisal, and work-life balance. However, merely putting HRM practises in place is not enough; HRM practices contribute to firm performance when the approach to people management taken by manag- ers is ethical (Purcell & Kinnie, 2008). Therefore, this paper relies on the term “managerial ethics” and focuses on lead- ers’ ethical decisions and their impact on followers. Guillén and González’s (2001) conceptual model of ethical dimen- sions views managerial ethics through the lens of leadership. Table 1 is an adaptation of Guillén and González’s (2001) work (p. 183), from the perspective of HRM practices. Previous studies discussed the connection between charismatic leadership and managerial ethics. Findings by Howell and Avolio (1992) suggest that creating loyal supporters and eliminating dissenters is one of the charac- teristics of charismatic leadership. Although some charis- matic leaders develop their followers intellectually, over long periods overdependence on a charismatic leader can mean losing more significant opportunities for follow- ers. For instance, those who could have started their own business using the skills they had gained, instead prefer Table 1 is an adaptation of Guillén and González’s (2001) work (p. 183), from the perspective of HRM practices. Formal power and leadership Task assignment Direct control and supervision Compulsory rules Resource allocation Process and routine standardisation Goal definition Information-decision systems Remuneration systems and procedures Assessment systems Training Recognition and reward systems Code of conduct Common mission statement The attractiveness of a firm to current and potential employees Common mission statement 1 3 850 D. Data Sources The interview target number was estimated at 40 inter- views, 20 per country, using Hinkin and Holtom’s (2009) response rates and sample representations model in organi- sational studies. A broad scope of in-depth semi-structured or “intensive” (Charmaz, 2014) interviews with company owners, senior, middle and junior management and for- mer members of staff was achieved. The questionnaire was constructed with an emphasis on the relationship between a leader and his/her followers during two stages of a com- pany’s life-cycle—the origin and maturation (Bryman, 1992; Floyd & Wooldridge, 1992; Kets de Vries, 1988; Küng, 2000; Lund, 2008). The questionnaire included questions about the changes in practices of managerial ethics and its impact on employees’ job performance and job satisfaction. Questionnaires were adjusted for company owners, senior managers, middle managers, junior and for- mer employees, depending on the degree of one’s involve- ment in decision-making and years of company service. Following Kotter (1982), questions about early childhood, adulthood and family-related questions were asked, when appropriate. Overall, the questions were constructed to capture micro-issues, such as motivations among leaders and followers (Adair, 2009) and macro-issues, such as the functioning of an organisation in a business environment. Names of interview participants and companies were made anonymous, and pseudonyms are used in this paper, to protect their privacy (Table 2). Based on the analysis of market transformations, the fol- lowing research criteria were used to select the companies for a case study: (a) a company is a media holding (as defined above); i (b) a company is privately owned by local businesspeople; (c) a company was founded after the fall of the Soviet Union in 1991; therefore, it is a post-Soviet firm; i (d) a company has already broken even and is in a good financial state. Following a multiple-case study procedure proposed by Yin (2014, p. 60), two companies were selected: one for Kyr- gyzstan, and the one for Russia. The country-comparative approach allows identifying similarities and differences in managerial ethics between a stronger economy (Russia) and a weaker one (Kyrgyzstan), the ‘centre’ of the former Soviet Union and its ‘periphery’. The two-case study design was chosen to consider a greater variety of external and inter- nal influences. The business environments in the Central, Fieldwork took place in 2014–2015 in two stages: the first stage was in Bishkek, Kyrgyzstan and the second stage in Ekaterinburg, Russia. Research Design Research findings emphasise the immense role of indi- vidual arrangements when it comes to managerial ethics in the post-communist space. And, therefore, the role of com- pany leadership in this setting remains strong. To illustrate that point, Shekshnia and Kets de Vries (2008) wrote about Among all approaches to studying managerial ethics and organisational leadership, this study is using a qualitative comparative case study based on in-depth, semi-structured interviews. Arguably, it is the most suitable choice for this 1 3 3 The Rise and Fading Away of Charisma. Leadership Transition and Managerial Ethics in the… 851 Eastern and South-Eastern Europe, Russia, the Caucasus and Central Asia do share post-communist traits, but only to a limited degree. Also, their media markets are distinct (Dobek-Ostrowska, 2010; Gross & Jakubowicz, 2013; Sparks & Reading, 1994; Vartanova, 2013). Market condi- tions and political situation placed the Russian media in a position where they receive vastly more foreign investment and state subsidies than the Kyrgyz ones (Dovbysh, 2019; Tokbaeva, 2019). On the other hand, the Kyrgyz media have enjoyed relatively more press freedom than their Russian counterparts—the countries are ranked 82nd and 149th, respectively (World Press Freedom Rating, 2020). There- fore, leadership patterns of media holding founders were examined and compared on the backdrop of similar yet dis- tinct media environments of regional Russia and Kyrgyzstan. Eastern and South-Eastern Europe, Russia, the Caucasus and Central Asia do share post-communist traits, but only to a limited degree. Also, their media markets are distinct (Dobek-Ostrowska, 2010; Gross & Jakubowicz, 2013; Sparks & Reading, 1994; Vartanova, 2013). Market condi- tions and political situation placed the Russian media in a position where they receive vastly more foreign investment and state subsidies than the Kyrgyz ones (Dovbysh, 2019; Tokbaeva, 2019). On the other hand, the Kyrgyz media have enjoyed relatively more press freedom than their Russian counterparts—the countries are ranked 82nd and 149th, respectively (World Press Freedom Rating, 2020). There- fore, leadership patterns of media holding founders were examined and compared on the backdrop of similar yet dis- tinct media environments of regional Russia and Kyrgyzstan. research of new practices of managerial ethics in countries where media has never been a business. Russia and Kyr- gyzstan, two post-soviet states, are an example of countries where media has never been a business until 1991. Inter- views allow capturing first-hand experiences and reflections about an ongoing process (Gummesson, 2000, p. Setting In this paper, leadership transition and managerial ethics are observed within a specific type of business entity—a pri- vately-owned media firm. The post-Soviet media landscape consists of state-owned media companies (e.g., Rossiya TV- Channel); media assets of diversified media groups (e.g. STS channel, a part of STS Media); foreign-owned media (e.g., Cosmopolitan-Russia magazine, a subsidiary of the Nordic media group Sanoma), and privately owned media firms. To reduce economic risks and increase political influence or resistance to external political influence (Jaksic et al., 2014; Rowland & Higgs, 2008), many media firms in the past 20 years have grouped into holdings, and thus, changed its leaders. Selling a successful media business to a corporate media group is standard practice. Therefore, it is crucial to concentrate on a privately owned firm, which was not sold, to capture the development of a founder’s leadership and a firm’s managerial ethics. Only a dozen media holdings in Russia and three ones in Kyrgyzstan are still owned by the same person or group who bootstrapped them in the 1990s or 2000s (Tokbaeva, 2018, 2019). Research Design 35; Kvale, 2007) such as leadership transition. At the same time, the case study framework helps to situate this process in its con- text and explain the reasons behind its peculiarities. Type of informants Type of informants Countries in which information was collected Kyrgyzstan, Russia observations and document analysis, which allowed to minimise potential bias (Cassell, 2009). in a soviet working-class family. He was a teenager when the Soviet Union collapsed. After the break-up of the Soviet Union, Alibek got a degree in software engineering. He started his private business in his late 20 s to support his family, a wife and a child. In 2001 he borrowed money and ventured to publish his first title. He served as a journalist, editor and layout editor all in one. Alibek distributed papers himself also. His attempts to sell the paper at the bazaar (Central Asian marketplace) were not a success. He went bankrupt. Few months after in summer of 2001 he published another title—a crossword puzzle paper. He created cross- words himself using computer software and dictionaries. He took out a mortgage for a house, having borrowed from his wife’s family. By autumn of 2001, the circulation of the crossword puzzle paper rose twofold, and he could hire the first five people and rent an office in the basement with no windows. Alibek came up with an idea of a newspaper that would sell out across the whole country. The first issue of The Altyn was published in spring 2002. Data Analysis Textual analysis of interview data was performed using a grounded theory approach (Glaser & Strauss, 1999; Goulding, 2009), which is suitable for dealing with subjec- tive data from interviews. Following Hsieh and Shannon’s framework (2005), this research argued that the directed approach of textual analysis can effectively interpret data on leadership and managerial ethics in transitioning con- texts. The analytic codes that resulted from the textual analysis of interviews laid the basis for comparing and contrasting the cases of Russian and Kyrgyz media hold- ing. The empirical results and the analysis of findings will be presented in the next subsections of the paper. The new paper was not profitable. I had to support it by means of earnings from crossword puzzle paper. All the time I’d simply make the whole team work for The Altyn for free. And I couldn’t explain them logically why they’d have to do it. (From an interview with the owner) Empirical Results The sufficient evidence was collected for analysis of leader- ship and managerial ethics during both phases—company origin and company maturation. Empirical data from the Russian and the Kyrgyz one is laid out together, in a chrono- logical way. The discussion goes around the development of leadership within media companies and its transition from a charismatic one to a hybrid one. It also shows that, despite improving, managerial ethics issues in these companies remained unsolved. The new paper included crosswords and news about local and international celebrities. But it received a cold welcome from the readers. There were moods [among the team] that new paper rather had to be shut down. But I remember telling them that there will be the times when we will all make a living thanks to that paper [The Altyn]. And I expected that in two-to-three years we could build it up very well – it could become one of the top five news- papers in the country in terms of circulation. (From an interview with the owner) Data Sources Additional interviews were gathered by Skype or email during 2016; and then dur- ing 2019. 43 interviews were collected; 22 on Kyrgyzstan and 21 on Russia. The primary data from in-depth semi- structured interviews were supported through fieldwork 1 3 852 D. Tokbaeva Table 2   Overview of data collection Period of data collection December 2014–August 2019 Data sources In-depth semi-structured interviews, business publications, news agencies, corporate mate- rials, observations at companies and industry conferences Total number of interviews collected Forty-three interviews with 41 informants # informants with one interview conducted: 39 # informants with two interviews conducted: 2 Type of informants Company founders and owners, top executives, branch heads, middle management, junior management, former employees, industry analysts, press association members Countries in which information was collected Kyrgyzstan, Russia Table 2   Overview of data collection December 2014–August 2019 In-depth semi-structured interviews, business publications, news agencies, corporate mate- rials, observations at companies and industry conferences The Emergence of a Charismatic Leader We begin the narrative with the Kyrgyz case. Murat Alibek is the founder of Altyn Kul, a Kyrgyz media holding. This media holding currently includes the highest circulation paper The Altyn and one of the most visited websites in the Kyrgyz language in the world. He was born in Kyrgyzstan 3 853 The Rise and Fading Away of Charisma. Leadership Transition and Managerial Ethics in the… a very young, dynamic programming team. (From an interview with the owner) a very young, dynamic programming team. (From an interview with the owner) In 2008, the new paper finally broke even. Its circulation and advertising revenue have been steadily growing since then. Alibek demonstrated charismatic features in the early stages of the company growth by inspiring people and leading them (Bryman, 1992; Weber, 1922/1947), despite not being able to offer them stability. He later left that cooperative to start his own business together with his friend. Thus, the company was founded in 1992. It was all about entrepreneurship. It was in the air. And then after some time, I caught myself thinking that I had a new criterion of success. Success is no longer measured by the pages of code one’s written or the number of orders delivered, but the amount of returns on investment. That’s when I realised I started think- ing like a businessman. (From an interview with the owner) If I could not satisfy my staff financially, I would embrace them with hope and confidence instead, as I believed myself that success was eventually going to happen. (From an interview with the owner) If I could not satisfy my staff financially, I would embrace them with hope and confidence instead, as I believed myself that success was eventually going to happen. (From an interview with the owner) Alibek showed a business talent in identifying the market niche and creating a product that has eventually become the nation’s favourite paper in the Kyrgyz language. However, for the first seven years, from 2001 to 2008, the venture’s continuous existence relied on unpaid labour. Both skilled staff or the staff Alibek trained himself worked without per- manent or even fixed-term contracts, and the honorarium was paid occasionally according to oral conventions between Alibek and each employee. Moreover, this case is genuinely charismatic since the leader depended on his followers as much as they relied on him to guide them. The Emergence of a Charismatic Leader Needless to say, that employees themselves yielded the right to make the most critical business decisions in the company to Alibek, which, again, corresponds to the Weberian understanding of charismatic authority. Andreev’s business success so far may be explained through his ability to surround himself with loyal people and trans- late his vision through them to the rest of the staff. His busi- ness venture was a success, and the company could reinvest its surplus into its development. Around 2004 and 2006 the company could afford to try out several products to see which one of them would work. The company employees recalled that it was an exciting period when they had a say in the company’s business and saw their ideas put into prac- tice. By 2007, Zhuravl Media included a portfolio of B2B, B2C services, a business newspaper, a glossy magazine and several user-generated content websites. It was in 2007 that Zhuravl Media reached its maturation stage and realised that it could scale up its successful business model. During the 2000s, the company expanded, and sold its media franchise to partners across Russia and, later, Kazakhstan. There were times when I had doubts. I inspired my very first employees with confidence, I have then seen this confidence in their eyes, and it inspired me back. (From an interview with the owner) We now move on to the Russian case. Mikhail Andreev is a founder of Zhuravl-Media, a Russian media holding. Zhuravl-Media is one of the largest publishing houses in regional Russia. Its specialism is business press and busi- ness-to-client (B2C) services. Andreev was born in the Rus- sian Ural region in an educated soviet family. He graduated from the Mechanical Engineering Faculty of Ural State Technical University in 1983. When the Soviet Union col- lapsed, he was working at the analogue computer plant as a programmer. In 1992, he left the plant and joined a coopera- tive together with a group of friends, who were programmers like him. Alexei Kharitonov thinks that his programming education helped him to set up his first business, a book- let publishing company. Although he didn’t know anything about business, he was no stranger to setting precise tasks and solving complex problems. The Fading Away of Charisma and the Rise of a Legal‑Rational Leader For Andreev, the most recent problems were the competition from global services like Chinese e-commerce platform Ali- baba and a drop in local business activity and advertisement revenues caused by foreign sanctions. Alibaba is doing well on the Russian market. Andreev cancelled several depart- ments entirely and had to cut staff. However, most of Zhuravl Media’s heads of branches have stayed with the company for ten years and more. Some employees worked at the company for more than 15 years, and some for more than 20 years. They have accumulated knowledge of the market and the company’s competitive advantages. They are the members of the senior management team who sustain Andreev’s vision and share it with the middle and junior management. Those senior managers who didn’t share Andreev’s vision, left the company, regardless of the time they had served at the media holding, feeling a lack of career progression opportunities and lack of being valued. When the business started to get better and better, the long-term employees believed even more in our chances for the future. In turn, their mood affected recently hired employees. (From an interview with the owner) Speaking of human resource management practises at Altyn Kul, Alibek said he trusted his gut feeling when choosing “the right” people, and his decisions have been uncontested. I was very strict towards certain deeds. If someone influenced the work negatively, I always tried to get rid of that person. I wanted to create a solid basis [of peo- ple and ideas], which would not infect others. (From an interview with the owner) In an interview, Alibek also said that he associates the com- pany with himself. The ones who did not believe in the company, who influenced negatively, I would always say goodbye to them, no matter how skilled they were. They just did not suit me and the company policy. This is the way, such a team was formed, and it stays the same until now. (From an interview with the owner) I do not have an inspiration coming to the meetings anymore. When my department was vital, I was one of the main decision-makers in a company. We used to have lively discussions about everything together (with the company head), and I found him so intelli- gent. Then, when we lost several advertising contracts, I became just an operations man. The Fading Away of Charisma and the Rise of a Legal‑Rational Leader We begin with the Kyrgyz case. Altyn Kul employees describe Alibek as a determined, intelligent and optimis- tic person. In recent years, according to interviews with company employees and media experts, Alibek has become more rigorous and pragmatic. Yet, he gets informed about the situation and controls his subordinates through “having discussions with them”. He cultivated loyalty among many employees that have been working for the company for more than ten years. It’s all about choosing whom to hire. We used to have competent specialists, who were probably most inter- ested in salary. I understand that, but I did not have money in those days. As all companies do, I would choose the employee, whom I can satisfy financially. (From an interview with the owner) I worked at a plant, and I had a great team there. When- ever, for different reasons, some of the people I used to work with left [the plant] to create their cooperatives, they started inviting me in. I later joined them. We had Recently hired staff workers do not share the feeling of long- term employees that Alibek is the “father of the company”. 1 854 D. Tokbaeva By the time I had my baby, I was promoted to an edi- tor position. Then I asked to be a journalist instead of an editor [to reduce workload]. When my child got older, I returned to the editorial position. It is a com- mon practice [at our company]. (From an interview with one of the editors-in-chief) Instead, they believe they work for a reliable business with a stable salary and staff benefits. These are 20–22-year-old recent graduates of journalism faculties as well as current students who are working part-time at the media holding. Nevertheless, they are still affected by the organisation cul- ture sustained by middle management and senior manage- ment. The latter consists of the people who were among the first five whom Alibek had hired back in 2001. Moving on to the Russian case, Zhuravl Media employees describe Mikhail Andreev as the person who values listen- ing over talking. Most commonly, he listens to the staff’s suggestions and concerns and takes business decisions, based on what he has heard. If the 2000s were the period of growth and market expansion for Zhuravl Media, the 2010s struck the company with several crises, one after another. Findings This paper explored the connection between leadership shift and the changes in managerial ethics in post-communist organisations on the example of two media firms in Kyr- gyzstan and Russia. The findings are based on the adaption of Guillén and González’s (2001) conceptual model of man- agerial ethics in relation to HRM and Walter’s and Bruch’s (2009) model for assessing the impact of charismatic lead- ership on organisations. Figure 1 presents two variations of former charismatic leaders by showing that there are several outcomes to a leader’s behaviour when one’s charisma fades. Guillén and González’s (2001) profound model is exten- sive and covers managerial practices such as, for instance, resource allocation, that can not be evaluated through quali- tative research. Therefore, it was decided to build the current model of this research (Table 3) based on the following four managerial practices in HRM: a) workload management; b) task delegation; c) career progression of junior and middle managers; and d) employees’ rights for vacation and pater- nity and maternity leave. Speaking on this topic, the owner himself mentioned that the company tries to avoid getting involved in politics. Thank God, we’ve got people who serve as a buffer between us and the authorities. Apart from that, we and the state authorities exist in a parallel universe. (From an interview with the owner) Despite losses, the main divisions of the company, such as its B2B and B2C platforms, function smoothly. There is no cash-flow issue, and the company is financially healthy. It emerged from interviews with company representatives who were asked to evaluate which departments were doing well and which weren’t. The only department that is under pressure is the glossy magazine unit, and the atmosphere among the staff confirmed that. In all of the departments, the holding’s employees clearly understand the compa- ny’s vision, and there is compliance with that vision. The magazine employees are aware that their department may soon undergo cuts. However, even if reductions happen, Fig. 1   What does a charismatic leader transform to, once cha- risma fades? Evidence from the Kyrgyz and Russian cases A public persona (the Kyrgyz case) The company head converts accumulated business reputation into political influence and takes on engagements outside of the firm, having preserved the ownership. The Fading Away of Charisma and the Rise of a Legal‑Rational Leader (From an interview with the department head) Since 2012, Alibek has held several positions in the Kyrgyz government. According to article 22 of the Kyrgyz law on government service, a civil servant is not allowed to be an entrepreneur and a company head (St. 22 Zakon KR 2016). Although Alibek appointed other people in charge of opera- tions of the media holding, he remained the founding mem- ber, shareholder and advisor of the media holding. Until now, he stays in close contact with the staff by regularly appearing in the office. Once one’s service in the govern- ment ceases, one can restore one’s position as a company head in charter and memorandum. Andreev belongs to the first generation of media managers to understand the importance of the market data on any busi- ness in the Russian regions. He is pragmatic and understands that now the business times are not so favourable for Zhuravl Media as they used to be during the 2000s when advertising revenues were higher. Although these qualities seem essen- tial for general managers described by Kotter (1982), these qualities are not widespread in the post-Soviet space. It is the feature of the legal-rational style of leadership that it relies more on data rather than people and their feelings. The issues of task delegation and career progression of newly hired junior and middle managers remain unsolved in the legal-ration stage. What has improved in the legal- rational stage of leadership is the issue of employees’ rights for maternity and paternity leave. Although the Labour Codex of Kyrgyzstan ensures it, not all companies observe it. Therefore, companies like Altyn Kul who secure staffers’ employment after pregnancy as their competitive advantage over competitors. Now [as of 2015] we are in the period of patching up holes. The publishing industry is impetuously break- ing; the paper is dying right before our very eyes. 1 3 3 855 The Rise and Fading Away of Charisma. Leadership Transition and Managerial Ethics in the… New technologies like Instagram appear almost every month. Change of means of communication leads to a shift in marketing. (From an interview with the owner) New technologies like Instagram appear almost every month. Change of means of communication leads to a shift in marketing. (From an interview with the owner) employees will be offered compensations. Settlements did not exist in the charismatic phase of Zhuravl Media devel- opment. The Fading Away of Charisma and the Rise of a Legal‑Rational Leader Currently, the most ambitious department of the company is the IT department, which employs 60 people, and it is growing. Most likely, the IT managers will get a promotion shortly, since the entire media holding is shifting its focus to becoming fully digital. The leadership of Zhuravl Media grips control over major decisions, as standard for a charismatic phase, and at the same time negotiates with its employees, as typical for a legal-rational stage. The Russian case is a mixture or a hybrid of leadership styles, where the company owner feels more comfortable being in full control. Moreover, the company took special arrangements navigat- ing the turbulent economic and political times. Being part of a larger state holding could leverage the outcomes of politi- cal uncertainty (Jaksic et al., 2014). Also, state contracts for the media could help (Dovbysh, 2019), but the leadership decided to sustain its editorial independence. Our boss gathered us at a staff meeting and told us he does not have any connections in the government that would get him out of trouble if it happens. Therefore, he asked us not to touch on sensitive topics such as Ukraine and the church. We didn’t cover them at all anyway. (From an interview with the editor-in-chief) Findings However, increased bureaucratisation, lack of room for career progression, and lack of owner’s understanding of the ideas that come from the staff were identified as the reasons why those previ- ously loyal managers left. At the same time, one needs to acknowledge that the legal-rational authority has brought positive changes to human resource management, especially for junior-level employees. For instance, employees’ rights for vacation and p/maternity leave and workload manage- ment have been taken into consideration and been exercised. That can be explained through the need for the companies to compete with other employees for the talent pool and keep the qualified employees trained within the company. There- fore, from the long-range planning perspective, the measure to offer benefits and compensations for staff workers is an effective way of optimising business costs and sustain quali- fied talent. The findings indicated that skilled talent might seek employment elsewhere, for instance, in a metropolis, such as Moscow and, thus, contributing to “brain drain”. Successful Russian firms manage to retain their talent, yet such moods exist and were expressed in interviews. It is less so in Kyrgyzstan where none of the interviewees voiced a possibility of relocation. To keep the talent, private firms in Leadership style plays a considerable role in shaping managerial ethics, given the novelty of managerial ethics concept for post-communist business owners and employees (Bohatá, 1997; Rossouw, 2011). This research has found that when a leadership style shifts from charismatic to a legal- rational one in a post-communist firm, managerial ethics changes also. Table 3 summarises the findings of this study based on the extensive interview-based research with com- pany owners, department heads, middle and junior manage- ment as well as former employees and industry experts in Russia and Kyrgyzstan. The shift in leadership style brings more consideration towards workload management and employees’ rights for vacation and p/maternity leave. However, the leadership transition in both media holdings did not bring changes in managerial ethics. Ill-performed task delegation among employees demonstrates it. Furthermore, the implementa- tion of career progression of junior and middle managers in the post-communist firms, on the contrary, slowed down. During a charismatic phase, a charismatic leader rapidly pro- moted his/her loyal followers, even to senior positions. Findings Variations of former pure charismatic leaders Variations of former pure charismatic leaders A low-key leader (the Russian case) The company head is concentrated on business goals and does not loosen the grip of the power of the company. Despite the lack of new skills, prefers to educate himself to be in full control, rather than delegate tasks to others. The company head is not interested in public life. 1 3 856 D. Tokbaeva progression became unclear, especially to junior employees and mid-career managers. At the kickstart stage, businesses benefit from a charis- matic leader (Bryman, 1992; Conger & Kanungo, 1987). It is particularly so in emerging transitional markets, where cultural attitudes favour risk-taking and high power dis- tance between members of an organisation. Yet, when the companies break even and mature, regardless of context, charisma fades (Weber, 1922/1947) and transforms into a legal-rational leadership or, most likely, into a hybrid. Cur- rently, the leadership style of both the Russian and the Kyr- gyz media owners is a hybrid form of a charismatic and a legal-rational leadership style. The process of transformation does not happen at once and depends on a leader’s personal- ity. Once the company matured, the Kyrgyz company head turned into a public persona and took a post in the Kyrgyz government. In contrast, the Russian media company head prefers to be a low key leader (See Fig. 1 for details). In summary, this study found that the shift from a charis- matic leadership style to a legal-rational authority does not imply that all managerial ethics issues are solved. Therefore, neither charismatic nor legal-rational leadership styles are not entirely ethical; at least it is so in the post-communist context. On the other hand, the legal-rational authority may even intensify some issues that weren’t acute before. For instance, Zhuravl Media lost several loyal middle manag- ers and one top manager after the shift to a legal-rational style. They explained their choice to leave the company by lack of opportunities to progress, for instance, from a mid- dle managerial level to a senior managerial level, or from a senior managerial level to a co-owner level, during a legal- rational leadership phase. The conflicts may be described as personal choices of involved parties. Conclusion This paper contributes to a deeper understanding of the nature of post-communist leadership, management and governance. This research confirmed previous studies on charismatic bonding between leaders and followers as a requisite for a business to overcome the crisis by De Cre- mer and Van Dijke (2010) and Shamir and Howell (1999). Tied up to economic turmoil and uncertainty, whether it is caused by post-communist market transformations, the 2008 financial crisis, or the need to digitise the value chain for high performance, persona-based leadership style remains acute in the post-Soviet space. It is there for cultural rea- sons. Start-up and survival stages of business development require a passion-driven, risk-taking charismatic leadership style. However, with the growth of market concentration, as illustrated on the example of media markets in Russia and Kyrgyzstan in this paper, there is a tendency of a “start-up” hero finds him/herself turn into a legal-rational bureaucrat. Some leaders explain it by the need to concentrate on other (e.g. political goals), while still maintaining advisor and owner role in the business (the Kyrgyz case). In contrast, others prefer to remain low key while navigating the tighten- ing socio-political environment (the Russian case). There is a need for detailed case-studies in critical leadership in the post-Soviet business setting. The appearance of new ventures founded by self-made businesspeople after the fall of the Soviet Union and their consequent maturation is what makes a post-Soviet set- ting particularly interesting one from the managerial ethics perspective. Due to the unique mix of lack of institutional development and widespread reliance on informal networks in business, Weberian three types of authority—a tradi- tional authority, a charismatic authority, and a legal-rational authority—in this setting exist in multiple combinations or hybrid forms. The more post-communist countries detach themselves from communist practises and find their way of developing small, medium and large-scale businesses, the more hybrid variations of leadership styles come to place. Hybrids can be found across all business and national can- vases, hence a country-comparative approach is a suitable framework for leadership studies. It is a comparative case study of leadership and managerial ethics transition in two local media holdings in Russia and Kyrgyzstan. Both were founded after the fall of the Soviet Union by people with no connections to the previous communist elite networks and no prior experience in doing business. Conclusion The Kyrgyz media holding Altyn Kul broke even after seven years on the mar- ket, while it took the Russian media holding Zhuravl Media 12 years to do the same. The break-even point is a mile- stone which marks leadership transition from a charismatic style to a legal-rational style. This study has found that in post-communist firms, managerial ethics changes when a charismatic leadership phase shifts to a hybrid charismatic/ legal-rational phase. Workload management and employees’ rights for vacation and paternity and maternity leave have been positively affected in both Russian and Kyrgyz firms. However, the leadership transition in both media holdings Findings Yet, once the leadership style changed, the career progression opportunities narrowed down, and the practises for career Table 3   Managerial ethics in the two phases of leadership in two the post-communist media holdings in Russia and Kyrgyzstan Ethical dimension Managerial practices in HRM Charismatic leadership phase Legal-rational leadership phase Altyn Kul (Kyr- gyzstan) 2001–2008 Zhuravl Media (Russia) 1994–2007 Altyn Kul (Kyrgyzstan) 2008–2016 Zhuravl Media (Russia) 2007–2016 Formal power and leadership Effectiveness of decision-making and execution The attractiveness of a firm to employees Workload management Absent Absent Present Present Task delegation Absent Absent Absent Absent Career progression of junior and middle managers Present Present Absent Absent Employees’ rights for vacation and paternity and maternity leave Absent Absent Present Present o phases of leadership in two the post-communist media holdings in Russia and Kyrgyzstan 1 The Rise and Fading Away of Charisma. Leadership Transition and Managerial Ethics in the… 857 did not bring changes in task delegation, and it affected the career progression of junior and middle managers negatively. post-Soviet states need to address the confusion around task delegation and the ambiguity around career progression of junior and middle managers. Table 4   Weberian leadership styles and hybrids in various organisational settings in the post-Soviet space Charismatic leadership These are pure entrepreneurship initiatives in the early 1990s and 2000s bootstrapped by first-generation entrepreneurs without prior elite network connections. According to this research, this leadership style has now faded as some firms ceased to exist, and others transformed Traditional leadership These are the early 1990s and 2000s’ small businesses bootstrapped by people with sufficient communist elite network links who turned entrepreneurs (Ivlevs et al., 2020) Legal-rational leadership These are the managers of the former state enterprises turned into private companies (Kiser, 1989; Soulsby & Clark, 1996) Charismatic/legal-rational hybrid Current department heads in foreign-owned firms, MNCs, and owners and leaders of temporary and gig economy projects such as start-ups Charismatic/traditional hybrid These are owners and partners in all-sized family businesses Traditional/legal-rational hybrid These are the C-level and middle management in state-controlled corporations Implications for Policymakers and Governing Bodies Baumhart, R. C. (1961). How ethical are businessmen? Harvard Busi- ness Review, 39(4), 6–19. Regardless of that, our results reveal that the issues of mana- gerial ethics such as task delegation and career progression of junior and middle managers remain unsolved. The media holdings observed in this study are, in many ways, the for- ward-thinking pioneers. Yet, these firms have not figured out its approach towards managerial ethics and not considered ethics as a driver of business success. This study confirms the findings of Bohatá (1997) and Rossouw (2011) that the business world in Central and Eastern Europe, and Central Asia, has not taken a systemic approach towards managerial ethics. Training interventions by policymakers and govern- ing bodies such as national business associations and cham- bers of commerce is required. This study is limited to the comparative analysis of two cases of media firms in Russia and Kyrgyzstan. Therefore, other combinations of leadership styles, how leadership affects managerial ethics, and which solutions firms offer to its employees in various national and industry contexts, should be explored further. Bohatá, M. (1997). Business ethics in Central and Eastern Europe with the special focus on Czech Republic. Journal of Business Ethics, 16, 1571–1577. https://​doi.​org/​10.​1023/A:​10058​67132​040 Bryman, A. (1992). Charisma and leadership in organisations. SAGE Publications. Cassell, C. (2009). Interviews in organizational research. In D. A. Buchanan & A. Bryman (Eds.), The SAGE handbook of organi- zational research methods (pp. 500–515). SAGE. Charmaz, K. (2014). Constructing grounded theory (2nd ed.). SAGE. Conger, J. A., & Kanungo, R. N. (1987). Toward a behavioural the- ory of charismatic leadership in organisational settings. Acad- emy of Management Review, 12(4), 637–647. De Cremer, D., & Van Dijke, M. (2010). Charismatic leadership still important to Dutch employees. Erasmus University Rot- terdam. Retrieved August 25, 2019 from https://​www.​erim.​eur.​ nl/​behav​ioural-​ethics/​news/​detail/​1615-​chari​smatic-​leade​rship-​ still-​impor​tant-​to-​dutch-​emplo​yees/. Deshpande, S. P., Joseph, J., & Maximov, V. V. (2000). Perceptions of proper ethical conduct of male and female Russian managers. Journal of Business Ethics, 24(2), 179–183. https://​doi.​org/​10.​ 1023/A:​10060​02030​496 Dobek-Ostrowska, B. (2010). Comparative media systems: European and global perspectives. CEU Press. Donaldson, T. (1996). Values in tension: Ethics away from home. Harvard Business Review, 74(5), 48–62. Acknowledgements  The author is grateful to three  anonymous reviewers and editors for their valuable feedback. The author would like to thank colleagues for their feedback on earlier versions of the manuscript. Dovbysh, O. (2019). Commercial or public service actors? References Adair, J. (2009). Leadership and motivation: The fifty-five rule and the eight key principles of motivating others. Kogan Page. Declarations Conflict of Interest  Dinara Tokbaeva declares that she has no conflict of interest. Ethical Approval  This article does not contain any studies with human participants or animals performed by the author. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Nowadays many market niches are occupied by the play- ers who had survived on the market throughout the 1990s, 2000s and 2010s. At least, as the research has shown, this is the case for the media markets of Russia and Kyrgyzstan. It means that in the next 5–10 years, one will be able to observe and study more successions in post-communist firms. For example, the ownership may be transferred to family members, or non-family members can be appointed as CEOs. Therefore, Kotter’s work on professional manage- ment will be acute for the post-communist business context in and after the 2020s. Implications for International and Local Business This research has shown that a leader’s charisma in its pure form has faded, and will only exist in post-Soviet firms a hybrid form as soon as a firm breaks even, and business opti- misation is priotitised over survival. However, companies require different types of leaders at various stages of their development (See Table 4). Table 4   Weberian leadership styles and hybrids in various organisational settings in the post-Soviet space Charismatic leadership These are pure entrepreneurship initiatives in the early 1990s and 2000s bootstrapped by first-generation entrepreneurs without prior elite network connections. According to this research, this leadership style has now faded as some firms ceased to exist, and others transformed Traditional leadership These are the early 1990s and 2000s’ small businesses bootstrapped by people with sufficient communist elite network links who turned entrepreneurs (Ivlevs et al., 2020) Legal-rational leadership These are the managers of the former state enterprises turned into private companies (Kiser, 1989; Soulsby & Clark, 1996) Charismatic/legal-rational hybrid Current department heads in foreign-owned firms, MNCs, and owners and leaders of temporary and gig economy projects such as start-ups Charismatic/traditional hybrid These are owners and partners in all-sized family businesses Traditional/legal-rational hybrid These are the C-level and middle management in state-controlled corporations Table 4   Weberian leadership styles and hybrids in various organisational settings in the post-Soviet space Charismatic/traditional hybrid These are owners and partners in all-sized family businesses Traditional/legal-rational hybrid These are the C-level and middle management in state-controlled corporations 1 3 858 D. 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Tissue specific expression of 11BHSD and its effects on plasma corticosterone during the stress response

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General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Tissue specific expression of 11BHSD and its effects on plasma corticosterone during the stress response Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Journal of Experimental Biology Edinburgh Research Explorer Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 http://jeb.biologists.org/lookup/doi/10.1242/jeb.209346 Access the most recent version at First posted online on 3 December 2019 as 10.1242/jeb.209346 Corresponding author: Jonathan H. Pérez Journal of Experimental Biology • Accepted manuscript [email protected] [email protected] [email protected] [email protected] Key words: glucocorticoid, corticosterone, hypothalamic-pituitary-adrenal (HPA) axis, 11β- HSD, negative feedback, songbird, stress Tissue specific expression of 11BHSD and its effects on plasma corticosterone during the stress response Jonathan H. Pérez1,2,3, Ryan E. Swanson1, Hannah J. Lau1, Jeffrey Cheah1, Valerie R. Bishop3, Katherine R.S. Snell4, Angus M.A. Reid3,5, Simone L. Meddle3, John C. Wingfield1 & Jesse S. Krause1 Jonathan H. Pérez1,2,3, Ryan E. Swanson1, Hannah J. Lau1, Jeffrey Cheah1, Valerie R. Bishop3, Katherine R.S. Snell4, Angus M.A. Reid3,5, Simone L. Meddle3, John C. Wingfield1 & Jesse S. Krause1 1Department of Neurobiology, Physiology and Behavior, University of California Davis, One Shields Avenue, Davis, CA 95616, USA. 2The Institute of Biodiversity, Animal Health & Comparative Medicine, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK. 1Department of Neurobiology, Physiology and Behavior, University of California Davis, One Shields Avenue, Davis, CA 95616, USA. 2The Institute of Biodiversity, Animal Health & Comparative Medicine, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK. 3The Roslin Institute, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, UK. 4Center for Macroecology, Evolution and Climate; Natural History Museum of Denmark; University of Copenhagen; Universitetsparken 15; DK-2100 Copenhagen, Denmark. 5MRC HGU, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, EH4 2XU, Scotland, UK. 2The Institute of Biodiversity, Animal Health & Comparative Medicine, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK. 3The Roslin Institute, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, UK. 3The Roslin Institute, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, UK. 4Center for Macroecology, Evolution and Climate; Natural History Museum of Denmark; University of Copenhagen; Universitetsparken 15; DK-2100 Copenhagen, Denmark. 5MRC HGU, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, EH4 2XU, Scotland, UK. Journal of Experimental Biology • Accepted manuscript Summary Statement Peripheral enzymes are primarily responsible for enzymatic modulation of the glucocorticoid stress response in songbirds. © 2019. Published by The Company of Biologists Ltd. Introduction Glucocorticoids are critical for the physiological responses to environmental, both external and internal, perturbations. In response to acute stressors such as food shortage, inclement weather or predators, the organism responds by increasing the synthesis of glucocorticoids from the adrenal glands, giving rise to the classic “stress response”. In the short term, elevation of glucocorticoids is adaptive promoting changes in physiology and behavior to promote survival (e.g. Sapolsky et al., 2000; Romero, 2002; Krause et al., 2017). As prolonged or chronic elevation of glucocorticoids can result in a number of pathological conditions, the homeostatic regulation of glucocorticoid levels is essential for maximizing fitness. Regulation of glucocorticoid synthesis and secretion in response to stressors begins with integration of internal and external cues by the brain. Ultimately, cells in the paraventricular nucleus (PVN) of the hypothalamus are stimulated causing the release of corticotrophin-releasing factor (CRF) and arginine vasopressin or arginine vasotocin depending on species (e.g. Joëls et al., 2008). CRF triggers the release of adrenocorticotropic hormone (ACTH) from the corticotroph cells in the anterior pituitary gland into the systemic circulation. ACTH then acts to stimulate increased synthesis of glucocorticoids in the adrenal glands. Glucocorticoids not only signal to target cells, but also provide negative feedback to the hypothalamo-pituitary-adrenal (HPA) axis regulating further activation of the HPA axis and ultimately returning plasma hormone levels to baseline. These actions occur through two classes of receptors: the high affinity mineralocorticoid (MR) and low affinity glucocorticoid (GR) receptors. GR receptor tends to be bound at high circulating levels of glucocorticoids such as those encountered during a stress response so GR is considered to be the primary mediator of stress effects and negative feedback (Reul et al., 1987). Glucocorticoid signaling generates negative feedback by binding to CRF-neurons in the PVN as well as blocking hippocampal signaling to the CRF neurons, thus reducing CRF release and inhibiting ACTH release through binding at corticotrophs in the anterior pituitary gland (de Kloet, 2014). Journal of Experimental Biology • Accepted manuscript Recent work has suggested a short negative feedback loop within the adrenal gland itself, with locally produced glucocorticoids serving to suppress further glucocorticoid synthesis as levels rise (Walker et al., 2015). Thus, negative feedback regulation of the stress response can broadly be categorized as either central or peripheral depending upon the site of glucocorticoid signaling involved. Abstract The hypothalamic-pituitary-adrenal (HPA) axis is under complex regulatory control at multiple levels. Enzymatic regulation plays an important role in both circulating levels and target tissue exposure. Three key enzyme pathways are responsible for the immediate control of glucocorticoids. De novo synthesis of glucocorticoid from cholesterol involves a multistep enzymatic cascade. This cascade terminates with 11β-hydroxylase, responsible for the final conversion of 11 deoxy- precursors into active glucocorticoids. Additionally, 11β- hydroxysteroid dehydrogenase type 1 (11β-HSD1) controls regeneration of glucocorticoids from inactive metabolites, providing a secondary source of active glucocorticoids. Localized inactivation of glucocorticoids is under the control of Type 2 11β-HSD (11β-HSD2). The function of these enzymes is largely unexplored in wild species, particularly songbirds. Here we aim to explore the contribution of both clearance and generation of glucocorticoids to regulation of the hormonal stress response via use of pharmacological antagonists. Additionally, we mapped 11β-HSD gene expression. We found 11β-HSD1 primarily in liver, kidney, and adrenal glands though it was detectable across all tissue types. 11β-HSD2 was predominately expressed in the adrenal glands and kidney with moderate gonadal and liver expression. Inhibition of glucocorticoid generation by metyrapone was found to decrease levels peripherally, while both peripheral and central DETC administration resulted in elevated concentrations of corticosterone. These data suggest that during the stress response, peripheral antagonism of the 11β-HSD system has a greater impact on circulating glucocorticoid levels than central control. Further studies show aim to elucidate the respective roles of the 11β-HSD and 11β-hydroxylase enzymes. Journal of Experimental Biology • Accepted manuscript Introduction Introduction Given the importance of glucocorticoid receptors in mediating negative feedback control, the majority of research to date has focused on variation in distribution and concentration of MR and GR within the HPA axis (Breuner and Orchinik, 2001; Canoine et al., 2007; Harris et al., 2013; de Kloet, 2014; Krause et al., 2015; Cornelius et al., 2018). To date other mechanisms of stress axis modulation have received comparatively little attention. Of particular interest is enzymatic control of glucocorticoid synthesis/regeneration and localized inactivation. Adrenal generation of active glucocorticoids from cholesterol ends with the conversion of deoxy- forms (deoxycorticosterone or 11-deoxycortisol) to active glucocorticoid by the 11β hydroxylase enzyme. The liver also serves as a secondary source of glucocorticoids through the regeneration of inactive 11 keto-glucocorticoids to glucocorticoids by the enzyme 11β hydroxysteroid dehydrogenase type 1 (11β-HSD1). Indeed in humans regeneration of inactive 11 keto-glucocorticoids has been found to account for up to 40 % of glucocorticoid synthesis (Basu et al., 2004). 11β-HSD1 may also serve to mediate local tissue level exposure as it has been reported in an array of mammalian tissue types including the brain, liver, adipose tissue, fat, gonads, vasculature, multiple brain regions, uterus, and muscle (Diaz et al., 1998; Holmes and Seckl, 2006; Wyrwoll et al., 2011; reviewed in Chapman et al., 2013). Reports of 11β-HSD1 in avian species are limited with a single study reporting it as undetectable in the brain of zebra finches, Taeniopygia guttata, (Rensel et al., 2018). Of particular interest is enzymatic control of glucocorticoid synthesis/regeneration and localized inactivation. Adrenal generation of active glucocorticoids from cholesterol ends with the conversion of deoxy- forms (deoxycorticosterone or 11-deoxycortisol) to active glucocorticoid by the 11β hydroxylase enzyme. The liver also serves as a secondary source of glucocorticoids through the regeneration of inactive 11 keto-glucocorticoids to glucocorticoids by the enzyme 11β hydroxysteroid dehydrogenase type 1 (11β-HSD1). Whereas 11β-HSD1 and 11β hydroxylase act to synthesize and regenerate inactivated glucocorticoids respectively, 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) serves as a key regulator of local exposure to glucocorticoids at the tissue level by inactivating glucocorticoids to inert metabolites. This is best demonstrated by its well-documented action in the kidneys where inactivation of glucocorticoids by 11β-HSD2 allows aldosterone, instead of glucocorticoids, to bind to the non-selective MR receptors. Introduction Similar to 11β-HSD1 in birds, 11β-HSD2 has been reported to have a relatively limited neural distribution in mammals and is most closely associated with protection of developing tissue from excess glucocorticoid signaling and modulation of neural aldosterone signaling in the adult brain (Wyrwoll et al., 2011). In birds 11β-HSD2 has been described in the chicken (Gallus gallus; Klusoňová et al., 2008) and zebra finch (Katz et al., 2010; Rensel et al., 2018) in brain, liver, kidney, colon and gonads. The expression of 11β-HSD enzymes in the brain in particular suggests the potential for altering negative feedback control of the hormonal stress response. Similarly, peripheral 11β-HSD is expected to impact rates of clearance and regeneration of glucocorticoids. Together, these dual effects may provide a critical mechanism for maintenance of both plasticity and variation (seasonal and inter-individual) in the functional Journal of Experimental Biology • Accepted manuscript characteristics of the HPA axis. This is supported by studies in 11β-HSD1 knockout mice that have demonstrated increased glucocorticoid secretion in response to restraint stress (Harris et al., 2001). To date, the role of regulatory enzymes, both in the periphery and brain, in controlling glucocorticoid levels remains poorly understood in free living animals, particularly birds. Here we seek to understand the relative contribution of both peripheral and central inactivation (via 11β-HSD2) and activation (via 11β-HSD1 regeneration and 11β- hydroxylase synthesis) of glucocorticoids in modulating the hormonal stress response. In order to address these major knowledge gaps, we have taken a multi-step approach utilizing the Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) - a seasonally breeding songbird species with well-characterized stress physiology. First, we quantified mRNA expression of 11β-HSDs across both central and peripheral tissues in both sexes. To test the importance of both central and peripheral enzyme activity we conducted both peripheral and central ICV administration of pharmacological antagonists targeting both glucocorticoid (hereafter corticosterone, the major glucocorticoid in birds) generation and clearance. To test the contribution of corticosterone synthesis in modulating circulating plasma levels we utilized the well-characterized blocker of corticosterone synthesis metyrapone (MET), shown to block both synthesis by 11β hydroxylase and 11β-HSD1 regeneration of corticosterone. Simultaneously we utilized a previously identified selective inhibitor of 11β-HSD2 (Schweizer et al., 2003), sodium diethyldithiocarbamate trihydrate (DETC; inhibits 11β-HSD2) to block clearance of corticosterone. Journal of Experimental Biology • Accepted manuscript Materials and Methods Animals Gambel’s White-crowned Sparrows were captured on their wintering grounds near Davis, California USA (N 38° 33’, W 121°44’) between 2016 and 2017 using a combination of seed baited potter traps and Japanese mist nets. In December 2016, photosensitive field caught birds (n= 9 per sex) were euthanized by overdose of isoflurane and following confirmation of death brain, pituitary gland, gonad, kidney, liver, fat, gastrocnemius muscle, pectoralis muscle, heart, and adrenal glands were collected for subsequent RT-PCR analysis. Collected tissues were immediately fresh frozen on dry ice and stored at - 80°C then shipped to the Roslin Institute on dry ice, and then stored at - 70°C. Time to euthanasia from capture was 146 + 5 sec and a baseline blood sample (ca 70 µL) was taken from all animals within 87 + 7 sec of capture to determine baseline levels of corticosterone. Blood was collected by puncture of the alar vein with a 26 gauge needle and surface blood collected by heparinized microcapillary tube (41B501; Kimble Chase, Vineland, NJ USA). Twenty-eight males and twenty-two females were sampled in the field for Experiment 1 (Peripheral 11β-HSD2 Regulation) from February to March of 2017. In early April 2017 an additional 26 pre-breeding males were captured and transferred to captivity in aviary facilities at the University of California, Davis for use in Experiment 2 (Central Regulation). Sex was determined by PCR followed by gel electrophoresis per (Griffiths et al., 1998) for free living birds and by necropsy for captive birds. All procedures were approved by UC Davis Institutional Animal Care and Use Committee (IACUC), protocol # 19758 and followed UK ARRIVE (ASPA) guidelines. In all experiments birds were randomly assigned to treatment. Determining 11β-HSD mRNA Expression RNA was extracted from tissues using Zymo DIRECT-zol RNA miniprep kits (Zymo Research, Irvine CA. USA). Following RNA extraction, total concentration of RNA was determined via nanodrop. RNA input was equalized tissue-wise for reverse transcription to cDNA using a High Capacity cDNA Reverse Transcription Kit (Cat no. 4368814; Life Technologies, Carlsbad, CA USA) prior to quantification. Quantitative polymerase chain reaction (qPCR) using Brilliant III Ultra-Fast Sybr Green (Agilent Technologies, http://www.genomic.agilent.com, Santa Clara, CA USA) in Thermofast 96 well detection plates (AB1100, ThermoFisher, UK) with optical caps (4323032, ThermoFisher, UK) was used to measure 11β-HSD1 and 11β-HSD2 gene expression. Reactions were performed and samples counted on a Stratagene MX 3000 Machine and relative measurements calculated using MxPro software by extrapolation to a standard sample series of defined concentration (standard curve), as previously described (Reid and Dunn, 2018). Manual examination of reaction quality involved examination of dissociation curves for a single peak, check of standard curve correlation (>0.995) and confirmation of good reaction efficiency (90-110%). Following quality control, sample size for male gastrocnemius muscle was reduced to 8, and both gonad and pituitary gland to 6 samples for final analysis. All qPCR data were normalized to the geometric mean value of two reference genes; 3- RNA was extracted from tissues using Zymo DIRECT-zol RNA miniprep kits (Zymo Research, Irvine CA. USA). Following RNA extraction, total concentration of RNA was determined via nanodrop. RNA input was equalized tissue-wise for reverse transcription to cDNA using a High Capacity cDNA Reverse Transcription Kit (Cat no. 4368814; Life Technologies, Carlsbad, CA USA) prior to quantification. Quantitative polymerase chain reaction (qPCR) using Brilliant III Ultra-Fast Sybr Green (Agilent Technologies, Journal of Experimental Biology • Accepted manuscript Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) and NADH:ubiquinone oxidoreductase subunit A1 (NDUFA1). Primers were designed based on NCBI (https://www.ncbi.nlm.nih.gov) database entries for available passerine species: White- throated sparrow (Zonotrichia albicollis) and zebra finch (Taeniopygia guttata) Two potential sequences were examined for 11β-HSD1 based on a search of NCBI databases and were denoted 11β-HSD1-762 (XM_005495762.2) and 11β-HSD1-865 (XM_005492865.1). Based on sequencing and homology data, candidate 11β-HSD1-865 (hereafter referred to as 11β- HSD1) was determined to correctly represent 11β-HSD1 and used for subsequent tissue analysis. Single sets of primers were designed for 11βHSD2 (XM_014269709.1), YWHAZ (NM_001031343.1) and NDUFA1 (NM_001302115.1). All primers were validated via standard PCR of Z. leucophrys cDNA and amplicons sequenced to confirm identity. Antagonists 2-Methyl-1,2-di-3-pyridyl-1-propanone - 96% (MET, M2696; Sigma-Aldrich, USA) and sodium diethyldithiocarbamate trihydrate (DETC, D3506; Sigma-Aldrich, USA) were freshly prepared in Ringer's Lactated saline (0.9%) to provide an injection volume of 100-200 µL at desired dosages: MET high 30 mg kg-1, MET low 15 mg kg-1, DETC high 400 mg kg-1, and DETC low 200 mg kg-1. DETC has been shown to be a selective inhibitor of 11β-HSD2 with an IC50 of 6.3 ± 3.8 µM and no detectable activity with respect to both reduction and oxidation reactions catalyzed by 11β-HSD1 (Schweizer et al., 2003). Journal of Experimental Biology • Accepted manuscript Determining 11β-HSD mRNA Expression See Table 1 for details of primers used. Experiment 2: Effects of central administration of DETC and MET on corticosterone stress response in captive white-crowned sparrows Birds were initially housed in two large flight aviaries (3×2.5×2m) on a 11L:13D photoperiod for acclimation. A total of 24 birds were utilized in the experiment as described below. A 3:1 mixture of Mazuri Small Bird Maintenance Diet (#56A6; Mazuri, Richmond, IN USA) and mixed wild bird seed along with water and grit were provided ad libitum. After 30 days birds were transferred to individual cages (35×25×40 cm) and housed in groups of 3- 4 in sound chambers on a fixed photoperiod of 10L:14D for the remaining duration of the experiment. Birds were cannulated as previously described (Bentley et al., 2006). Birds were food deprived two hours prior to surgery, anesthetized with 2-4% isoflurane with supplemental oxygen (1 L min-1) and placed into a stereotaxic apparatus specially designed for songbirds (MyNeuroLab.com). The intersection of the mid-sagittal and transverse sinuses was located on the skull and served as a reference point. The 11 mm 26 gauge guide cannula (C315G; Plastics One, Roanoke, VA USA) was moved 2.3 mm anterior from the reference point, lowered 6mm below the surface of the skull, bonded in place with dental cement (NC9655090; Stoelting Wood Dale, IL USA) and allowed adequate curing time before the animal was removed from the stereotaxic frame. A 33 gauge dummy cannula (C315DC; Plastics One, Roanoke, Virginia) was inserted into the guide cannula to prevent the development of obstruction. Patency of the cannula was determined by administration of human angiotensin II (A9525-1mg; Sigma-Aldrich, USA) which rapidly promotes thirst and drinking behavior within 2 min of infusion (Wada et al., 1975; Richardson and Boswell, 1993). Only birds that drank within 2 min following the administration of 1µg of Angiotensin II in 2 µL of sterile LRS were included in experimental treatments. Journal of Experimental Biology • Accepted manuscript Birds were divided into three groups (n = 8 per group/round), which initially received one of the following treatments: control (0.9% sterile saline), MET (20 µg), or DETC (200 µg) administered in a 2 µL bolus infusion over 2 min by infusion pump (PHD 2000; Harvard Apparatus, Holliston, MA USA). Central administration was carried out using a 10 µL Hamilton syringe attached to a clear piece of polyethylene tubing marked to show 1 µL volumes. The injection cannula (C315I ; Plastics One, Roanoke, VA USA) protruded 0.5 mm past the end of the guide cannula allowing central administration into the third ventricle. Experiment 1: Effects of peripheral injection of DETC and MET on the corticosterone stress response in free-living white-crowned sparrows Immediately following capture (within 3 min) a baseline blood sample (ca. 70 µL) was obtained as described above. Birds were weighed by Pesola spring scale to the nearest 0.1 g to determine appropriate drug dosage. Birds were randomly assigned to one of 5 treatment groups receiving either: high dose MET (30 mg kg-1; n = 11), low MET (15 mg kg- 1; n =12), high dose DETC (400 mg kg-1; n =13), low dose DETC (200 mg kg-1; n=9), or control (100 µL of Lactated Ringers solution; 13) via IP injection. Birds were held under a standardized capture restraint protocol for 60 min with additional blood samples collected at 10, 30 and 60 mins in an opaque cloth bag (Astheimer et al., 1992). Each bird was banded with a unique US Fish and Wildlife Service band prior to release. Experiment 2: Effects of central administration of DETC and MET on corticosterone stress response in captive white-crowned sparrows Birds were assigned to initial treatments such that each chamber received a mix of treatments. Birds were subsequently rotated through each treatment condition so all birds experienced each treatment over the 3 week experimental period. Upon opening of each sound chamber a baseline blood sample (approximately 50 µL) was collected from the alar vein within 3 min or less (as previously described), prior to administration of the appropriate each treatment over the 3 week experimental period. Upon opening of each sound chamber a baseline blood sample (approximately 50 µL) was collected from the alar vein within 3 min or less (as previously described), prior to administration of the appropriate each treatment over the 3 week experimental period. Upon opening of each sound chamber a baseline blood sample (approximately 50 µL) was collected from the alar vein within 3 min or less (as previously described), prior to administration of the appropriate intracerebroventricular (ICV) infusion. Additional 50 µL blood samples were collected at 10, 30 and 60 mins post disturbance. Two birds per chamber were infused and sampled each day, such that all birds were sampled in a two day period each week. Sampling order was reversed each week. intracerebroventricular (ICV) infusion. Additional 50 µL blood samples were collected at 10, 30 and 60 mins post disturbance. Two birds per chamber were infused and sampled each day, such that all birds were sampled in a two day period each week. Sampling order was reversed each week. Blood sample processing All blood samples were stored on ice until processing. Plasma was separated from red blood cells by centrifuging at 10,000 rpm for 5 min. The plasma was then aspirated via a Hamilton syringe and placed into microcentrifuge tubes and stored at -30° C until corticosterone quantification. Corticosterone Radioimmunoassay Journal of Experimental Biology • Accepted manuscript Corticosterone levels were measured by a radioimmunoassay as previously described by Wingfield et al. (1992). Briefly, 15 µL of plasma from baseline samples and 10 µL of the post capture time points were assayed. Recovery efficiency was estimated by adding 2000 CPM of tritiated corticosterone (Perkin Elmer NET399250UC, Waltham, MA USA) to each sample prior to extraction. Corticosterone was extracted from the samples by incubating with 4 mL of re-distilled dichloromethane with regular vortexing (D154-4; Fisher Chemical, Pittsburgh, PA USA). The aqueous phase was then extracted into a clean 10 mL test tube and the samples were dried in a water bath at 35°C under nitrogen gas, prior to being reconstituted using 550 µL of phosphate buffered saline gelatin (PBSG). The reconstituted samples were separated into 200 µL duplicate aliquots for quantification and 100 µL retained for determination of recovery efficacy. Recovery samples were combined with 2 mL of scintillation fluid (Ultima Gold: 6013329; Perkin Elmer, Waltham, MA USA) and counted to determine the percent recovery for each sample. 100 µL of tritiated corticosterone and 100 µL antiserum (07–120016, lot 3R3-PB-20E; MP Biomedical, Santa Ana, CA USA) were added to each duplicate assay tube and incubated overnight at 4°C. 500 µL of dextran-coated charcoal was added to each duplicate and after exactly 12 min, samples were centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was decanted into scintillation vials and combined with 4 mL of scintillation fluid (Perkin Elmer Ultima Gold: 6013329, Waltham, MA USA). Samples were placed on a Beckman 6500 liquid scintillation counter and each vial was counted for 5 min or within 2% accuracy. The corticosterone values were determined from a standard curve and adjusted using the corresponding recovery percentage. Mean recoveries were 82.7% and intra-assay (calculated using C.V. between duplicates) and inter-assay variations were 7.25% and 10.87%, respectively. The detection limit of the assays was 8.85 ± 0.49 pg per tube (~0.7 ng mL-1 per tube). Statistical Analyses All statistical analyses were performed in R (R Core Development Team, 2018) using packages: pracma (Borchers, 2017), ggplot2 (Wickham, 2009), lme4 (Bates et al., 2014), lmerTest (Kunznetsova et al., 2014), emmeans (Lenth, 2019) and tidyr (Wickham and Henry, 2018). Normalized gene expression data was analyzed by ANOVA and post-hoc testing performed using Tukey’s Honestly Significant Difference tests. Sex differences between tissues were not tested based on the absence of main or interaction effects of sex. For the peripheral injection study the data low and high doses of DETC and MET were compared via linear mixed effects model with a dose by handling time interaction individually to determine any dose effect. As no significant main effect (DETC: F1,66 = 1.77, p = 0.188; MET: F1,35 = 0.18, p = 0.674) of dose nor any interaction with restraint time (DETC: F1,64 = 0.001, p = 0.975; MET: F1,60 = 0.09, p = 0.764) was found, the dosages were combined for each drug for all subsequent analyses. Subsequently a fully parameterized linear mixed effects model of Treatment, Restraint Time, and Sex and all interactions was tested and no effect of sex nor interaction was detected. This model returned as rank deficient thus a second model with the main effect of Treatment, Restraint Time and Sex as well at the interaction of Treatment and restraint time was tested and again sex was found to be non-significant (F2,51= 1.83, p = 0.172) and thus sex was excluded from further analyses. A final base model of the interaction of Restraint Time and Treatment (MET, DETC, and saline) with both band number (unique identifier) and corticosterone assay number included as random intercepts to account for repeated sampling and intra-assay variation respectively, was used. The dosage of the drug had no effect for MET (F1,22 = 0.12, P = 0.85) nor for DETC (F1,18 = 2.58, P = 0.12) in field samples, thus in both cases dosages were combined to increase statistical power. Following detection of significant effect in the linear mixed effects model, post hoc tests were performed using estimated marginal means with Tukey’s Honestly Significant Difference and Journal of Experimental Biology • Accepted manuscript Journal of Experimental Biology • Accepted manuscript Kenward-Roger estimation of degrees of freedom in the emmeans package. Integrated corticosterone was determined by calculation of area under the curve using the function trapz in the pracma package in R. 11β -HSD Gene Expression 11β -HSD Gene Expression 11β-HSD1 and 11β-HSD2 were detected across tissues (Figs 1,2). 11β-HSD1 expression differed significantly between tissues (Fig. 1; F10, 169 = 54.94, p < 0.001) and tissue level expression patterns varied by sex (F10, 169 = 2.60, p = 0.006). Expression of 11βHSD1 was highest in liver for both sexes (Fig. 1), though sexes differed significantly from each other (F1,169 = 0.339, p < 0.001). 11β-HSD1 expression was detected in the hippocampus, hypothalamus and anterior pituitary gland, but was close to the lower limit of detection in all three tissues (Fig. 1). 11β-HSD2 expression also differed significantly between tissues (Fig. 2; F10,170 = 63.24, p < 0.001), but no effect of sex (F1,170 = 1.33, p = 0.251) nor sex by tissue interaction was detected (F10,170 = 1.41, p = 0.181). As with 11β-HSD1 expression, 11β- HSD2 was low but present in the hippocampus, hypothalamus and anterior pituitary (Fig. 2). Journal of Experimental Biology • Accepted manuscript Journal of Experimental Biology • Accepted manuscript Statistical Analyses Integrated corticosterone was analyzed by linear mixed effects model with fixed effect of Drug and random effects matched to experimental design (see Table 2 for final models). All data are reported as the mean ± standard error of the mean (s.e.m). Experiment 1: Effects of peripheral antagonist injections on corticosterone concentrations during the stress response in free living white-crowned sparrows The final model consisted of restraint time, treatment and their interaction with the random effect of bird. Corticosterone was found to increase independent of treatment with duration of restraint stress (Fig. 3A; F1,200 = 103.98, p < 0.001). Treatment also significantly altered plasma corticosterone levels (F2,200 = 7.22, p < 0.001). DETC treatment resulted in significant elevation of corticosterone at 10 min compared to MET (DETC-MET: t171 = 5.47, p < 0.001), at 30 min post capture compared to both Saline and MET groups (DETC-Saline: t169 = 4.36, p < 0.001; DETC-MET: t174 = 9.23, p < 0.001), and at 60 minutes post capture compared to MET (t174 = 3.04, p = 0.007). Peripheral injection of MET resulted in decreased corticosterone compared to Saline at 30 minutes post capture (t171 = -3.36, p = 0.003). Total integrated corticosterone secreted over the hour restraint period, as calculated by area under the curve, was affected by treatment (Fig. 3B; F2,45 = 25.9, p < 0.001). DETC significantly increased corticosterone compared to Saline (t45 = 3.15, p = 0.008) and MET (t45 = 7.17, p< 0.001). MET treatment reduced total corticosterone secreted compared to Saline controls (t45 = -2.88, p = 0.017). Experiment 2: Effects of central ICV administration of antagonists on corticosterone stress response in captive white-crowned sparrows Corticosterone concentrations increased over the 60 min restraint period across treatments (Fig. 3C; F1, 192 = 45.8, p <0.001). The increase of corticosterone concentrations over time was found to be dependent upon drug type infused (F2, 192 = 4.14, p = 0.02). There were no differences between treatments detected at the 0 and 10 min time points. DETC treated birds had significantly higher corticosterone at 30 (Fig.3B; DETC-Saline: t193 = 4.81, p < 0.001; DETC-MET: t188 = 3.38, p =0.04) and 60 min post restraint (DETC-Saline: t193 = 3.37, p = 0.02; DETC-MET: t188 = 3.53, p =0.026). Corticosterone concentration in response to MET infusion did not differ from Saline controls at any time point (p > 0.05). Total corticosterone secreted showed a trend towards being affected by drug infused (Fig. 3D; F2,31 = 2.68, p = 0.084). However, post-hoc testing detected no difference between treatment groups in total corticosterone secreted, though DETC infusion (t36 =1.79, p = 0.19) trends towards increasing corticosterone as compared to saline when inspected graphically. Journal of Experimental Biology • Accepted manuscript Journal of Experimental Biology • Accepted manuscript 11β-HSD gene expression Detection of 11β-HSD expression across tissues supports a functional role in regulation of tissue specific and circulating corticosterone levels in birds. Consistent with previous reports in birds we found 11β-HSD1 expression to be highest in the liver (Rensel et al., 2018) and 11β-HSD2 to be highest in the kidney and detectable levels in the gonads, liver and brain (Klusoňová et al., 2008; Katz et al., 2010; Rensel et al., 2018). This expression across body tissues supports the established major role of 11β-HSD1 in the hepatic generation of active glucocorticoids from circulating precursors (Rensel et al., 2018) and 11β- HSD2 protection of renal aldosterone signaling via protection of MR from corticosterone binding. For the first time we report expression of both 11β-HSD1 and 11β-HSD2 in the adrenal glands of a bird. While, the presence of 11β-HSD1 in the adrenal gland is expected, given its key role in corticosterone biosynthesis, the presence of 11β-HSD2 is surprising as it inactivates corticosterone. We found 11β-HSD2 levels here to be 2-3 times higher than in the kidney (the second-highest site of expression). Protection of the adrenal glands from toxic levels of corticosterone and modulation of local autocrine or paracrine feedback loops by 11β-HSD2, may explain these findings. This is supported by data from rats and humans showing that adrenal 11β-HSD2 expression is concentrated primarily in the zona fasciculata, where glucocorticoid synthesis occurs, with lower expression into the zona reticularis and medulla (Roland and Funder, 1996; Mazzocchi et al., 1998) and none in the capsule and zona glomerulosa (site of mineralocorticoid synthesis). Mazzocchi and colleagues (1998) also found 11β-HSD2 activity in human adrenal preparations to be indirectly responsive to exogenous ACTH. This modulation of adrenal 11β-HSD2 and its distribution suggests the possibility of dynamic modulation of glucocorticoid production within the adrenal gland itself over the course of the stress response. Such modulation of glucocorticoid secretion by adrenal 11β-HSD2 remains to be tested in avian systems. Hypothalamic expression of both 11β-HSD enzymes were low compared to expression in peripheral tissues. These results are consistent with previous studies in zebra finches (Katz et al., 2010; Rensel et al., 2018). Zebra finch 11β-HSD2 expression was found to be widespread across the brain, and has been suggested to be driven by the widespread neural expression of MR in this species as compared to mammals and other birds (Katz et al., 2010). 11β-HSD gene expression The present study lacks the necessary data to test this hypothesis and whether it is a unique feature of zebra finches. Journal of Experimental Biology • Accepted manuscript Peripheral and central MET administration Our findings suggest that MET's efficacy may be context and species dependent as inly peripheral administration had minimal detectable effect. Previous studies have demonstrated the efficacy of MET in inhibiting corticosterone synthesis in rodents, with a dose of 40 mg/kg generating robust suppression in rats (Herman et al., 1992). Previous studies in birds, utilizing implants to deliver MET in a time released manner, had no prolonged effect on corticosterone levels in house sparrows (Gray et al., 1990; Aharon- Rotman et al., 2017). Though this may be taken to suggest that MET simply lacks efficacy in avian species, the lowest levels of circulating corticosterone was found to occur two days after implant placement with levels rising over the course of the study (Aharon-Rotman et al., 2017). This suggests that MET implants are able to alter basal corticosterone levels, but that these alterations are unable to overcome homeostatic control in the long run. These results combined with the findings of the present study support a limited efficacy of MET in disrupting generation of corticosterone in birds. The single short term bolus injection approach used in this study may have enabled us to better detect METs effects. However, our data also suggest that MET has a very limited ability to alter corticosterone levels in birds. Further interpretation of MET’s specific actions is limited. Further studies are necessary to disentangle the role of 11β hydroxylase action from that of 11β-HSD1. Inhibition of 11β-HSD2 by peripheral and central DETC administration Peripheral administration of DETC effectively blocked 11β-HSD2 action, as evidenced by the increase in circulating corticosterone (Fig.3A&B) as predicted by previous cell based assays utilizing DETC (Atanasov et al., 2003; Schweizer et al., 2003). This suggests that peripheral 11β-HSD2, in addition to providing localized protection at the level of the target tissue, also serves to modulate circulating levels of corticosterone. Of particular interest in this respect is the high level of 11β-HSD2 expressed in the adrenal glands, previously unreported in songbirds. Contrary to our a priori predictions, central DETC administration also resulted in elevated circulating corticosterone levels over the restraint period (Fig.3C). Blocking of hypothalamic 11β-HSD2 was expected to lead to a local elevation of corticosterone, thereby increasing negative feedback and ultimately lowering the total amount of corticosterone secreted or at least increasing the speed at which corticosterone returned to baseline. Instead we see a trend towards increased total corticosterone secreted in response to DETC infusion into the 3V (Fig. 3D). Examination of the present data in light of the broader literature, including the ability of DETC to cross the blood brain barrier (Frank et al., 1995), supports two broad hypotheses. First, the escape of centrally injected DETC into the periphery to act upon the major sites of corticosterone inactivation, the kidney and liver. While physically possible, the small volume of DETC administered via ICV (representing a comparatively tiny absolute DETC dosage in comparison to the peripheral injections), makes escape of centrally administered DETC to the periphery a highly unlikely explanation. Alternatively, DETC within the brain may inhibit as Journal of Experimental Biology • Accepted manuscript Journal of Experimental Biology • Accepted manuscript yet uncharacterized sites of neural stress axis regulation, that in the absence of normal 11β- HSD2 protective action trigger positive feedback supporting the further release of corticosterone. Future studies utilizing labelled corticosterone to determine tissue level processing in response to antagonist treatment will be necessary to elucidate the role 11β- HSD2 in regulation of the stress axis. Furthermore, conditional knockout models may provide a robust alternative, but are presently not widely available in avian systems. Role of 11β-HSD2 enzymes in regulation of the stress response Our findings support a critical role for 11β-HSD2 in the regulation of circulating levels of corticosterone. The high peripheral gene expression and clear reduction in systemic clearance observed in response to peripheral blocking of 11β-HSD2 suggest that peripheral 11β-HSD2 action contributes more to regulation of the hormonal stress response than central 11β-HSD2 activity, if this indeed exists. It is critical to recall that the presence of 11β-HSD2 in multiple peripheral tissues may complicate interpretation of plasma levels in response to antagonist treatment due to potentially opposing effects of 11β-HSD2 blockage between tissues. The complexity of 11β-HSD action has been previously highlighted by studies in 11β-HSD1 knockout mice, which display compensatory adrenal hyperplasia and increased basal levels of corticosterone, despite the presumed absence of hepatic corticosterone reactivation from cortisone (Kotelevtsev et al., 1997). Additionally, in this study it was found that the adrenal glands continued to effectively secrete corticosterone in 11β-HSD1 knockout animals, which also displayed increased adrenal sensitivity to ACTH stimulation. While the results of our present study support clear involvement of peripheral tissue 11β-HSD2 in regulation of circulating corticosterone levels, the study design prevents the disentanglement of the contribution of 11β-HSD2 from specific tissues. Development of novel targeted approaches to separate these tissue specific effects will be critical to advancing our understanding of peripheral corticosterone metabolism. Journal of Experimental Biology • Accepted manuscript 11β-HSD1 and 11β-HSD2 expression were low in the brain and this is consistent with previous studies in chickens and zebra finches. However, the contribution of 11β-HSD activity in modulating neural negative feedback to the hypothalamus remains unclear. In rats, 11β-HSD2 has been co-localized with GR and MR in the brain, strongly suggests important local regulatory action by these enzymes (Whorwood et al., 1992). Similar data is lacking in free living species; such co-localization data is needed to clarify the potential of 11β-HSD enzymes to modulate corticosterone activity. Existing data from avian studies of neural distribution of MR and GR receptors may provide limited insight, but must be interpreted with caution. Both MR and GR appear to be generally widely distributed within the avian brain (Senft et al., 2016; Rensel et al., 2018). In white-crowned sparrows, GR was strongly expressed in hypothalamus but concentrated primarily in the PVN and pre-optic areas (POA) (Krause et al., 2015). Funding This work was supported by the National Science Foundation Division of Integrative Organismal Systems [IOS 1558049 to JCW] and Roslin Institute strategic grant funding from the Biotechnology and Biological Sciences Research Council [BB/P013759/1 to SLM]. J.C. Wingfield would like to acknowledge the University of California, Davis Endowed Chair in Physiology. The Danish Council for Independent Research supported the MATCH project [1323-00048B to KS] and Danish National Research Foundation supported Center for Macroecology, Evolution and Climate [DNRF96 to KS]. Acknowledgements We would like to thank Thomas Blackmon, Katrina Macalello, and Rebecca Stanley for their assistance with animal care and sampling. List of Abbreviations 11β-HSD: 11 beta-hydroxysteroid dehydrogenase; HPA axis: Hypothalamic-Pituitary- Adrenal axis; MR: mineralocorticoid receptor; GR: glucocorticoid; DETC: sodium diethyldithiocarbamate trihydrate; MET: 2-Methyl-1,2-di-3-pyridyl-1-propanone; ICV: intracerebroventricular; 3V: third ventricle; PVN: paraventricular nucleus; ACTH: adrenocorticotropin; CRF: corticotrophin releasing factor. adrenocorticotropin; CRF: corticotrophin releasing factor. Competing Interests The authors have no competing interests to declare. Author Contributions Conceptualization: JHP, JSK, RES, HJL JCW SLM. Sample collection: JHP, JSK, KS, RES, HJL. Avian surgery: JHP and JSK. Molecular biology: JSK, JHP, AR, VRB, JC. Data Analysis: JHP and RES. Original draft and primary writer: JHP and RES. Review & Editing: JHP, JSK, KS, SLM, AR, JCW. Project management: JHP, JSK, JCW, SLM. Funding Acquisition: JCW and SLM. Journal of Experimental Biology • Accepted manuscript Role of 11β-HSD2 enzymes in regulation of the stress response However, caution must be taken in extrapolating as these data are drawn from observation of breeding as opposed to wintering (present study) white-crowned sparrows and brain-wide localization of 11β-HSD1 and 11β-HSD2 remains lacking in this species. Regional co-localization by RT-PCR of both MR and GR expression with 11β-HSD2 expression has been found in zebra finches (Rensel et al., 2018). While this supports an active role for 11β-HSD2 in modulating local corticosterone concentrations within the avian brain, the lack of neuroanatomical co-localization precludes determination of a functional relationship between the two proteins. The present work adds to a growing body of literature that supports a significant role for 11β-HSD2 regulatory enzyme action in mediating localized tissue exposure and basal glucocorticoid levels, in addition to modulation in response to stressors. While complete elucidation of role of 11β-HSD2 enzymatic actions in HPA axis modulation remains unexplained, it is clear that 11β-HSD2 is vital in the regulation of tissue specific exposure to glucocorticoids. Future work addressing tissue and brain region specific functional contributions of 11β-HSD enzymes are required to fully explain the roles played in HPA axis regulation. Journal of Experimental Biology • Accepted manuscript Data Availability All data is available directly upon request from the authors. References Distinct ontogeny of glucocorticoid and mineralocorticoid receptor and 11β-hydroxysteroid dehydrogenase types I and II mRNAs in the fetal rat brain suggest a complex control of glucocorticoid actions. 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Interactions of Corticosterone with Feeding, Activity and Metabolism in Passerine Birds. Ornis Scandinavica 23, 355-365. Atanasov, A. G., Tam, S., Röcken, J. M., Baker, M. E. and Odermatt, A. (2003). Inhibition of 11β-hydroxysteroid dehydrogenase type 2 by dithiocarbamates. Biochemical and Biophysical Research Communications 308, 257-262. Biophysical Research Communications 308, 257-262. Basu, R., Singh, R. J., Basu, A., Chittilapilly, E. G., Johnson, C. M., Toffolo, G., Cobelli, C. and Rizza, R. A. (2004). Splanchnic Cortisol Production Occurs in Humans. Evidence for Conversion of Cortisone to Cortisol Via the 11-β Hydroxysteroid Dehydrogenase (11β-HSD) Type 1 Pathway 53, 2051-2059. Bates, D., Maechler, M., Bolker, B. and Walker, S. (2014). lme4: Linear mixed-effects models using Eigen and S4. R package version 1.1-7. Bentley, G. E., Jensen, J. P., Kaur, G. J., Wacker, D. W., Tsutsui, K. and Wingfield, J. C. (2006). 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Social information changes stress hormone receptor expression in the songbird brain. Hormones and Behavior 97, 31-38. de Kloet E R (2014) From receptor balance to rational glucocorticoid therapy de Kloet, E. R. (2014). From receptor balance to rational glucocorticoid therapy. E d i l 155 2754 2769 de Kloet, E. R. (2014). From receptor balance to rational glucocorticoid therapy. Endocrinology 155, 2754-2769. Diaz, R., Brown, R. W. and Seckl, J. R. (1998). References R. et al. (1997). 11β-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress. Proceedings of the National Academy of Sciences 94, 14924. Krause, J. S., Pérez, J. H., Meddle, S. L. and Wingfield, J. C. (2017). Effects of short-term fasting on stress physiology, body condition, and locomotor activity in wintering male white- crowned sparrows. Physiology & Behavior 177, 282-290. Krause, J. S., McGuigan, M. A., Bishop, V. R., Wingfield, J. C. and Meddle, S. L. (2015). Decreases in mineralocorticoid but not glucocorticoid receptor mRNA expression during the short Arctic breeding season in free-Living Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii). Journal of Neuroendocrinology 27, 66-75. Kunznetsova, A., Brockhoff, P. B. and Christensen, R. H. B. (2014). lmerTest: Tests for random and fixed effects for linear mixed effects models (lmer objects of lme4 package). R package version 2.0-11. Lenth, R. (2019). emmeans: Estimated Marginal Means, aka Least-Squares Means. . In R Package version 1.41. Mazzocchi, G., Rossi, G. P., Neri, G., Malendowicz, L. K., Albertin, G. and Nussdorfer, G. G. (1998). 11β-Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex. The FASEB Journal 12, 1533-1539. R Core Development Team. (2018). R: a language and environment for statistical computing. In R Foundation for Statistical Computing. Vienna, Austria. Reid, A. M. A. and Dunn, I. C. (2018). Gastrointestinal distribution of chicken gastrin- cholecystokinin family transcript expression and response to short-term nutritive state. Gen. Comp. Endocrinol. 255, 64-70. Rensel, M. A., Ding, J. A., Pradhan, D. S. and Schlinger, B. A. (2018). 11β-HSD Types 1 and 2 in the Songbird Brain. Frontiers in Endocrinology 9. Journal of Experimental Biology • Accepted manuscript Reul, J., Van den Bosch, F. and De Kloet, E. (1987). Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. Journal of Endocrinology 115, 459-467. Richardson, R. D. and Boswell, T. (1993). A method for third ventricular cannulation of small passerine birds. Physiology & Behavior 53, 209-213. Roland, B. L. and Funder, J. W. (1996). Localization of 11beta-hydroxysteroid dehydrogenase type 2 in rat tissues: in situ studies. Endocrinology 137, 1123-1128. Romero, M. L. (2002). Seasonal changes in plasma glucocorticoid concentrations in free- living vertebrates. General and Comparative Endocrinology 128, 1-24. Sapolsky, R. M., Romero, L. M. and Munck, A. U. (2000). How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. References Endocr Rev 21. Schweizer, R. A., Atanasov, A. G., Frey, B. M. and Odermatt, A. (2003). A rapid screening assay for inhibitors of 11β-hydroxysteroid dehydrogenases (11β-HSD): flavanone selectively inhibits 11β-HSD1 reductase activity. Molecular and cellular endocrinology 212, 41-49. Senft, R. A., Meddle, S. L. and Baugh, A. T. (2016). Distribution and Abundance of Glucocorticoid and Mineralocorticoid Receptors throughout the Brain of the Great Tit (Parus major). PLOS ONE 11, e0148516. Wada, M., Kobayashi, H. and Farner, D. S. (1975). Induction of drinking in the White- crowned Sparrow, Zonotrichia leucophrys gambelii, by intracranial injection of angiotensin II. General and Comparative Endocrinology 26, 192-197. Walker, J. J., Spiga, F., Gupta, R., Zhao, Z., Lightman, S. L. and Terry, J. R. (2015). Rapid intra-adrenal feedback regulation of glucocorticoid synthesis. Journal of The Royal Society Interface 12. y Whorwood, C. B., Franklyn, J. A., Sheppard, M. C. and Stewart, P. M. (1992). Tissue localization of 11β-hydroxysteroid dehydrogenase and its relationship to the glucocorticoid receptor. The Journal of Steroid Biochemistry and Molecular Biology 41, 21-28. Wickham, H. (2009). ggplot2: Elegant Graphics for Data Analysis.: Springer-Verlag New York. Wickham, H. and Henry, L. (2018). tidyr: Easily Tidy Data with 'spread()' and 'gather()' Functions. R package version 0.8.2. https://CRAN.R-project.org/package=tidyr. Wyrwoll, C. S., Holmes, M. C. and Seckl, J. R. (2011). 11β-hydroxysteroid dehydrogenases and the brain: from zero to hero, a decade of progress. Frontiers in neuroendocrinology 32, 265-286. Whorwood, C. B., Franklyn, J. A., Sheppard, M. C. and Stewart, P. M. (1992). Tissue localization of 11β-hydroxysteroid dehydrogenase and its relationship to the glucocorticoid receptor. The Journal of Steroid Biochemistry and Molecular Biology 41, 21-28. Wickham, H. (2009). ggplot2: Elegant Graphics for Data Analysis.: Springer-Verlag New York. Wickham, H. and Henry, L. (2018). tidyr: Easily Tidy Data with 'spread()' and 'gather()' Functions. R package version 0.8.2. https://CRAN.R-project.org/package=tidyr. Wyrwoll, C. S., Holmes, M. C. and Seckl, J. R. (2011). 11β-hydroxysteroid dehydrogenases and the brain: from zero to hero, a decade of progress. Frontiers in neuroendocrinology 32, 265-286. Journal of Experimental Biology • Accepted manuscript Table 1. qPCR Primers utilized in this study. Target Accession No. Forward Primer Reverse Primer 11β-HSD1 XM_005492865.1 5’GCTCATCCTCAACCACATCG 5’CCATCTAGGGCGAACTTGGT 11β-HSD2 XM_014269709.1 5’ATATCCAGGCCCACACCAAC 5’CACGTTGTCCCTGTTTTGTAGT YHWAZ NM_001031343.1 5'GTGGAGCAATCACAACAGGC 5'GCGTGCGTCTTTGTATGACTC NDUFA1 NM_001302115.1 5'ATGTGGTACGAGATCCTGCC 5'TTCTCCAGACCCTTGGACAC Tables Table 1. qPCR Primers utilized in this study. Target Accession No. Forward Primer Reverse Primer 11β-HSD1 XM_005492865.1 5’GCTCATCCTCAACCACATCG 5’CCATCTAGGGCGAACTTGGT 11β-HSD2 XM_014269709.1 5’ATATCCAGGCCCACACCAAC 5’CACGTTGTCCCTGTTTTGTAGT YHWAZ NM_001031343.1 5'GTGGAGCAATCACAACAGGC 5'GCGTGCGTCTTTGTATGACTC NDUFA1 NM_001302115.1 5'ATGTGGTACGAGATCCTGCC 5'TTCTCCAGACCCTTGGACAC Reverse Primer Journal of Experimental Biology • Accepted manuscript Table 2. Summary of final mixed effects models used in statistical analyses. Model Fixed Effects Random Effects Peripheral Time Series Time X Drug Bird ID, Assay Peripheral Area Under the Curve Drug Assay Central Tme Series Time X Drug Bird ID, Assay Central Area Under the Curve Drug Bird ID, Assay Table 2. Summary of final mixed effects models used in statistical analyses. Journal of Experimental Biology • Accepted manuscript Figures Effects of a single bolus peripheral injections of MET (combined 15 & 30 mg/kg; n = 21) and DETC (combined 200 & 400 mg/kg; n = 21) versus controls (100 µL of Lactated Ringers solution; n=12) on plasma corticosterone concentrations over a A) one hour handling restraint sampling period and B) integrated hormonal response using integrated area under the curve (AUC). Dose had no effect on corticosterone so samples were pooled. Effects of central infusion of Lactated Ringers solution (n = 30), MET (90 nmol; n = 11) and DETC (900 nmol; n = 13) into the third ventricle on corticosterone concentrations over a C) one hour sampling period and D) Integrated area under the curve over the same period. An initial blood sample was taken and then birds were immediately injected with the drug. Data analyses by linear mixed effects model, with Tukey’s HSD post-hoc testing. Letters indicate significant differences (P<0.05) between treatments at given time point. Values represent means ± SEM. Fig. 3. The effects pharmacological specific inhibition of CORT synthesis using MET and inhibition of 11β-HSD2 clearance of CORT using DETC on plasma concentrations of corticosterone. Effects of a single bolus peripheral injections of MET (combined 15 & 30 mg/kg; n = 21) and DETC (combined 200 & 400 mg/kg; n = 21) versus controls (100 µL of Journal of Experimental Biology • Accepted manuscript ig. 3. The effects pharmacological specific inhibition of CORT synthesis using MET and Fig. 3. The effects pharmacological specific inhibition of CORT synthesis using MET and inhibition of 11β-HSD2 clearance of CORT using DETC on plasma concentrations of corticosterone. Effects of a single bolus peripheral injections of MET (combined 15 & 30 Fig. 3. The effects pharmacological specific inhibition of CORT synthesis using MET and inhibition of 11β-HSD2 clearance of CORT using DETC on plasma concentrations of corticosterone. Effects of a single bolus peripheral injections of MET (combined 15 & 30 mg/kg; n = 21) and DETC (combined 200 & 400 mg/kg; n = 21) versus controls (100 µL of Lactated Ringers solution; n=12) on plasma corticosterone concentrations over a A) one hour handling restraint sampling period and B) integrated hormonal response using integrated area under the curve (AUC). Dose had no effect on corticosterone so samples were pooled. Effects f l i f i f d i l i l d Fig. 3. Figures Figures Fig. 1. Relative expression of 11βHSD1 mRNA as measured by qPCR for female (black) and male (grey) non-breeding Gambel’s white-crowned sparrows across multiple tissues. Letters that are different from one another indicate significant differences (p < 0.05) between tissues as determined by post-hoc testing (Tukey’s HSD). N = 9 per sex per tissue, except for male gastrocnemius muscle n= 8, gonad n= 6, and anterior pituitary n= 6. Expression was standardized against the geometric mean of YWHAZ and NDUFA reference gene expression. Values are expressed as means + SEM. Journal of Experimental Biology • Accepted manuscript Fig. 1. Relative expression of 11βHSD1 mRNA as measured by qPCR for female (black) and male (grey) non-breeding Gambel’s white-crowned sparrows across multiple tissues. Letters that are different from one another indicate significant differences (p < 0.05) between tissues as determined by post-hoc testing (Tukey’s HSD). N = 9 per sex per tissue, except for male gastrocnemius muscle n= 8, gonad n= 6, and anterior pituitary n= 6. Expression was standardized against the geometric mean of YWHAZ and NDUFA reference gene expression. Values are expressed as means + SEM. Fig. 2. Relative expression of putative 11βHSD2 mRNA as measured by qPCR for female (black) and male (grey) non-breeding Gambel’s white-crowned sparrows across multiple tissues. Letters that are different from one another indicate significant differences (p < 0.05) differences between tissues as determined by post-hoc testing. N = 9 per sex per tissue, except for male gastrocnemius muscle n= 8, gonad n= 7, and anterior pituitary n= 6. Expression was standardized against YWHAZ and NDUFA reference gene expression. Values are expressed as means + SEM. Journal of Experimental Biology • Accepted manuscript Fig. 2. Relative expression of putative 11βHSD2 mRNA as measured by qPCR for female (black) and male (grey) non-breeding Gambel’s white-crowned sparrows across multiple tissues. Letters that are different from one another indicate significant differences (p < 0.05) differences between tissues as determined by post-hoc testing. N = 9 per sex per tissue, except for male gastrocnemius muscle n= 8, gonad n= 7, and anterior pituitary n= 6. Expression was standardized against YWHAZ and NDUFA reference gene expression. Values are expressed as means + SEM. Fig. 3. The effects pharmacological specific inhibition of CORT synthesis using MET and inhibition of 11β-HSD2 clearance of CORT using DETC on plasma concentrations of corticosterone. Figures The effects pharmacological specific inhibition of CORT synthesis using MET and inhibition of 11β-HSD2 clearance of CORT using DETC on plasma concentrations of corticosterone. Effects of a single bolus peripheral injections of MET (combined 15 & 30 mg/kg; n = 21) and DETC (combined 200 & 400 mg/kg; n = 21) versus controls (100 µL of Lactated Ringers solution; n=12) on plasma corticosterone concentrations over a A) one hour handling restraint sampling period and B) integrated hormonal response using integrated area under the curve (AUC). Dose had no effect on corticosterone so samples were pooled. Effects of central infusion of Lactated Ringers solution (n = 30), MET (90 nmol; n = 11) and DETC (900 nmol; n = 13) into the third ventricle on corticosterone concentrations over a C) one hour sampling period and D) Integrated area under the curve over the same period. An initial blood sample was taken and then birds were immediately injected with the drug. Data analyses by linear mixed effects model, with Tukey’s HSD post-hoc testing. Letters indicate significant differences (P<0.05) between treatments at given time point. Values represent means ± SEM. Fig. 3. The effects pharmacological specific inhibition of CORT synthesis using MET and inhibition of 11β-HSD2 clearance of CORT using DETC on plasma concentrations of corticosterone. Effects of a single bolus peripheral injections of MET (combined 15 & 30 mg/kg; n = 21) and DETC (combined 200 & 400 mg/kg; n = 21) versus controls (100 µL of Lactated Ringers solution; n=12) on plasma corticosterone concentrations over a A) one hour handling restraint sampling period and B) integrated hormonal response using integrated area under the curve (AUC). Dose had no effect on corticosterone so samples were pooled. Effects of central infusion of Lactated Ringers solution (n = 30), MET (90 nmol; n = 11) and DETC (900 nmol; n = 13) into the third ventricle on corticosterone concentrations over a C) one hour sampling period and D) Integrated area under the curve over the same period. An initial blood sample was taken and then birds were immediately injected with the drug. Data analyses by linear mixed effects model, with Tukey’s HSD post-hoc testing. Letters indicate significant differences (P<0.05) between treatments at given time point. Values represent means ± SEM. bition of 11β-HSD2 clearance of CORT using DETC on plasma concentrations of costerone. Figures Effects of a single bolus peripheral injections of MET (combined 15 & 30 kg; n = 21) and DETC (combined 200 & 400 mg/kg; n = 21) versus controls (100 µL of Journal of Experimental Biology • Accepted manuscript

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The effect of listening to music on human transcriptome

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The effect of listening to music on human transcriptome Chakravarthi Kanduri1 , Pirre Raijas2 , Minna Ahvenainen1 , Anju K. Philips1 , Liisa Ukkola-Vuoti1 , Harri Lähdesmäki3 and Irma Järvelä1 1 Department of Medical Genetics, University of Helsinki, Finland 2 DocMus Department, University of the Arts Helsinki, Helsinki, Finland 3 Department of Information and Computer Science, Aalto University, AALTO, Finland ABSTRACT Submitted 9 January 2015 Accepted 18 February 2015 Published 12 March 2015 Corresponding author Irma Järvelä, [email protected] Academic editor Keith Crandall Additional Information and Declarations can be found on page 12 Although brain imaging studies have demonstrated that listening to music alters human brain structure and function, the molecular mechanisms mediating those effects remain unknown. With the advent of genomics and bioinformatics approaches, these effects of music can now be studied in a more detailed fashion. To verify whether listening to classical music has any effect on human transcriptome, we performed genome-wide transcriptional profiling from the peripheral blood of participants after listening to classical music (n = 48), and after a control study without music exposure (n = 15). As musical experience is known to influence the responses to music, we compared the transcriptional responses of musically experienced and inexperienced participants separately with those of the controls. Comparisons were made based on two subphenotypes of musical experience: musical aptitude and music education. In musically experiencd participants, we observed the differential expression of 45 genes (27 up- and 18 down-regulated) and 97 genes (75 up- and 22 down-regulated) respectively based on subphenotype comparisons (rank product non-parametric statistics, pfp 0.05, >1.2-fold change over time across conditions). Gene ontological overrepresentation analysis (hypergeometric test, FDR < 0.05) revealed that the up-regulated genes are primarily known to be involved in the secretion and transport of dopamine, neuron projection, protein sumoylation, long-term potentiation and dephosphorylation. Down-regulated genes are known to be involved in ATP synthase-coupled proton transport, cytolysis, and positive regulation of caspase, peptidase and endopeptidase activities. One of the most up-regulated genes, alpha-synuclein (SNCA), is located in the best linkage region of musical aptitude on chromosome 4q22.1 and is regulated by GATA2, which is known to be associated with musical aptitude. Several genes reported to regulate song perception and production in songbirds displayed altered activities, suggesting a possible evolutionary conservation of sound perception between species. We observed no significant findings in musically inexperienced participants. DOI 10.7717/peerj.830 Copyright 2015 Kanduri et al. Distributed under Creative Commons CC-BY 4.0 Subjects Genetics, Genomics Keywords Music, RNA, Gene expression profiling, Dopamine, Long-term potentiation, Genomics, Peripheral blood, SNCA OPEN ACCESS How to cite this article Kanduri et al. (2015), The effect of listening to music on human transcriptome. PeerJ 3:e830; DOI 10.7717/peerj.830 INTRODUCTION Listening to music is common in all societies. A plethora of neurophysiological studies have demonstrated that listening to and/or playing music has multiple measurable effects on human brain structure and function (Elbert et al., 1995; Blood & Zatorre, 2001; Sutoo & Akiyama, 2004; Koelsch, 2011; Salimpoor et al., 2011; Salimpoor et al., 2013; Herholz & Zatorre, 2012; Chanda & Levitin, 2013). In investigations using positron emission tomography (PET), music listening has been reported to cause physiological changes in cerebral blood flow, cardiovascular and muscle function, and enhanced dopamine secretion in the human brain (Sutoo & Akiyama, 2004; Angelucci et al., 2007; Salimpoor et al., 2011). Music has been demonstrated to regulate emotions and evoke pleasure, primarily through its action on the brain’s reward centres like the limbic and mesolimbic structures including the nucleus accumbens, hypothalamus, subcallosal cingulate gyrus, pre-frontal anterior cingulate, and hippocampus (Brown, Martinez & Parsons, 2004; Koelsch, 2011; Koelsch, 2014; Salimpoor et al., 2011; Salimpoor et al., 2013). Music has also been used as a therapeutic tool in clinical settings (AMTA, 2014; Conrad, 2010; Holmes, 2012). However, the molecular mechanisms and biological pathways mediating the effects of music remain unknown. Genomics and bioinformatics offer methods (Lander, 2011) to explore the biology and evolution of music and sounds at the molecular level. To date, few genome-wide scans have been performed in musical traits in humans (Asher et al., 2008; Pulli et al., 2008; Park et al., 2012; Oikkonen et al., 2014). Genome-wide expression analysis can be applied to study human traits in an unbiased, hypothesis-free fashion based on their molecular properties, rather than anatomic regions. Here, we have utilized a combination of genomic and bioinformatic methods to analyze the effect of classical music on the peripheral whole blood transcriptome. Peripheral blood was used, as brain samples are inaccessible in humans. MATERIALS AND METHODS Ethics statement The Ethics Committee of Helsinki University Central Hospital approved this study. Written informed consent was obtained from all the participants. Participants and phenotypes A total of 48 individuals (aged 18–73; mean 42.5) participated in the study (Table S1). All the 48 participants were characterized for musical aptitude and music education. Musical aptitude was measured using three tests: the auditory structuring ability test (Karma music test) (Karma, 2007), and the Seashore tests for pitch and for time (Seashore, Lewis & Saetveit, 1960). Details of the tests have been described in Oikkonen et al. (2014). A combined score of the three tests (COMB score; range 0–150) was used to define musical aptitude. COMB scores were classified as either high or low based on the upper and lower quartiles of score distribution. Data on music education were collected using a questionnaire. The self-reported levels of music education (referred to as edu classes 1–4), received through studies or degrees from music schools, institutes, or universities, were Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 2/17 Table 1 Phenotype characteristics of the sample set (total n = 48). Phenotype n* Edu classes 1–2 Edu classes 3–4 Low COMB scores High COMB scores Male Female 19 29 12 12 22 26 Notes. * n represents the number of participants within each category. classified as follows: 1 represents no music education; 2 represents music education of less than two years; 3 represents music education of more than ten years; and 4 represents a professional musician. The approximate time of systematic music education and training was 21.42 years on average, in participants of edu classes 3–4. Table 1 shows the sample’s phenotype characteristics. Exposure to music To our knowledge, no previous studies have systematically studied the effect of listening to music on genome-wide transcriptional profiles of humans. We have previously shown that music-listening habits vary a lot among listeners (Ukkola-Vuoti et al., 2011). To start with, we chose to study the effect of classical music, Wolfgang Amadeus Mozart’s Violin Concerto No. 3 in G major, K.216 because it is relatively familiar in the western culture. As the human brain perceives complex sounds in a millisecond-level time frame (Wang et al., 2009; Kayser, Logothetis & Panzeri, 2010), we expected that the 20 min-listening session (duration that the concerto lasts) will induce an effect of music on human transcriptome. In studies on the effect of pain in humans, very short durations of pain induction (8 to 150 s) have been used (Hubbard et al., 2011). The participants were unaware of the type of music that was intended for the listening session. Peripheral blood samples were collected from all the 48 participants just before and after the listening session. From here on, participants who listened to music are referred to as listeners throughout the text. Control study The same 48 participants were invited to a control study. Of these, 15 participants could attend. The participants were advised to avoid listening to music and hard exercise the day before the control study. The control study was performed in a ‘music-free’ environment, where the participants had an opportunity to converse, read a magazine, or take a walk outside (no exercise) during the session. Peripheral blood samples were collected from the participants just before and after 20 min in the control session (the same duration as in the listening session). From here on, participants of the control session are referred to as controls throughout the text. Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 3/17 Genome-wide expression profiling For this procedure, 2 × 2.5 ml samples of peripheral blood were drawn into PAXgene blood RNA tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) as per the kit instructions, in both of the sessions. Total RNA was isolated using the PAXgene blood miRNA Kit (PreAnalytiX GmbH, Hombrechtikon, Switzerland) as per the kit manual. Purified RNA samples were measured for concentration and purity on the NanoDrop 1000 v.3.7 (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Globin mRNA was depleted from our samples using Ambion’s Human GLOBINclearTM kit (Applied Biosystems, Carlsbad, California, USA) as per the kit insert. The samples were measured on the NanoDrop 1000 to determine the sample concentration and purity and for integrity on the 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) before being diluted to 50 ng/µl using RNase-free water. A total of 2 µg of RNA was assayed on the Illumina HumanHT-12 v4 bead array (Illumina Inc., San Diego, California, USA), which targets more than 47,000 probes. The gene expression profiling assays for the listening and control sessions were conducted in two separate batches. To account for the batch effect corrections, six samples from the listening session were assayed together with the control session samples. Intensity data were exported through Bead Studio software. The data reported in this article have been deposited in the Gene Expression Omnibus database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48624). Data preprocessing The Lumi package was used to read and preprocess the signal intensity data. Specifically, pre-processing included background correction, variance stabilizing transformation, and quantile normalization. Data from both the listening (n = 48) and control (n = 15) sessions were normalized separately. Five samples from the control session were excluded from further analyses owing to data quality. In addition, we used the ComBat method (Johnson, Li & Rabinovic, 2007) to adjust for batch effects and determine if this correction affected the pre-post fold-changes across conditions. However, we did not find significant differences between the fold-changes of corrected and uncorrected data over time across conditions. Therefore, we chose to proceed with the uncorrected data owing to the strengths of our analysis methods as described below. After normalization, duplicate and un-annotated probes were excluded using the genefilter package (R package version 1.40.0). Before extracting the expression values from the normalized data, Illumina’s detection p-values (threshold: 0.01) were used to filter out probes with low intensities corresponding to the background signal. Finally, only those probes that were expressed in at least half of all of the arrays (listening and control sessions) were chosen for the study. Differential expression analysis The choice of an appropriate statistical test for the identification of differentially expressed genes depends upon several aspects of the data including the underlying distribution, homogeneity, and the sample size. As the statistical tests for normality are known to be sensitive to sample size, we used a normal Q-Q plot to get a glimpse of the distribution of Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 4/17 the data. For this, we randomly visualized the distribution of transcriptional responses of control samples (n = 10) for several transcripts (Fig. S1) using normal Q-Q plots. We observed that the data appeared to deviate from normality in several instances. As the central limit theorem does not always hold true for small sample sizes, a cautious approach here would be to employ a non-parametric test (better being safe than sorry). Non-parametric tests are deemed to be appropriate analysis tools when the distribution of data is difficult to characterize, because they make less stringent distributional assumptions. Therefore, we chose to use the rank product (Breitling et al., 2004) non-parametric test statistic, which is relatively powerful especially for small sample sizes, and when the data is heterogeneous and does not meet normality. Rank product-based methods outperformed several other methods including empirical Bayes statistic (limma) and SAM, when the sample size is small and when the data is non-homogeneous (Jeffery, Higgins & Culhane, 2006). Also, a comparison of eight gene ranking methods using Microarray Quality Control datasets (golden-standard) has demonstrated the high sensitivity and specificity of the rank product method (Kadota & Shimizu, 2011). To identify the differentially expressed genes, we compared the magnitude of pre-post changes in gene expression across conditions using the rank product method implemented in the RankProd Bioconductor package. Based on the estimated percentage of false predictions (pfp), RankProd employs a non-parametric statistic to identify genes that are consistently ranked high among the most up- or down-regulated genes in replicate experiments. Instead of analyzing the actual expression value, this method utilizes the ranks of genes in each sample. The strength of rank product method allows us to compare and combine the datasets of the listening and control studies. After the identification of differentially expressed genes using a pfp of 0.05 in RankProd, we selected only those genes that exceeded an effect-size cut-off (>1.2-fold change over time across conditions, and at least a pre-post change of 10% in gene expression in the listening session). Here, we would like to point out a couple of aspects of these selection criteria. First, pfp employed by RankProd is equivalent to the standard false discovery rate (FDR). Second, there exists a widespread misconception that only two-fold changes are significant (Hoheisel, 2006) and that false notion is based on the very initial publications of microarray studies, which used a twofold change criteria for a particular group of experiments because of biological relevance. Fold-change thresholds are completely arbitrary and in the majority of the cases they depend upon the underlying biological question. For example, studies that investigated the effect of gene-environment interactions (socio-environmental effect (Cole et al., 2007), yogic meditation effect (Black et al., 2012)) used unorthodox fold-change thresholds. We further performed successive functional annotation analyses using GeneTrail (http://genetrail.bioinf.uni-sb.de/) and IPA (Ingenuity® Systems, www.ingenuity.com). The gene ontological overrepresentation analysis in GeneTrail uses a hypergeometric distribution test along with a conservative multiple testing correction method (FDR < 0.05) to assess whether genes belonging to certain functional categories are enriched in the dataset. In addition to the standard gene ontology analyses, we performed upstream transcription regulator analysis, which essentially predicts all the upstream transcription Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 5/17 regulators (transcription factors, receptors, cytokines, microRNA, and kinases) that could have possibly mediated the observed differential expression. Based on the overlap between known targets of a transcription regulator and the set of differentially expressed genes, an overlap p-value is computed using Fisher’s exact test (p < 0.01). Further, the activation states of the predicted transcription regulators are also inferred using an activation Z-score, which is based on literature-derived knowledge on the direction of regulation (either activating or inhibiting). RESULTS Transcriptional responses after a control session First we assessed the homogeneity of transcriptional responses in the control session. For this, we analyzed whether the transcriptional responses of participants belonging to edu classes 3–4 (n = 4) and edu classes 1–2 (n = 6) differed in the control session. Only 4 genes were found to be differentially expressed, suggesting no major differences in the transcriptional responses between groups to a ‘non-musical activity.’ Therefore, we used the expression data of the whole ‘music-free’ control group as a reference for the comparative analyses. We used Spearman’s rank-based correlation to check for each gene, whether the transcriptional responses correlated with the age or sex of the participants in either the listening session (n = 48) or the control sessions (n = 10). After multiple testing corrections, we found no significant effects of age or sex on the transcriptional responses. Transcriptional response after listening to music Based on neuroscientific studies, the brains of musicians and non-musicians differ structurally and functionally (Elbert et al., 1995; Gaser & Schlaug, 2003). This led us to ask whether the transcriptional responses of musically experienced participants would differ from those of musically inexperienced participants when listening to music. Therefore, we compared the transcriptional responses of listening to music separately for musically experienced and inexperienced participants vs controls. Comparisons were made based on two subphenotypes of musical experience: musical aptitude and music education. First, we compared the magnitude of pre-post fold-changes in the genome-wide transcriptional profiles of listeners of edu classes 3–4 (n = 29) and controls (n = 10). Using RankProd non-parametric statistics and stringent selection criteria, we identified 45 differentially expressed genes (27 up-regulated and 18 down-regulated). Next, we compared the genome-wide transcriptional profiles of listeners with high COMB scores (n = 12) and controls (n = 10). Similar statistical analysis identified 97 differentially expressed genes (75 up-regulated and 22 down-regulated). The differentially expressed genes from both the comparisons are listed in Table S2, and a comparison of the pre-post changes in both conditions is shown in Fig. 1. Functional annotation Based on gene ontology analyses (Table S3), the genes up-regulated in the listeners of edu classes 3–4 are known to be primarily involved in the regulation, secretion and Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 6/17 Figure 1 Differential gene expression in experienced listeners vs ‘music-free’ controls. Heatplot representations of mean expression values preand post-music listening session and control sessions. The red-yellow-green palette represents low-moderate-high expression values. (A) Educated listeners vs controls, (B) Competent listeners vs controls. transport of the neurotransmitter dopamine (e.g., SNCA, RTN4, and SLC6A8), protein sumoylation (SUMO2 and HDAC4) and neuron projection (SNCA, RTN4, DICER1 and MYC). Down-regulated genes are known to affect functions such as mitochondrial ATP synthase-coupled proton transport and cytolysis (e.g., ATP5J, ATP5L, GZMH, and GZMA). Several of the genes, including the dopamine secretion-related genes (SNCA, RTN4) up-regulated in listeners of edu classes 3–4, were also found to be up-regulated in listeners with high COMB scores. Here, we should note that the COMB scores are strongly correlated with music edu classes (Spearman’s rho 0.5644; p-value 2.931e–05). In listeners with high COMB scores, gene ontology classification revealed that the up-regulated genes are involved in functions such as long-term synaptic potentiation (NPTN and SNCA), dephosphorylation and regulation of cell communication. Down-regulated genes are known to be involved in functions such as positive regulation of caspase, peptidase and endopeptidase activities (Table S3). We further performed Entrez gene annotation and an extensive literature survey for all the genes that are differentially expressed after listening to music (in listeners of both edu Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 7/17 Table 2 Putative biological functions of the differentially expressed genes after listening to music. Biological function Gene Direction of regulation Dopamine secretion, transport, signaling Synaptic neurotransmission (Vesicular exocytosis, endocytosis) Synaptic function Learning and memory, cognitive performance Song learning and singing in songbirds Auditory cortical activation Absolute pitch Neuroprotection Neurogenesis Neuronal apoptosis ATP synthase coupled proton transport SNCA, RTN4, RGS2, SLC6A8 SNCA, STXBP2, FKBP8, SYNJ1, LYST, SUMO2, HDAC4, DUSP6 SNCA, NPTN, FKBP8, NRGN, HDAC4 SNCA, NRGN, NPTN, FKBP8, RTN4, SLC6A8, NEDD9 SNCA, NRGN, RGS2, MYC, UBE2B HDAC4, LRRFIP1 FAM49B, HDAC4 SNCA, RTN4, FKBP8, SLC6A8, KLF4 KLF4, SMNDC1, S100A12 CASP8, GZMH, GZMA, IFI6, PYCARD, TNFRSF10B, HSPE1 ATP5J, ATP5L Up Up Up Up Up Up Up Up Up Down Down classes 3–4 and high COMB scores). This revealed that the up-regulated genes are known to be associated with dopamine signaling, synaptic neurotransmission, synaptic function, learning, memory and cognitive performance, song learning and singing in songbirds, auditory cortical activation, absolute pitch, neuroprotection and neurogenesis (Tables 2 and S4). On the other hand, down-regulated genes are known to cause mammalian neuronal apoptosis, immoderate oxidative phoshorylation and deficits in dopaminergic neurotransmission, which are the characteristics of neurodegeneration (Tables 2 and S4). Detailed information about the putative biological functions of the differentially expressed genes is provided in Table S4. Upstream regulator analysis To obtain insight into the molecules that might mediate the observed differences in gene expression, we performed an upstream transcription regulator analysis using IPA (Table S5). This analysis revealed that the up-regulated genes in listeners of edu classes 3–4 significantly overlap the known target genes of the glucocorticoid receptor NR3C1 (p-value 0.001), and progesterone receptor PGR (p-value 0.0008), whereas the down-regulated genes did not display any significant overlap. In listeners with high COMB scores, upstream regulator analysis again identified the up-regulation of target genes of the glucocorticoid receptor gene (NR3C1) and also the target genes of several other transcription regulators such as TP53, MYC, HOXA9, CD24 and chorionic gonadotropins. Down-regulated genes significantly overlapped the known target genes of pro-inflammatory transcription regulators such as tumor necrosis factor (TNF), a member of its superfamily (TNFSF10), interferon gamma (IFNG), a member of its gene cluster (IFNA2), the microtubule-associated protein tau (MAPT)and the nuclear factor kappa B family gene (NFKB1A; Table S5). Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 8/17 Transcriptional responses of participants with no significant experience Furthermore, we repeated similar analyses to compare the magnitude of pre-post fold-changes over time in listeners of edu classes 1–2 vs controls and listeners with low COMB scores vs controls. Using the same analysis methods and selection criteria to identify the differentially expressed genes, we identified 8 and 22 differentially expressed genes, respectively in the comparisons. However, functional characterization of those genes did not reveal any significant findings. DISCUSSION The findings of this study suggest that listening to classical music has an effect on human transcriptome. The up-regulation of genes related to dopamine secretion and signaling is in agreement with the previous neuroimaging-based evidences (Salimpoor et al., 2011). Particularly, alpha-synuclein (α-synuclein; SNCA), one of the most up-regulated genes, is involved in dopamine (DA) neuronal homeostasis (Murphy et al., 2000; Oczkowska et al., 2013). Interestingly, SNCA is located on chromosome 4q22.1, the most significant region of linkage for musical aptitude (Pulli et al., 2008; Oikkonen et al., 2014) and regulated by GATA2 (Scherzer et al., 2008), which is associated with musical aptitude (Oikkonen et al., 2014) (Fig. 2). These data provide convergent evidence about the molecular basis of musical traits from both DNA and RNA studies. Another finding from the upstream regulator analysis suggests that listening to music primarily increased the expression of the target genes of the glucocorticoid receptor (NR3C1). Notably, dopaminoceptive neuronal glucocorticoid receptor has been described as a key molecule in the regulation of addictive behavior. By reducing dopamine re-uptake, NR3C1 increases the synaptic concentration of dopamine, which leads to rewarding and reinforcing properties (Ambroggi et al., 2009) that have previously been linked to listening to music (Blood & Zatorre, 2001; Koelsch, 2011; Koelsch, 2014; Salimpoor et al., 2011; Salimpoor et al., 2013). The up-regulation of genes related to synaptic vesicular exocytosis, endocytosis, neurotransmission and plasticity seems perfectly rational here because the majority of these biological processes are essential for the secretion and signaling of neurotransmitters (Südhof & Rizo, 2011; Saheki & De Camilli, 2012). As listening to music has been known to induce the secretion and signaling of a neurotransmitter, dopamine (Sutoo & Akiyama, 2004; Salimpoor et al., 2011), we can speculate the role of these up-regulated genes in facilitating dopaminergic neurotransmission after listening to music. Moreover, some of the up-regulated genes have evident roles in enhancing cognitive functions like long-term potentiation (LTP) and memory. In previous behavioral studies, music education and training have proven to have beneficial effects on cognitive development, cognitive performance, verbal and long-term memories (Rammsayer & Brandler, 2003; Schlaug et al., 2005; Sluming et al., 2007; Wong et al., 2007; Roden, Kreutz & Bongard, 2012; Rodrigues, Loureiro & Caramelli, 2013). Several of the differentially expressed genes have been demonstrated to be responsible for song learning and singing in songbirds (Wada et al., 2006), which suggests a possible evolutionary conservation in biological processes related to sound perception. Further- Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 9/17 Figure 2 Schematic representation of chromosome 4. The α-synuclein gene (SNCA) that was found to be up-regulated after music perception in this study is located in the best linkage region of musical aptitude as shown by Pulli et al. (2008), Park et al. (2012) and Oikkonen et al. (2014). GATA2, which is located in the best genome-wide association region of musical aptitude (Oikkonen et al., 2014) and regulates the SNCA, is also shown. more, the up-regulation of genes associated with human auditory cortical activation (Renvall et al., 2012) and absolute pitch (Theusch, Basu & Gitschier, 2009; Gervain et al., 2013) are logical, because listening to music involves both of those auditory phenomena. Auditory perception processes have been known to exhibit convergent evolution across species. Notably, the human auditory center is identical to those of the first primates who inhabited the planet millions of years ago (Langner , 1992; Montealegre-Z et al., 2012). In addition, widespread adaptive convergent sequence evolution has been found recently in hearing-related genes in echolocating bats and dolphins (Parker et al., 2013). Similarly, convergent sequence evolution has also been identified in vocal-learning birds and mammals (Zhang et al., 2014). More recently, convergent gene expression specializations have been detected in songbirds and humans in the regions of brain that are essential for auditory perception and speech production (Pfenning et al., 2014). Thus, the genes detected by Pfenning et al. (2014), in general, represent the genes belonging to auditory perception pathway in both songbirds and humans. Here, genes belonging to the auditory perception pathway (∼2-fold enrichment; p-value: 0.028; two-sided Fisher’s exact test) were found to be enriched among the genes that are differentially expressed after listening to music. This suggests that our results serve as a relevant molecular background for music perception in humans. The widely-documented neuroprotective role of some of the up-regulated genes and the down-regulation of several neurodegeneration-inducing genes support the notion of a neuroprotective role for music and may provide a working mechanism for the use of music therapy, especially in treating neurodegenerative diseases (AMTA, 2014; Conrad, 2010; Holmes, 2012). Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 10/17 In this study, significant transcriptional responses were observed only in individuals who either have substantial periods of music education/training or have relatively higher musical aptitude scores. This suggests that certain musical abilities (either innate or acquired through music education) may influence the transcriptional responses of listening to music. Previous works have shown that the familiarity of music attained through music education or repeated music exposure is known to largely influence the rewarding aspects of listening to music (Sarkamo et al., 2008; Salimpoor et al., 2009; Van den Bosch, Salimpoor & Zatorre, 2013; Schubert, Hargreaves & North, 2014). Here, we acknowledge that the effect of music exposure on human gene expression could be very subjective and may vary depending on several factors such as age, sex, culture, previous listening habits, music education and training and personal liking of music, as recently discussed (Wong et al., 2007; Salimpoor et al., 2009; Van den Bosch, Salimpoor & Zatorre, 2013; Mikutta et al., 2014). To be able to comprehensively characterize the transcriptomic alterations of music-listening, further studies are required to assess the effect of listening to different genres of music, at different ages, in different ethnicities and in individuals with varying music education levels and listening habits, with varying durations of listening. The participants, who listened to music in this study, were unaware of the type of music that was intended for the listening session. Similarly, without further details about the intended activity, the participants were invited to attend a ‘music-free’ control session. Here, it is crucial to discuss the association between ‘anticipation’ and ‘listening to music.’ Anticipation is always involved in listening to music (Salimpoor et al., 2011). The unveiling of series of tones with time evokes anticipatory responses because of the cognitive expectations and prediction cues (Huron, 2006; Meyer, 2008). Functional neuroimaging studies have successfully distinguished the anatomical regions that respond to anticipation and consumption of music (Salimpoor et al., 2011). However, distinguishing the human gene expression signatures of anticipatory and consummatory responses of music is not yet feasible. Therefore, even if we perform a blinded experiment here, we might not be able to exclude the effect of anticipation or expectation. As brain samples are inacesssibile in this type of study, we used peripheral blood as a window to the study the effects of listening to music. Peripheral blood is known to share more than 80% of the transciptome and significant gene expression similarities with other tissues including multiple regions of brain (Liew et al., 2006; Sullivan, Fan & Perou, 2006; Tylee, Kawaguchi & Glatt, 2013). Thus, peripheral blood could certainly provide surrogate information concerning gene expression in brain tissue for a subset of genes (Davies et al., 2009). For instance, the molecular alterations in dopamine metabolism and mitochondrial function, which are the potential hallmarks of Parkinson’s disease, have been detected in peripheral blood (Scherzer et al., 2007). Notably, genes that are responsive to physiological stimuli (which are earlier thought to be tissue-specific) and genes involved in neuroendocrine pathways (e.g., hormone receptors, neurotransmitter receptors) are expressed in the peripheral blood. Because of these characteristics, peripheral blood has been used as a proxy in several studies when a specific tissue is not available (e.g., human brain), especially in behavioral and neurodegenerative studies (Mohr & Liew, 2007). In Kanduri et al. (2015), PeerJ, DOI 10.7717/peerj.830 11/17 the wake of all these findings, a subset of the molecular mechanisms identified here may legitimately reflect the transcriptomic alterations in brain after listening to music. ACKNOWLEDGEMENTS We thank all of the participants for their generous cooperation. We thank Päivi Onkamo for her constructive comments about the manuscript. We are grateful to Petri Myllynen, Sanna Pyy, Laura Salmela, Sonja Suhonen, Jaana Oikkonen and Kai Karma for expert technical help. We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics for the generation of the Gene Expression data. ADDITIONAL INFORMATION AND DECLARATIONS Funding The Academy of Finland (grant reference #13371) and the Biocentrum Helsinki Foundation supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Grant Disclosures The following grant information was disclosed by the authors: The Academy of Finland: #13371. Biocentrum Helsinki Foundation. Competing Interests The authors declare there are no competing interests. Author Contributions • Chakravarthi Kanduri analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Pirre Raijas and Liisa Ukkola-Vuoti contributed reagents/materials/analysis tools, reviewed drafts of the paper. • Minna Ahvenainen and Anju K. Philips performed the experiments, reviewed drafts of the paper. • Harri Lähdesmäki conceived and designed the experiments, reviewed drafts of the paper. • Irma Järvelä conceived and designed the experiments, wrote the paper, reviewed drafts of the paper. Human Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The Ethics Committee of Helsinki University Central Hospital approved this study (permission number 233/13/03/2013). Written informed consent was obtained from all the participants. Kanduri et al. 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A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew

Frontiers in plant science

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Citation for published version (APA): Zhang, W-J., Hanisch, S., Kwaaitaal, M. A. C. J., Pedersen, C., & Thordal-Christensen, H. (2013). A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew. Frontiers in Plant Science, 4, [127]. https://doi.org/10.3389/fpls.2013.00127 Download date: 24. Oct. 2024 A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew Zhang, Wen-Jing; Hanisch, Susanne; Kwaaitaal, Mark Adrianus Cornelis J; Pedersen, Carsten; Thordal-Christensen, Hans Zhang, Wen-Jing; Hanisch, Susanne; Kwaaitaal, Mark Adrianus Cornelis J; Pedersen, Carsten; Thordal-Christensen, Hans university of copenhagen university of copenhagen A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew Zhang, Wen-Jing; Hanisch, Susanne; Kwaaitaal, Mark Adrianus Cornelis J; Pedersen, Carsten; Thordal-Christensen, Hans university of copenhagen INTRODUCTION this process is the RxLR-dEER motif, located a few amino acids downstream of the SP cleavage site in many oomycete effectors. By an unknown process, this motif guides the effectors to be trans- portedacrossmembranesandallowsthemtoenterthehostcytosol (Whisson et al., 2007). Many filamentous plant pathogenic fungi and oomycetes rely on placing a feeding structure, a so-called haustorium inside host cells in order to exploit host resources and to transfer effector proteins to the host cytosol. By unknown mechanisms, these pathogens trigger the host cells to generate an extrahaustorial membrane (EHM), which allows the host cells to stay alive despite the severe haustorial invasions (Gan et al., 2012). In between the haustorium and the EHM, a sealed compartment, called the extrahaustorial matrix (EHMx) is present. Many of these pathogens, such as powdery mildew fungi, have genetically lost certain general life- sustaining processes during their evolution (Spanu et al., 2010). This prevents them from living on dead biological material, mak- ing them strict biotrophs. In the meantime, they secrete hundreds of effectors from the haustoria, mediated by signal peptides (SPs) and default secretion. Many of these effectors are transferred to the host cytosol, where they play important roles in pathogenicity by assisting in nutrient acquisition, suppression of defense and reprogramming cellular processes (Bozkurt et al., 2012; Pedersen et al., 2012; Saunders et al., 2012). An inherent problem, which is poorly understood, concerns how the effectors escape the EHM- delimited haustorial compartment to access the plant cytosol. This requires a mechanism to cross membranes, such as a protein trans- mitting pore. Essentially, the only currently established element of The endoplasmic reticulum (ER) is a major organelle in eukaryotic cells, which forms an extended network, function- ing in, e.g., protein processing and sorting. Voegele et al. (2009) have previously suggested that the ER plays a role in trans- fer of effector to the plant cytosol. In the ER, proper folding and modification of proteins is assisted and validated by the ER quality control (ER-QC) machinery. If proteins finally fail the quality check, they are recognized by the ER-associated protein degradation (ERAD) machinery and retrotranslocated into the cytosol to be degraded by proteasomes (Nakatsukasa and Brod- sky, 2008). Effectors may exploit this retrotranslocon pore in order to get access to the plant cytosol. Zhang, Wen-Jing; Hanisch, Susanne; Kwaaitaal, Mark Adrianus Cornelis J; Pedersen, Carsten; Thordal-Christensen, Hans Published in: Frontiers in Plant Science Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Citation for published version (APA): Zhang, W-J., Hanisch, S., Kwaaitaal, M. A. C. J., Pedersen, C., & Thordal-Christensen, H. (2013). A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew. Frontiers in Plant Science, 4, [127]. https://doi.org/10.3389/fpls.2013.00127 Download date: 24. Oct. 2024 ORIGINAL RESEARCH ARTICLE published: 16 May 2013 published: 16 May 2013 doi: 10.3389/fpls.2013.00127 A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew Wen-Jing Zhang†, Susanne Hanisch†, Mark Kwaaitaal, Carsten Pedersen and HansThordal-Christensen* Section for Plant and Soil Science, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark *Correspondence: Correspondence: Hans Thordal-Christensen, Section for Plant and Soil Science, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. e-mail: [email protected] †Wen-Jing Zhang and Susanne Hanisch have contributed equally to this work. †Wen-Jing Zhang and Susanne Hanisch have contributed equally to this work. Keywords: powdery mildew, haustorium, extrahaustorial membrane (EHM), endoplasmic reticulum-associated degradation (ERAD), Sec61 complex, susceptibility factor Reviewed by: Reviewed by: Ralph Huckelhoven, Technische Universitaet Muenchen, Germany Thomas Liebrand, Wageningen University, Netherlands *Correspondence: Hans Thordal-Christensen, Section for Plant and Soil Science, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. e-mail: [email protected] †Wen-Jing Zhang and Susanne Hanisch ha e contrib ted eq all to Reviewed by: Ralph Huckelhoven, Technische Universitaet Muenchen, Germany Thomas Liebrand, Wageningen University, Netherlands Reviewed by: Ralph Huckelhoven, Technische Universitaet Muenchen, Germany Thomas Liebrand, Wageningen University, Netherlands *Correspondence: Hans Thordal-Christensen, Section for Plant and Soil Science, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. e-mail: [email protected] †Wen-Jing Zhang and Susanne Hanisch have contributed equally to Keywords: powdery mildew, haustorium, extrahaustorial membrane (EHM), endoplasmic reticulum-associated degradation (ERAD), Sec61 complex, susceptibility factor Edited by: Biotrophic pathogens, like the powdery mildew fungi, require living plant cells for their growth and reproduction. During infection, a specialized structure called the haustorium is formed by the fungus.The haustorium is surrounded by a plant cell-derived extrahaustorial membrane (EHM). Over the EHM, the fungus obtains nutrients from and secretes effector proteins into the plant cell. In the plant cell these effectors interfere with cellular processes such as pathogen defense and membrane trafficking. However, the mechanisms behind effector delivery are largely unknown. This paper provides a model for and new insights into a putative transfer mechanism of effectors into the plant cell. We show that silencing of the barley Sec61βa transcript results in decreased susceptibility to the powdery mildew fungus. HvSec61βa is a component of both the endoplasmic reticulum (ER) translocon and retrotranslocon pores, the latter being part of the ER-associated protein degradation machinery. We provide support for a model suggesting that the retrotranslocon function of HvSec61βa is required for successful powdery mildew fungal infection. HvSec61βa-GFP and a luminal ER marker were co-localized to the ER, which was found to be in close proximity to the EHM around the haustorial body, but not the haustorial fingers. This differential EHM proximity suggests that the ER, including HvSec61βa, may be actively recruited by the haustorium, potentially to provide efficient effector transfer to the cytosol. Effector transport across this EHM-ER interface may occur by a vesicle-mediated process, while the Sec61 retrotranslocon pore potentially provides an escape route for these proteins to reach the cytosol. Corné M. Pieterse, Utrecht University, Netherlands INTRODUCTION Different multicom- ponent retrotranslocon pores have been described in yeast and mammals, in which, e.g., Derlin, Hrd, and Sec61 proteins are major elements (Kawaguchi and Ng, 2007; Nakatsukasa and Brod- sky, 2008). The ERAD substrates are ubiquitinated during the retrotranslocation process by retrotranslocon-associated ubiqui- tin ligases, and this targets them for proteasomal degradation as soon as they reach the cytosol (Carvalho et al., 2006, 2010). May 2013 | Volume 4 | Article 127 | 1 www.frontiersin.org Plant Sec61β in fungal pathogenicity Zhang et al. was TOPO-cloned into the pENTR/D-TOPO vector (Invitro- gen). Positive clones were validated by sequencing. Using Gate- way LR cloning, according to the manufacturer’s instructions (Invitrogen), the insert was transferred to the 35S-promoter driven destination vector, pIPKTA30N (Douchkov et al., 2005), to generate the final RNAi construct. To generate the Sec61βa- GFP construct for localization, the full-length Sec61βa cod- ing sequence, without stop-codon, was amplified with the primer pair HvSec61βa_KZK_GWY_FW (GGGGACAAGTTTG- TACAAAAAAGCAGGCACCATGGTGGCTAATGGTGACGCCC CT) and HvSec61βa_ns_GWY_Rv (GGGGACCACTTTGTA- CAAGAAAGCTGGGTTATTAGGGGTGCGGTACAGCTTGCC) on the pENTR clone described above, and using a BP clonase reaction it was cloned into the pDONR201 vector (Invitrogen). Positive clones were confirmed by sequencing. Using a Gateway LR clonase reaction according to the manufacturer’s instructions (Invitrogen), the insert was transferred to the 35S-promoter- driven destination vector, P2GWF7 (Curtis and Grossniklaus, 2003). All final clones were verified by restriction enzyme digestion. The Sec61 pore can translocate proteins bi-directionally, and it is primarily known as the translocon pore, mediating the pro- cess of SP-dependent protein translocation into the ER. The Sec61 pore is a doughnut-shaped heterotrimeric complex, con- sisting of the subunits, Sec61α, Sec61β, and Sec61γ. SP and Sec61-dependent translocation into the ER can occur either co- or post-translationally (Zimmermann et al., 2011). The ERAD pathway has in several cases been shown to be recruited by oppor- tunistic pathogens for transfer of polypeptides into the host cell cytosol. For example, cholera toxin, shiga toxin, and Pseudomonas aeruginosa exotoxin enter the cytosol through retrotranslocon pores, but escape from ubiquitination and proteasome-mediated degradation (Rodighiero et al., 2002; Blanke, 2006). Retrotranslo- cation of cholera toxin occurs through the Sec61 retrotranslocon pore, and depletion of the Sec61 complex prevented the retro- translocation of this toxin into the cytosol (Schmitz et al., 2000; Teter et al., 2002). Here we aimed at studying the role of the Sec61 pore in plant susceptibility to the powdery mildew fungus. PARTICLE BOMBARDMENT Transformation of gene constructs into epidermal cells of 7- day-old barley leaves was conducted by particle bombardment, essentially as described by Douchkov et al. (2005). For transient induced gene silencing (TIGS) studies, the constructs were co- transformed with a β-glucuronidase (GUS) reporter construct, followed by inoculation with Bgh 2 days later (inoculation den- sity around 200 conidia per mm2). Three days after inoculation, the leaves were GUS-stained, and the relative susceptibility index was calculated by dividing the number of GUS-stained epidermal cells containing a haustorium by the total number of GUS-stained cells. The data were normalized to the empty vector (pIPKTA30N) control. The experiments were repeated at least three times. A cell viability test was performed by co-transformation of the HvSec61βa RNAi construct or the empty vector control, pIP- KTA30N, with the anthocyanin biosynthesis-activating construct, pBC17 (Schweizer et al., 2000). Two days after transformation, the leaves were inoculated with Bgh at a density of 200 coni- dia per mm2, and after another 3 days, the anthocyanin-stained cells were counted. Constructs for marker proteins, fused with fluorescent proteins, were transformed and inoculated with Bgh 1 day later, and examined by confocal microscopy 2 days after transformation. INTRODUCTION Barley (Hordeum vulgare) has two Sec61α, two Sec61β, and one Sec61γ protein1 (Deng et al., 2007; Mayer et al., 2012), and to unravel the role of the pore, we made use of the fact that the Sec61β component is essential for retrotranslocon activity for various substrates, but less important for translocon activity under non-stressed conditions (Finke et al., 1996; Van den Berg et al., 2004; Liao and Carpen- ter, 2007; Kelkar and Dobberstein, 2009; Zhao and Jantti, 2009; Wang et al., 2010; Guerriero and Brodsky, 2012). We show that silencing of HvSec61βa reduced the susceptibility of barley epider- mal cells to the powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh). In addition, the HvSec61βa-GFP-labeled ER network is differentially associated to the body, and not the fingers of the powdery mildew fungal haustorium. To explain the role of the Sec61βa in pathogenicity, we propose a model in which the fun- gus actively recruits the ER in order to exploit the Sec61 pore for pathogenicity. MATERIALS AND METHODS PLANTS AND FUNGI Barley (Hordeum vulgare) cv. Golden Promise plants were used for transient transformation and subsequent studies with and without powdery mildew fungal inoculation. The barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh), isolate DH14, was maintained on susceptible barley, cv. Golden Promise, grown at 20◦C, 16 h light (150 μE/sm2)/8 h dark, by weekly inocu- lum transfer. These growth conditions were used throughout the studies. 1http://webblast.ipk-gatersleben.de/barley/viroblast.php CONFOCAL MICROSCOPY A Leica SP5-X confocal laser scanning microscope, mounted with a 63 × 1.2 numerical aperture water-immersion objective, was used. For fluorescent protein detection and localization, GFP was excited at 488 nm, and the fluorescence emis- sion was detected between 518 and 540 nm. mCherry flu- orescence was excited at 543 nm and fluorescence emission was detected between 590 and 640 nm. 3D projections were created using the Image Surfer 1.2 software2 (Feng et al., 2007). 2www.imagesurfer.org HvSec61βa IS A POTENTIAL SUSCEPTIBILITY FACTOR FOR THE BARLEY POWDERY MILDEW FUNGUS In barley two Sec61β genes have been identified, which are named HvSec61βa and HvSec61βb. Interestingly, the HvSec61βa tran- script accumulates in leaves after attack by Bgh3 (Dash et al.,2012). Therefore, we selected to analyze the role of HvSec61βa in the bar- ley/Bgh interaction, and performed single cell TIGS of this gene. A 35S-promoter-driven RNAi construct, covering the full-length coding region of this gene, was generated and transiently trans- formed together with a GUS reporter-gene construct into barley epidermal cells (Douchkov et al., 2005). After 2 days, the leaves were inoculated with Bgh and transformed cells were stained for GUS activity 3 days thereafter. Infection success of Bgh was evalu- ated microscopically by scoring the total number of GUS-stained cells and the number of GUS-stained cells containing one or more haustoria. Subsequently, the data were normalized to the empty vector control. The RNAi construct of HvSec61βa resulted in more than 40% reduction in susceptibility to Bgh (Figure 1A). As a pos- itive control, the relative susceptibility of cells transformed with an Mlo-RNAi construct was included (Douchkov et al., 2005). These cells were 70% less susceptible than the control cells. In order to confirm that the RNAi construct in fact results in silenc- ing of HvSec61βa, we co-transformed barley epidermal cells with the RNAi construct of HvSec61βa and a 35S promoter-driven HvSec61βa-GFP fusion construct. Five days after transformation together with a reference construct for cytosolic mCherry expres- sion, confocal imaging revealed that the RNAi construct prevented appearance of GFP signal, while it did not affect the signal from mCherry in the same cell (Figure 1B). The reduced HvSec61βa- GFP signal indicated that the HvSec61βa RNAi silencing construct indeed induced degradation of HvSec61βa encoding mRNA and In order to analyze whether the reduced susceptibility could be due to reduced viability of the cells in which HvSec61βa was silenced, a second experiment was performed. Co-transformation was performed with an anthocyanin biosynthesis gene activation construct, pBC17, causing the transformed cells to accumulate the red anthocyanin pigment as long as they stay alive (Schweizer et al., 2000). Two days after transformation, the leaves were inoc- ulated with a high density of Bgh conidia (≈200 per mm2). Similar numbers of anthocyanin accumulating cells were detected in HvSec61βa-silenced and non-silenced cells after Bgh infection (Figure 1C). Therefore, this result confirmed that the HvSec61βa RNAi construct did not affect the viability of the barley cells after inoculation. CLONING To generate a gene-specific RNA interference (RNAi) con- struct to silence HvSec61βa (AK252927.1), its coding sequence was PCR-amplified using the primer pair Sec61βa_F1 (CAC- CATGGTGGCTAATGGTGACG) and Sec61βa_R1 (GGGGT- GCGGTACAGCTTTC) on cDNA generated from mRNA iso- lated from 7-day-old barley leaf material. The PCR product May 2013 | Volume 4 | Article 127 | 2 Frontiers in Plant Science | Plant-Microbe Interaction Plant Sec61β in fungal pathogenicity Zhang et al. likely as well impaired endogenous HvSec61βa transcript and pro- tein accumulation. Thus, the observed increased resistance of HvSec61βa-silenced cells indicates a potential role of HvSec61βa as a susceptibility factor for efficient Bgh infection. HvSec61β LOCALIZATION IN UNINFECTED AND INFECTED BARLEY CELLS Next we aimed to subcellularly localize HvSec61βa to search for clues for the powdery mildew-related function of this protein. Sec61β is a small ∼8 kDa protein with a single transmembrane domain, and GFP-tagging has previously been used for its local- ization (Rolls et al., 1999; Voeltz et al., 2006). Therefore, we co-expressed our 35S promoter-driven HvSec61βa-GFP fusion construct together with a 35S promoter-driven SP-mCherry- HDEL construct (Nelson et al., 2007) in infected and uninfected barley epidermal cells. The SP targets mCherry to the ER and the ER retrieval motif HDEL (His-Asp-Glu-Leu) at the C-terminus retains it in the lumen of the ER (Gomord et al., 1997). Confo- cal images of epidermal single cells expressing HvSec61βa-GFP and SP-mCherry-HDEL were recorded 48 h after particle bom- bardment (Figure 2). Intense GFP signal was observed in the ER cortical network throughout the cells expressing GFP-tagged 3http://www.plexdb.org/plex.php?database=Barley FIGURE 1 | Silencing of HvSec61βa reduces susceptibility to Bgh without affecting plant cell viability. (A) Susceptibility after co-transformation of empty vector control, HvSec61βa-RNAi and Mlo-RNAi constructs with a GUS-reporter construct into barley epidermal cells, followed by inoculation with Bgh 3 days later. The relative susceptibility was calculated as described in Materials and Methods. Mlo-RNAi was used as a positive control. Error bars represent standard deviation (SD). **, P < 0.01 (Student’s t-test). (B) HvSec61βa-RNAi reduced the GFP signal originating from HvSec61βa-GFP, but not the fluorescence signal from cytosolic mCherry 5 days after transformation. Micrographs show maximum intensity projections. (C) Number of pBC17-transformed cells accumulating anthocyanin, reflecting cell viability, after co-bombardment with either an empty vector control or the HvSec61βa-RNAi construct on similarly sized pieces of leaf. Two days after bombardment, leaves were inoculated with Bgh, and the number of anthocyanin-accumulating cells was scored 3 days later (n = 4). f ti i M 2013 | V l 4 | A ti l 127 | 3 HvSec61βa-GFP, but not the fluorescence signal from cytosolic mCherry 5 days after transformation. Micrographs show maximum intensity projections. (C) Number of pBC17-transformed cells accumulating anthocyanin, reflecting cell viability, after co-bombardment with either an empty vector control or the HvSec61βa-RNAi construct on similarly sized pieces of leaf. Two days after bombardment, leaves were inoculated with Bgh, and the number of anthocyanin-accumulating cells was scored 3 days later (n = 4). FIGURE 1 | Silencing of HvSec61βa reduces susceptibility to Bgh without affecting plant cell viability. HvSec61β LOCALIZATION IN UNINFECTED AND INFECTED BARLEY CELLS Similar to the mCherry ER-luminal marker (Figure 3), the HvSec61βa-GFP signal was present in the ER network around the Bgh haustorial body as well (Figure 4). Contiguous accumulation of HvSec61βa-GFP was detected around the nucleus, which was FIGURE 3 |The SP-mCherry-HDEL ER marker localizes around the Bgh haustorial body. (A,B) Confocal images of infected barley epidermal cell 48 h after inoculation with Bgh. The fluorescent signal of SP-mCherry-HDEL (A) localizes to the ER and surrounds the haustorium inhomogeneously. No fluorescence signal of SP-mCherry-HDEL was observed along the haustorial fingers (arrow). The merged image (B) displays the haustorium structure in bright field, overlaid with the fluorescence signal. To visualize the ER tubules around the haustorial body, a 3D projection of a z-series of confocal images (C) was generated (Image Surfer 1.2). Scale bar, 10 μm. FIGURE 2 | HvSec61βa co-localizes with an ER luminal marker. (A) Maximum intensity projection of a z-series of confocal images of a barley epidermal cell expressing HvSec61βa-GFP reveals the ER localization of HvSec61βa-GFP with the typical distribution within the reticular ER network. (B) In the same epidermal cell, the 35S promoter-driven SP-mCherry-HDEL construct is expressed and labels the ER. (C) The merged image shows that the HvSec61βa-GFP and SP-mCherry-HDEL signals largely overlap. Scale bar, 20 μm. HvSec61βa (Figure 2A). In addition, the HvSec61βa-GFP sig- nal largely colocalized with mCherry signal from the luminal ER marker (Figures 2B,C). The colocalisation is near perfect in the tubular parts of the ER, while the cisternal parts have relatively more mCherry signal. This likely reflects that HvSec61βa-GFP is membrane bound, and that the soluble mCherry luminal marker dominates the more voluminous cisternal ER. In conclusion, our observations indicate that HvSec61βa is localized to all parts of the ER. HvSec61βa (Figure 2A). In addition, the HvSec61βa-GFP sig- nal largely colocalized with mCherry signal from the luminal ER marker (Figures 2B,C). The colocalisation is near perfect in the tubular parts of the ER, while the cisternal parts have relatively more mCherry signal. This likely reflects that HvSec61βa-GFP is membrane bound, and that the soluble mCherry luminal marker dominates the more voluminous cisternal ER. In conclusion, our observations indicate that HvSec61βa is localized to all parts of the ER. FIGURE 3 |The SP-mCherry-HDEL ER marker localizes around the Bgh haustorial body. (A,B) Confocal images of infected barley epidermal cell 48 h after inoculation with Bgh. HvSec61β LOCALIZATION IN UNINFECTED AND INFECTED BARLEY CELLS (A) Susceptibility after f i f l H S 6 RNAi d Ml FIGURE 1 | Silencing of HvSec61βa reduces susceptibility to Bgh without affecting plant cell viability. (A) Susceptibility after co-transformation of empty vector control, HvSec61βa-RNAi and Mlo-RNAi constructs with a GUS-reporter construct into barley epidermal cells, followed by inoculation with Bgh 3 days later. The relative susceptibility was calculated as described in Materials and Methods. Mlo-RNAi was used as a positive control. Error bars represent standard deviation (SD). **, P < 0.01 (Student’s t-test). (B) HvSec61βa-RNAi reduced the GFP signal originating from co-transformation of empty vector control, HvSec61βa-RNAi and Mlo-RNAi constructs with a GUS-reporter construct into barley epidermal cells, followed by inoculation with Bgh 3 days later. The relative susceptibility was calculated as described in Materials and Methods. Mlo-RNAi was used as a positive control. Error bars represent standard deviation (SD). **, P < 0.01 (Student’s t-test). (B) HvSec61βa-RNAi reduced the GFP signal originating from May 2013 | Volume 4 | Article 127 | 3 | 3 www.frontiersin.org Plant Sec61β in fungal pathogenicity Zhang et al. FIGURE 2 | HvSec61βa co-localizes with an ER luminal marker. (A) Maximum intensity projection of a z-series of confocal images of a barley epidermal cell expressing HvSec61βa-GFP reveals the ER localization of HvSec61βa-GFP with the typical distribution within the reticular ER network. (B) In the same epidermal cell, the 35S promoter-driven SP-mCherry-HDEL construct is expressed and labels the ER. (C) The merged image shows that the HvSec61βa-GFP and SP-mCherry-HDEL signals largely overlap. Scale bar, 20 μm. host cells re-localize organelles and specific proteins, which results in their accumulation at the pathogen attack site (Takemoto et al., 2003; Koh et al., 2005; Caillaud et al., 2012). We used the 35S promoter-driven SP-mCherry-HDEL construct to study the local- ization of the ER after attack by Bgh. Confocal imaging of an infected barley cell revealed that the mCherry ER-luminal marker was located around the body of the Bgh haustorium. Meanwhile, this ER marker was most often not present around the hausto- rial fingers (Figures 3A,B). In a 3D projection (Figure 3C) of the mCherry fluorescent signal, this distinction between the hausto- rial body and fingers is clearly visible. These observations revealed that the ER network is in close proximity to the EHM around the haustorial body. Frontiers in Plant Science | Plant-Microbe Interaction HvSec61β LOCALIZATION IN UNINFECTED AND INFECTED BARLEY CELLS The fluorescent signal of SP-mCherry-HDEL (A) localizes to the ER and surrounds the haustorium inhomogeneously. No fluorescence signal of SP-mCherry-HDEL was observed along the haustorial fingers (arrow). The merged image (B) displays the haustorium structure in bright field, overlaid with the fluorescence signal. To visualize the ER tubules around the haustorial body, a 3D projection of a z-series of confocal images (C) was generated (Image Surfer 1.2). Scale bar, 10 μm. Since we confirmed the ER localisation of HvSec61βa-GFP in barley and have observed increased resistance after silencing this gene, we were interested in knowing how the ER changes its location after pathogen attack. It is often described that infected Frontiers in Plant Science | Plant-Microbe Interaction May 2013 | Volume 4 | Article 127 | 4 Plant Sec61β in fungal pathogenicity Zhang et al. either is a negative regulator of defense or a susceptibility factor required for disease. Sec61β is, as described above, associated with protein-transmitting pores in the ER. While it has been barely studied in plants, yeast data suggest that one of its activities is to be part of a post-translational translocon complex, but that this role is not essential under non-stressed conditions (Finke et al., 1996). Furthermore, Sec61β has also been associated with protein retrotranslocation from the ER (Kawaguchi and Ng, 2007; Nakat- sukasa and Brodsky, 2008; Willer et al., 2008), and the question is, which of these activities is important in barley cells attacked by Bgh. observed close to the haustorium, supporting the re-localization of this organelle upon pathogen attack (Figures 4A–E), as pre- viously described (Schmelzer, 2002). As for the ER-luminal marker, HvSec61βa-GFP confirmed that the ER and EHM are in close proximity around the haustorial body. In summary, these confocal microscopy results suggest that the HvSec61βa- GFP-labeled ER is differentially recruited to the proximity of the EHM around the haustorial body, but not around the fingers. DISCUSSION An important chaperone that counter acts UPR is the ER- luminal protein, BiP, which is taken up post-translationally through the translocon complex in a Sec61β-dependent manner (Finke et al., 1996). Therefore, a model could be that HvSec61βa silencing causes ER-deprivation of BiP, in turn resulting in UPR as well as increased resistance. An Arabidopsis BiP knock-out line has previously been suggested to be prone for UPR. How- ever, in disagreement with the model, the BiP knock-out line had reduced resistance (Wang et al., 2005). This may indirectly sug- gest that reduced BiP import into the ER is not the cause of the Sec61βa phenotype we observe, while Bgh resistance increases in this situation. We therefore favor a function for Sec61βa in protein retrotranslocation in the interaction with the powdery mildew fungus. In the meantime, we had an indication of active recruitment of ER by the fungus, supporting that HvSec61βa functions as a susceptibility component. We observed a close association of the ER, labeled by HvSec61βa-GFP, and the Bgh haustorial body. The ER has also in other cases been found to be closely associated with haustoria (Koh et al., 2005; Micali et al., 2011). However, only Blumeria haustoria differentiate in two parts and provide a chance to distinguish variations in ER association. Interestingly, there is little ER association with the haustorial fingers, which could suggest that the ER proximity to the haustorial body is not due to ER being present wherever there is cytosol. Therefore, it is possible that the fungus controls the ER-haustorium associ- ation. Voegele et al. (2009) proposed that effector proteins are transferred to the cytosol via the ER. Effectors need to cross a membrane in order to reach the host cytosol, and the ER retro- translocon pore offers an escape route for this. The resistance phenotype seen after HvSec61βa silencing is in agreement with a model, where this protein is necessary for pore function. As illustrated in Figure 5, we suggest that vesicle trafficking trans- fers the effectors from the EHMx to the ER in order for them subsequently to employ the retrotranslocon to enter the cytosol. While we consider the model in Figure 5 to describe the most likely mode of action of Sec61βa in plant powdery mildew inter- actions, other scenarios are possible. DISCUSSION Silencing of HvSec61βa would result in inhibition of secretion if this protein is generally required for co- or post-translational The fact that silencing of HvSec61βa causes the barley cells to become resistant to powdery mildew suggests that HvSec61βa FIGURE 4 | HvSec61βa-GFP localizes around the Bgh haustorial body. Confocal image of an epidermal cell, transformed with the HvSec61βa-GFP construct, taken 48 h after Bgh inoculation. (A–C) Three different focal planes from an image series of an infected cell with a haustorium. HvSec61βa-GFP localizes to the ER around the nucleus (arrow head, A) and surrounds the haustorium in an ER-like tubular pattern (asterisk, A). (C–E) GFP fluorescence (C), bright field (BF) (D) and merged image (E) show HvSec61βa-GFP localization at the surface of the haustorial body. HvSec61βa-GFP labels the tubular ER network, which is further illustrated in the 3D projection (F) (Image Surfer 1.2). Scale bar, 10 μm. haustorium in an ER-like tubular pattern (asterisk, A). (C–E) GFP fluorescence (C), bright field (BF) (D) and merged image (E) show HvSec61βa-GFP localization at the surface of the haustorial body. HvSec61βa-GFP labels the tubular ER network, which is further illustrated in the 3D projection (F) (Image Surfer 1.2). Scale bar, 10 μm. FIGURE 4 | HvSec61βa-GFP localizes around the Bgh haustorial body. Confocal image of an epidermal cell, transformed with the HvSec61βa-GFP construct, taken 48 h after Bgh inoculation. (A–C) Three different focal planes from an image series of an infected cell with a haustorium. HvSec61βa-GFP localizes to the ER around the nucleus (arrow head, A) and surrounds the haustorium in an ER-like tubular pattern (asterisk, A). (C–E) GFP fluorescence (C), bright field (BF) (D) and merged image (E) show HvSec61βa-GFP localization at the surface of the haustorial body. HvSec61βa-GFP labels the tubular ER network, which is further illustrated in the 3D projection (F) (Image Surfer 1.2). Scale bar, 10 μm. May 2013 | Volume 4 | Article 127 | 5 | 5 www.frontiersin.org Plant Sec61β in fungal pathogenicity Zhang et al. protein translocation into the ER. This can hardly explain our phenotype, as inhibition of secretion in barley results in increased susceptibility to Bgh (Ostertag et al., 2013). A more likely expla- nation might be found in a specific HvSec61βa-function in post-translational translocation. This could involve the so-called “unfolded protein response” (UPR), which results from ER stress due to accumulation of unfolded proteins. DISCUSSION During UPR, ER chap- erones and components of the ERAD system are up-regulated to prevent the cell from undergoing programmed cell death (Travers et al., 2000). Similarly, ER stress induced by, e.g., tunicamycin (an N-glycosylation inhibitor) increases transcript levels of genes encoding proteins of the ER-QC machinery and the secretory pathway (Martinez and Chrispeels, 2003; Huttner and Strasser, 2012). Recently, a functional link has been established between UPR and pathogen defense in plants. Arabidopsis plants mutated in the IRE1a gene, encoding a key positive regulator of UPR, were found to have reduced resistance to bacteria (Moreno et al., 2012). An important chaperone that counter acts UPR is the ER- luminal protein, BiP, which is taken up post-translationally through the translocon complex in a Sec61β-dependent manner (Finke et al., 1996). Therefore, a model could be that HvSec61βa silencing causes ER-deprivation of BiP, in turn resulting in UPR as well as increased resistance. An Arabidopsis BiP knock-out line has previously been suggested to be prone for UPR. How- ever, in disagreement with the model, the BiP knock-out line had reduced resistance (Wang et al., 2005). This may indirectly sug- gest that reduced BiP import into the ER is not the cause of the Sec61βa phenotype we observe, while Bgh resistance increases in protein translocation into the ER. This can hardly explain our phenotype, as inhibition of secretion in barley results in increased susceptibility to Bgh (Ostertag et al., 2013). A more likely expla- nation might be found in a specific HvSec61βa-function in post-translational translocation. This could involve the so-called “unfolded protein response” (UPR), which results from ER stress due to accumulation of unfolded proteins. During UPR, ER chap- erones and components of the ERAD system are up-regulated to prevent the cell from undergoing programmed cell death (Travers et al., 2000). Similarly, ER stress induced by, e.g., tunicamycin (an N-glycosylation inhibitor) increases transcript levels of genes encoding proteins of the ER-QC machinery and the secretory pathway (Martinez and Chrispeels, 2003; Huttner and Strasser, 2012). Recently, a functional link has been established between UPR and pathogen defense in plants. Arabidopsis plants mutated in the IRE1a gene, encoding a key positive regulator of UPR, were found to have reduced resistance to bacteria (Moreno et al., 2012). REFERENCES S., Ha, E., McKeon, F., and Rapoport, T. A. (1999). A visual screen of a GFP- fusion library identifies a new type of nuclear envelope membrane protein. J. Cell Biol. 146, 29–44. Carvalho, P., Goder, V., and Rapoport, T. A. (2006). Distinct ubiquitin-ligase complexes define convergent path- ways for the degradation of ER pro- teins. Cell 126, 361–373. eds S. Perotto and F. Baluska (Berlin: Springer-Verlag), 183–212. Gomord, V., Denmat, L. A., Fitchette- Laine, A. 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DISCUSSION An unexpected function has for instance been described for Drosophila Sec61β, which is FIGURE 5 | Schematic model for a possible Sec61-dependent route of effector release into the host cytosol. Effectors are hypothesized to be transferred from the extrahaustorial matrix to the cytosol through Sec61 retrotranslocon pores in the ER. Trafficking from the matrix to the ER is envisaged to take place in vesicles dependent or independent of Golgi. Adapted from Voegele et al. (2009). retrotranslocon pores in the ER. Trafficking from the matrix to the ER is envisaged to take place in vesicles dependent or independent of Golgi. Adapted from Voegele et al. (2009). FIGURE 5 | Schematic model for a possible Sec61-dependent route of effector release into the host cytosol. Effectors are hypothesized to be transferred from the extrahaustorial matrix to the cytosol through Sec61 retrotranslocon pores in the ER. Trafficking from the matrix to the ER is envisaged to take place in vesicles dependent or independent of Golgi. Adapted from Voegele et al. (2009). May 2013 | Volume 4 | Article 127 | 6 Frontiers in Plant Science | Plant-Microbe Interaction | 6 Plant Sec61β in fungal pathogenicity Zhang et al. modification of the gene can be exploited for a disease resistance purpose. important for the secretion of the Gurken protein (Kelkar and Dobberstein, 2009). After silencing of Sec61β, Gurken left the ER as it normally did in control cells, but subsequently became stalled in the Golgi. Since a control protein still was observed to be secreted after silencing of Sec61β, this suggests that Sec61β is required for the Golgi-processing of a subset of the secreted proteins, including Gurken (Kelkar and Dobberstein, 2009). While this cannot be excluded to be due to a retrotranslo- con defect, it may indicate that Sec61β also has a function in secretion, which is unrelated to the Sec61 protein pore. Future work will determine which function makes Sec61β important for plant’s susceptibility to the powdery mildew fungus, and whether ACKNOWLEDGMENTS We are grateful to Dr. Patrick Schweizer for providing the pIPKTA30N vector, to Prof. Robert Dudler for providing the pBC17 construct and to Prof. Andreas Nebenführ for the 35S- SP-mCherry-HDEL construct. This work was supported by the Danish Strategic Research Council and The Faculty of Science, University of Copenhagen. Fluorescence imaging was performed using the facilities of the Center for Advanced Bioimaging (CAB) Denmark, University of Copenhagen. Feng, D., Marshburn, D., Jen, D., Wein- berg, R. J., Taylor, R. M. II, and Burette, A. (2007). Stepping into the third dimension. J. Neurosci. 27, 12757–12760. REFERENCES doi: 10.3389/fpls.2012.00067 Nakatsukasa, K., and Brodsky, J. L. (2008). The recognition and retro- translocation of misfolded proteins from the endoplasmic reticulum. Traffic 9, 861–870. Deng, W., Nickle, D. 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Induction of protein secretory pathway is required for systemic acquired resistance. Sci- ence 308, 1036–1040. This article was submitted to Frontiers in This article was submitted to Frontiers in Plant-Microbe Interaction, a specialty of Frontiers in Plant Science. This article was submitted to Frontiers in Plant-Microbe Interaction, a specialty of Travers, K. J., Patil, C. K., Wodicka, L., Lockhart, D. J., Weissman, J. S., and Walter, P. (2000). Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101, 249–258. Frontiers in Plant Science. Wang, Y. N., Yamaguchi, H., Huo, L., Du, Y., Lee, H. J., Lee, H. H., et al. (2010). The translo- con Sec61beta localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus. J. Biol. Chem. 285, 38720–38729. Zimmermann, R., Eyrisch, S., Ahmad, M., and Helms, V. (2011). Protein translocation across the ER mem- brane. Biochim. Biophys. Acta 1808, 912–924. Copyright © 2013 Zhang, Hanisch, Kwaaitaal, Pedersen and Thordal- Christensen. Conflict of Interest Statement: The authors declare that the research was Frontiers in Plant Science | Plant-Microbe Interaction May 2013 | Volume 4 | Article 127 | 8 REFERENCES This is an open-access arti- cle distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are cred- ited and subject to any copyright notices concerning any third-party graphics etc. Van den Berg, B., Clemons, W. M. Jr., Collinson, I., Modis, Y., Hartmann, E., Harrison, S. C., et al. (2004). X- ray structure of a protein-conducting channel. Nature 427, 36–44. Conflict of Interest Statement: The authors declare that the research was Whisson, S. C., Boevink, P. C., Moleleki, L., Avrova, A. O., Morales, J. May 2013 | Volume 4 | Article 127 | 8

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High-Dimensional Mixed-Frequency IV Regression

Journal of business & economic statistics

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∗Department of Economics, University of North Carolina at Chapel Hill – Gardner Hall, CB 3305 Chapel Hill, NC 27599-3305. Email: [email protected]. High-Dimensional Mixed-Frequency IV Regression Supplementary Material Andrii Babii∗ May 14, 2021 1 Additional proofs Proof of Theorem 3.2. The proof follows from the proof of Theorem 3.1 with additional complications related to the estimation of nuisance parameters. We first show that the estimation of nuisance parameters has a second-order asymptotic effect. To that end, first note that ˆrd −ˆKdβ = 1 T T X t=1 {Ut + τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)} n ˜Ψ(., Wt, Xt) + τ3(., Xt) −ˆτ3(., Xt) o ≜1 T T X t=1 Ut ˜Ψ(., Wt, Xt) + S1 + S2 with with S1 = 1 T T X t=1 n Ut(τ3(., Xt) −ˆτ3(., Xt)) + ˜Ψ(., Wt, Xt) (τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)) o , S2 = 1 T T X t=1 {τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)} {τ3(., Xt) −ˆτ3(., Xt)} . S2 = 1 T X t=1 {τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)} {τ3(., Xt) −ˆτ3(., Xt)} . By the Cauchy-Schwartz inequality By the Cauchy-Schwartz inequality By the Cauchy-Schwartz inequality |S2(u)|2 ≤1 T T X t=1 {τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)⟩}2 1 T T X t=1 {τ3(u, Xt) −ˆτ3(u, Xt)}2 . Integrating over u ∥S2∥2 ≲ 1 T T X t=1 {τ1(Xt) −ˆτ1(Xt)}2 + ∥ˆτ2(., Xt) −τ2(., Xt)∥2 ! 1 T T X t=1 ∥ˆτ3(., Xt) −τ3(., Xt)∥2 = oP(T −1), where the second line follows under Assumptions 3.3 (i) and (iii). Additional proofs Since E[Ut|Xt] = 0 and E[˜Ψ(., Wt, Xt)|Xt] = 0, conditionally on the sample used to compute ˆτk, k = 1, 2, 3, S1 is a Online Appendix - 1 Online Appendix - 1 sum of centered stochastic processes, whence sum of centered stochastic processes, whence E∥S1∥2 ≲E 1 T T X t=1 ˜Ψ(., Wt, Xt) (τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)) 2 + E 1 T T X t=1 Ut(τ3(., Xt) −ˆτ3(., Xt)) 2 = T −1E∥˜Ψ(., Wt, Xt) (τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)) ∥2 + T −1E∥Ut(τ3(., Xt) −ˆτ3(., Xt))∥2 ≲T −1 ∥ˆτ1 −τ1∥2 PX + ∥ˆτ2 −τ2∥2 λ⊗PX + ∥ˆτ3 −τ3∥2 λ⊗PX
= oP(T −1), E∥S1∥2 ≲E 1 T T X t=1 ˜Ψ(., Wt, Xt) (τ1(Xt) −ˆτ1(Xt) + ⟨β, ˆτ2(., Xt) −τ2(., Xt)) 2 + E 1 T T X t=1 Ut(τ3(., Xt) −ˆτ3(., Xt)) 2 1 ˜ 2 where the first equality follows under stationarity and the martingale difference sequence Assumptions 3.3 (i) and (ii), the next bound under Assumptions 3.3 (i), and the last line under Assumption 3.3 (iii). Second, decompose Second, decompose ˆrd −rd = 1 T T X t=1 (˜Yt + τ1(Xt) −ˆτ1(Xt))(˜Ψ(., Wt, Xt) + τ3(., Xt) −ˆτ3(., Xt)) −E[˜Yt ˜Ψ(., Wt, Xt)] ≜1 T T X t=1 ˜Yt ˜Ψ(., Wt, Xt) −E[˜Yt ˜Ψ(., Wt, Xt)] + Sr 1 + Sr 2 + Sr 3 with Sr 1 = 1 T T X t=1 ˜Ψ(., Wt, Xt)(τ1(Xt) −ˆτ1(Xt)), Sr 2 = 1 T T X t=1 ˜Yt(τ3(., Xt) −ˆτ3(., Xt)), Sr 3 = 1 T T X t=1 (τ1(Xt) −ˆτ1(Xt))(τ3(., Xt) −ˆτ3(., Xt)) Similarly, to the treatment of S2, by the Cauchy-Schwartz inequality ∥Sr 3∥2 ≤1 T T X t=1 (τ1(Xt) −ˆτ1(Xt))2 1 T T X t=1 ∥τ3(., Xt) −ˆτ3(., Xt)∥2 o (T −1) = oP(T −1), where the second line follows under Assumption 3.3 (i) and (iii). Similarly, to the treatment of S1, we also obtain E∥Sr 1+Sr 2∥2 = oP(T −1), where the expected value is taken conditionally on the sample used to estimate the nuisance parameters. Additional proofs Online Appendix - 2 Online Appendix - 2 Lastly, decompose Lastly, decompose (ˆkd −kd)φ = 1 T T X t=1 ( ˜Zt + τ2(., Xt) −ˆτ2(., Xt))(˜Ψ(., Wt, Xt) + τ3(., Xt) −ˆτ3(., Xt)) −E[ ˜Zt, ˜Ψ(., Wt, X ≜1 T T X t=1 ˜Zt ˜Ψ(., Wt, Xt) −E[⟨˜Zt, φ⟩˜Ψ(., Wt, Xt)] + SK 1 + SK 2 + SK 3 with with SK 1 = 1 T T X t=1 ˜Ψ(., Wt, Xt)(τ2(., Xt) −ˆτ2(., Xt)), SK 2 = 1 T T X t=1 ˜Zt(τ3(., Xt) −ˆτ3(., Xt)), SK 3 = 1 T T X t=1 (τ2(., Xt) −ˆτ2(., Xt))(τ3(., Xt) −ˆτ3(., Xt By the same argument as before, we can show that ∥SK 1 ∥2 = oP(T −1) and E∥SK 2 + SK 3 ∥2 = oP(T −1) conditionally on the sample used to estimate nuisance parameters. Therefore, conditionally, on the sample used to estimate nuisance parameters, the estimation of these components has asymptotically negligible effect, which also holds unconditionally, e.g., see Chernozhukov, Chetverikov, Demirer, Duflo, Hansen, Newey, and Robins (2018), Lemma 6.1. Therefore, the result follows from the same decomposition as in the proof of Theorem 3.1 with ˆr replaced by ˆrd and ˆK replaced by ˆKd. Proof of Theorem 3.4. Under maintained assumptions, by Theorem 3.3, ˆβm(s) −β(s) = 1 T T X t=1 Ut[(αI + K∗K)−1K∗Ψ(., Wt)](s) + RT(s), where ∥RT∥≲P 1 αT + αγ∧(1/2) √ αT + αγ + ∆κ m α3/2. Then under Assumption 3.4 (ii) ˆβm(s) −β(s) = 1 T T X t=1 Ut[(αI + K∗K)−1K∗Ψ(., Wt)](s) + RT(s), where ∥RT∥≲P 1 αT + αγ∧(1/2) √ αT + αγ + ∆κ m α3/2. Then under Assumption 3.4 (ii) where ∥RT∥≲P 1 αT + αγ∧(1/2) √ αT + αγ + ∆κ m α3/2. Then under Assumption 3.4 (ii) where ∥RT∥≲P 1 αT + αγ∧(1/2) √ αT + αγ + ∆κ m α3/2. Then under Assumption 3.4 (ii) ∥ˆβm −β∥2 = 1 T T X t=1 Ut(αI + K∗K)−1K∗Ψ(., Wt) 2 + 2 * 1 T T X t=1 Ut(αI + K∗K)−1K∗Ψ(., Wt), RT + + ∥RT∥2 ≜IT + IIT + oP  1 αT  . Additional proofs Online Appendix - 3 Online Appendix - 3 By the Cauchy-Schwartz inequality By the Cauchy-Schwartz inequality By the Cauchy-Schwartz inequality |IIT| = 2  * 1 T T X t=1 Ut[(αI + K∗K)−1K∗Ψ(., Wt)](s), RT + ≤2 1 T T X t=1 Ut[(αI + K∗K)−1K∗Ψ(., Wt)](s) ∥RT∥ ≲P 1 √ αT  1 αT + αγ∧(1/2) √ αT + αγ + ∆κ m α3/2  = oP  1 αT  , where the last line follows under Assumption 3.4 (ii) by Lemma A.1.1 since (UtΨ(., Wt))t∈Z is covariance stationary with absolutely summable covariances under Assumptions 3.1. Next, write αTIT ≤α∥(αI + K∗K)−1K∗∥2 1 √ T T X t=1 UtΨ(., Wt) 2 ≤1 4 1 √ T T X t=1 UtΨ(., Wt) 2 . (1994) Under Assumption 3.4 (i), by the Hilbert space CLT, see Politis and Romano (1994), Theorem 2.3, 1 √ T PT t=1 UtΨ(., Wt) converges in L2 to a zero-mean Gaussian process with covariance operator R. Therefore, by the Karhunen-Lo`eve decomposition 1 √ T T X t=1 UtΨ(., Wt) 2 d−→ X j≥1 λjZ2 j ; see Shorack and Wellner (2009), Chapter 5. see Shorack and Wellner (2009), Chapter 5. see Shorack and Wellner (2009), Chapter 5. see Shorack and Wellner (2009), Chapter 5. Online Appendix - 4 Online Appendix - 4 References Chernozhukov, V., D. Chetverikov, M. Demirer, E. Duflo, C. Hansen, W. Newey, and J. Robins (2018): “Double/debiased machine learning for treatment and structural parameters,” Econometrics Journal, 21(1), C1–C68. Politis, D. N., and J. P. Romano (1994): “Limit theorems for weakly dependent Hilbert space valued random variables with application to the stationary bootstrap,” Statistica Sinica, pp. 461–476. Shorack, G. R., and J. A. Wellner (2009): Empirical Processes with Applications to Statistics, vol. 59. SIAM. Online Appendix - 5 Online Appendix - 5

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An exploration of the experiences of professionals supporting patients approaching the end of life in medicines management at home. A qualitative study.

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An exploration of the experiences of professionals supporting patients approaching the end of life in medicines management at home. A qualitative study. leanor Wilson  (  [email protected] ) University of Nottingham School of Health Sciences https://orcid org/0000-0003-0419-5901 An exploration of the experiences of professionals supporting patients approaching the end of life in medicines management at home. A qualitative study. Eleanor Wilson  (  [email protected] ) University of Nottingham School of Health Sciences https://orcid org/0000-0003-0419-5901 An exploration of the experiences of professionals supporting patients approaching the end of life in medicines management at home. A qualitative study. An exploration of the experiences of professionals supporting patients approaching the end of life in medicines management at home. A qualitative study. Eleanor Wilson  (  [email protected] ) University of Nottingham School of Health Sciences https://orcid.org/0000-0003-0419-5901 Glenys Caswell  University of Nottingham School of Health Sciences Asam Latif  University of Nottingham School of Health Sciences Claire Anderson  University of Nottingham School of Pharmacy Christina Faull  LOROS Hospice Kristian Pollock  University of Nottingham School of Health Sciences Research article Keywords: Managing medication, healthcare professionals, end of life care, patients, qualitative research, pharmacy, dose administration aids, Posted Date: March 4th, 2020 DOI: https://doi.org/10.21203/rs.2.13207/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Palliative Care on May 11th 2020 See the published version at Eleanor Wilson  (  [email protected] ) University of Nottingham School of Health Sciences https://orcid.org/0000-0003-0419-5901 Background Professional focus on medicine prescribing and adherence has sometimes been accompanied by a lack of awareness of the concerns which patients frequently have about their medicines, and the burden and practical difficulties involved in taking them 1. Notenboom et al. 2 demonstrate some of the pragmatic challenges patients face when charged with managing medications in the home. Study participants cited 211 problems with using oral prescription medications, with 95% of participants identifying at least one issue. Participants reported employing 184 strategies to manage these practical problems. Seventeen percent of participants experienced a medication issue that led to clinical deterioration. The participants in the study identified a range of issues around reading and understanding the instructions for use (also see 3), difficulties handling packaging, and physical issues around taking medicines 2. In the UK guidelines have been developed for professionals that focus on involving patients and their family members in decisions about medications and prescribing 4. Alsaeed et al.’s 5 literature review highlights a number of areas in which healthcare professionals (HCPs) could help to improve patients’ medicines management at home. While the focus of the review was on patients with dementia and their caregivers, many of their recommendations are more broadly applicable. Good communication, between patient and family caregiver (FCG), between families and HCPs, and between HCPs is fundamental to identifying and managing any issues with medication 6. Providing clear information about medications and undertaking regular reviews were also central elements that promote and support medicines management 5 (also see 1). There are a number of dose administration aids [see Figure 1] and technologies available for patients and FCGs to support management and administration. Multi-compartment compliance aids can be extremely useful for some but do have a number of limitations 7, 8 and there is currently insufficient evidence to support their widespread use 4, 9, 10. Research into older age, including the impact of dementia, is contributing to our knowledge about the role patients and FCGs must take on in order to manage medications 1, 2, 11, 12. This work has identified that patients often have to cope with complex regimes and require considerable support from FCGs. Managing medications when someone is seriously ill and dying at home can generate additional issues. However, little attention has been paid to the context of palliative and end of life care. Research article n, healthcare professionals, end of life care, patients, qualitative research, pharmacy, dose administration aids, cense:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full Lic Version of Record: A version of this preprint was published at BMC Palliative Care on May 11th, 2020. See the published version at https://doi.org/10.1186/s12904-020-0537-z. Page 1/10 Page 1/10 Background As patients become increasingly unwell, they often require frequent and irregular changes to their medication regimes. This may include stopping some long-term preventative medicines such as statins, but increasing others such as those for pain and nausea [see Figure 2 on Polypharmacy and deprescribing]. Patients may begin to struggle with swallowing standard tablets and medications may be dispensed in other forms such as patches, liquids or sublingually. There is increasing evidence that family caregivers experience a number issues in adjusting doses and responding to rapid drug changes to control difficult and distressing symptoms in the period leading up to the patient’s death 13. Abstract Background: The management of medicines towards the end of life can place increasing burdens and responsibilities on patients and families. This has received little attention yet can be a source of great difficulty and distress patients and families. Dose administration aids can be useful for some patients but there is no evidence for their wide spread use or the implications for their use as patients become increasing unwell. The study aimed to explore how healthcare professionals describe the support they provide for patients to manage medications at home at end of life. Methods: Qualitative interview study with thematic analysis. Participants were a purposive sample of 40 community healthcare professionals (including GPs, pharmacists, and specialist palliative care and community nurses) from across two English counties. Results: Healthcare professionals reported a variety of ways in which they tried to support patients to take medications as prescribed. While the paper presents some solutions and strategies reported by professional respondents it was clear from both professional and patient/family caregiver accounts in the wider study that rather few professionals provided this kind of support. Standard solutions offered included: rationalising the number of medications; providing different formulations; explaining what medications were for and how best to take them. Dose administration aids were also regularly provided, and while useful for some, they posed a number of practical difficulties for palliative care. More challenging circumstances such as substance misuse and memory loss required more innovative strategies such as supporting ways to record medication taking; balancing restricted access to controlled drugs and appropriate pain management and supporting patient choice in medication use. Conclusions: The burdens and responsibilities of managing medicines at home for patients approaching the end of life has not been widely recognised or understood. This paper considers some of the strategies reported by professionals in the study, and points to the great potential for a more widely proactive stance in supporting patients and family carers to understand and take their medicines effectively. By adopting tailored, and sometimes, ‘outside the box’ thinking professionals can identify immediate, simple solutions to the problems patients and families experience with managing medicines. Methods Study design Study design Qualitative study underpinned by a social constructionist perspective involving semi-structured in-depth interviews. Interviews were situated accounts of HCPs’ perspectives and understanding. In their different roles HCPs engage with patients and families in a variety of ways, in clinics, pharmacy/community settings and in their own homes allowing them to identify practical day-to-day issues and offer support. This paper draws on a set of findings from a wider NIHR funded UK study to explore the management of medications for patients who are approaching end of life. We report on how HCPs described the way in which they supported patients and families to manage medications in their homes. Patient and family perspectives will be reported elsewhere. S tti The study is set in two English counties. Both are diverse in levels of affluence, age, ethnicity and population density, with both rural and urban areas. In both regions there are generalist and specialist teams providing palliative and end of life care. Participants Page 2/10 Participants included 40 community professionals from a range of roles, including: General practitioners (GP), specialist palliative care nurses, community nurses and both GP- and community-based pharmacists (see Table 1). Participants were recruited using purposive and snowballing techniques across the two counties via GP practices, specialist palliative care services and via email to a number of key contacts who were asked to distribute the information to their networks. Participants included 40 community professionals from a range of roles, including: General practitioners (GP), specialist palliative care nurses, community nurses and both GP- and community-based pharmacists (see Table 1). Participants were recruited using purposive and snowballing techniques across the two counties via GP practices, specialist palliative care services and via email to a number of key contacts who were asked to distribute the information to their networks. Results The majority of HCPs in this study acknowledged that patients did not always take medications as prescribed and many reported trying to explore how to resolve this with patients and families. Dosette boxes, and more commonly ‘blister packs’, were prepared by pharmacists, and often delivered directly to patients’ homes. These were offered and perceived as a problem solving intervention to help patients to organise and remember when to take their medicines. In a number of cases these boxes could be helpful and appropriate. However, HCPs recognised that some patients and families needed more comprehensive and innovative approaches. Here we discuss the strategies reported by HCP participants, including some who described ways of thinking ‘outside the box’ in order to help support patients overcome the practical difficulties of managing their medications in the home environment. Awareness of the challenges faced by patients and family caregivers was not evident in all professional accounts as is illustrated in this insight from a community nurse in her explanation of the underpinning issues: It is very difficult for patients and I think, as healthcare professionals, sometimes, we can be a little bit blasé about writing prescriptions and not realising… the impact that will have [for the patient] at home when they’re faced with their twenty pills in the morning, and they’ve got to work out which one to take off, and which one to put back on, and obviously, we produce changes, they get used to the little round white one then it turns blue, and they don’t know what’s going on. So, I think it’s very important that when we’re prescribing, we spend an appropriate amount of time making sure that people understand what we’ve given them, what it’s for, what we expect it to do, but also, think about, …you’re in the home environment  … you can check for the last six months’ stockpile that’s in the kitchen cupboard and things like that, to help people out. (HCP19_Community nurse) I’ve got a lady at the minute …, she’s probably on twenty three medications. You go in [to her house and look at her medication boxes] ‘oh great, they’re taking all the medication’, and then you look on the floor, and you see there’s pills all over the place. So, I don’t delude myself that any of them are taking medications as prescribed. Analysis All interviews were transcribed verbatim and fully anonymised before transcripts were uploaded to Nvivo12© and coded. Using a process of thematic analysis EW, GC and KP reviewed codes and undertook condensing and sorting. The legitimacy of categories was reviewed with the project steering group and considered in the context of the data collection methods. Several transcripts were also read and coded by our PPI co-applicant (Alan Caswell) for an additional perspective, transparency and comparison. Each transcript was coded by two team members and three were triple coded. Once coding was complete, nodes were further interrogated and refined to generate node summaries of key themes illustrated by quotations from transcripts. A number of key findings emerged during this process. These have also been compared with patient, family caregiver and bereaved family caregiver finding as part of the wider study. In June 2019 findings were presented at two workshops for HCPs (some of whom participated in the interviews) for feedback and comment. This paper focuses on the ways in which HCPs reported supporting patients and families to manage medications in the home. Data collection and recording Interviews took place between June 2017 and October 2018. The majority of participants were interviewed individually at their place of work and during work hours. However, we were receptive to workload constraints and participant preferences so undertook four interviews over the telephone, one joint interview, and three group interviews (involving three or more participants). Interviews lasted between 19 and 69 minutes. All participants gave written or, in the case of telephone interviews, verbal consent. An interview schedule was used as a guide allowing the flexibility to tailor and adapt each interview to the participant and their responses. All interviews were audio recorded and notes written up after the interview took place. The focus of the interviews was to explore what HCPs saw the issues to be for patients and families managing medications at home as the patient approached the end of life, and how they sought to support such patients and families in managing their medications. HCPs were asked to think of a current or recent case which had posed issues for medicines management or where this had worked well. They were then asked to situate this case within their normal practice to establish how common or rare particular issues might be. The interview guide can be found in Appendix A. All interviews were conducted by EW, GC and KP. All have considerable training and experience in sensitive topics, qualitative methods and palliative and end of life care. Results (HCP05_Community Nurse) As the GP in the following quote indicates, a simple response of writing out a guide to a patient’s medication was not something they had done before nor intended to do again, despite the apparent benefits for the patient. They also note that the patient had to present to them in some considerable distress to prompt this option: So she came to see me in a complete sort of meltdown once, being just very upset and ... didn’t have any sort of memory about how to take her medications at all … that was just due to being very worked up I think rather than anything else going on. And I wrote down, I gave her a little sheet that said take this at this time in this dose basically, typed that out for her. And …she doesn’t want to give up her independence, it’s very, very important to her, so not being able to manage her own medications is for her a disaster… Yeah, she brought it back and showed me and said I’m using this now thank you. I’ve never done that for anyone else before. …That’s the only person I’ve ever done that for and it was only because she was so distressed by it and it is this unique set of circumstances. (HCP25_GP) Effectiveness and challenges of well established solutions to medicines management: Page 3/10 Dosettes and blister packs were often seen as an effective strategy in eliminating any confusion about what tablets to take when. HCPs viewed them as an important tool and appropriate way to support patient’s wishes to continue to manage at home independently. A few respondents observed that this system was sometimes put in place for the convenience of paid Home Care Workers enlisted to prompt patients to take medication in the home, rather than the patient. However, this could potentially undermine agency and competence, detaching patients from the process by ‘making people further removed from taking responsibility of their own medication’ (HCP05_Community nurse). They also made it difficult, if not impossible, for patients to identify individual tablets. Dosette boxes and blister pack were recognised to pose two key issues for palliative care. One is that they do not accommodate all types of medication. Consequently, patients might have to take additional medications, such as liquids or tablets prescribed to be taken ‘as needed’, alongside the allocated pills from the box. Results Secondly, box contents are usually made up for a month, and cannot be easily altered if medication is changed during this period. HCPs also identified potential problems with pain medications where patients who are unaware of, or unable to identify the contents, may risk taking more pain relief than they need if they are also taking ‘as needed’ doses on top of those included in their dosette box. And I think actually pain does vary day to day in a patient, what they do or are not doing. And I’m very hesitant actually to put it in dosette boxes as well for that reason. And also, if you do get side effects, you get more drowsy for any other reasons, you really want to reduce this quickly and not taking the next dose and so forth. So you don’t have this variety, because in a dosette box I can’t, most of the time, see which of the pills is which, let alone the patient, and you can’t take it out and then say, ‘Ooh I think I’d better miss that because I’m feeling drowsy’. (HCP25_GP) Some healthcare professionals recognised the limitations of the pre-prepared dosette box for patients at the end of life and the differing needs of individual patients. Reviewing and rationalising medications was often seen as the first step to supporting effective medicines management. HCPs, especially nurses making home visits, recognised that a number of issues with medications stemmed from patients not fully understanding what their medications were, what they were for, and how they should be taken. However, they also noted despite the involvement of multiple health professionals few took responsibility for overarching management of medications and many often did not ask the right questions or investigate the underlying causes of patients’ issues with medication: I suppose we always think that the pharmacists are doing a lot of the explaining and a lot of the discussion … [but] I’ve never really had that conversation with a pharmacist and saying, Actually, do you just give them the Morphine, or do you tell them anything else? (HCP11_GP) He was like a rabbit in the headlights, he didn’t know what to do, he’d just got all these tablets there, he didn’t know what to do, he didn’t understand what half of them were for. Results So his way was to not take them, …and people were going along the lines of, ‘oh those, those medicines haven’t worked, let’s try something else’, but actually, those medicines had never been taken. …And nobody had taken the time to discuss it with him. (HCP14_Specialist Palliative Care nurse) This was a particular issue when changes were made to medication regimes, as is common in end of life care. For these issues, taking some time to explain, and implementing a simple memory aid system to support the explanation, could be extremely effective. So I give [out] a lot of laminated prompt cards, that just lays out, you know,’ your aspirin is being taken for this, it should be taken with food in the morning’, and then they can follow that. And that actually helps way more than a dosette (HCP21_Pharmacist) HCPs also reported using items such as lockable tins for patients who could not safely manage their own medications, or where there was potential for misuse or misappropriation of medication, and dispensers with timing devices or alarms for those with impaired memory. Some reported changing the route of administration for a patient when swallowing tablets became an issue or when the number of tablets added to the burden of medicines management. One community nurse felt alternative administration routes also supported adherence as they did not hold the same negative connotations as ‘pills’ and ‘tablets’. Yet HCPs also noted that patients and families did not always know that medication came in different formulations or that they could request this. Actually, a lot of relatives and patients aren’t aware they can change the form of the medication and have something that’s either dispersible or in liquid form. …as I’m doing my nursing assessment, I’ll talk about medication as one of the things I’m discussing. And, so I’m saying, ‘Are you managing to swallow your medication okay?’  At that point, unless they were asked a direct question, patients don’t often flag it up to you, so, largely, ‘no’. ‘It takes me half an hour in the morning, to swallow one tablet’. And they don’t realise, actually something simple like putting it in some yogurt and having it as a bolus, might help it, help swallow it, or they won’t realise that, actually, that type of medication, you can have in a different form and have it in a syrup. Discussion Patients living at home at the end of life often have complex medication regimes with large numbers of medications, different routes of administration, and high doses of controlled drugs. In line with the international literature, the HCPs participating in this study recognised the burdens of polypharmacy at end of life 15, 17, 20-22, but did not always seem to acknowledge how the burdens of medication management added to the ‘work’ patients and FCG’s were undertaking when someone is seriously ill in the home environment 23. FCGs are also more likely to be older, and have reduced abilities and co-morbidities of their own 24. Until recently much of the literature on medication management focused on ‘adherence’ and ‘compliance’ and the implication for health outcomes 5, 25-28. There is now a small but growing body of work that recognises the difficulties for patients 2, their desire to limit the amount of medications they take as far as possible 17, 29, 30 and the burdens regimes can place on FCGs when a patient is dying at home 13, 21, 31. Indeed, the international literature indicates that in other countries, particularly the United States and Australia, FCGs are regularly undertaking even more advanced tasks in administering sub-cutaneous medications prescribed for end of life symptoms 32-36. While there have been some moves towards this in the UK the feasibility of this approach is still being assessed 37, 38. This paper has explored ways in which HCPs in a UK study reported on well established solutions to medicines management and more innovative ‘outside the box’ thinking in order to support patients and families with medication needs. Some expressed resignation that patients often failed to, could not or did not want to take their medications as prescribed. Not all HCPs recognise that a strategy of verbal explanation on its own was not always effective in helping patients to understand and manage their medications, particularly at the end of life. Accounts, particularly from specialist palliative care nurses, noted that when multiple health professionals were involved there was little coordination or designated responsibility for medications management. HCPs in this study often focused on the prescription element of the process, aiming to ensure appropriate symptom control by undertaking simple changes to regimes and routes of administration. However, while these may present as obvious and standard initial options not all HCPs in the study thought to implement them. Results (HCP06_Community nurse)  ( ) Thinking outside the (dosette) box Participants described cases where they had needed to take a more innovative and active role in supporting the patient to manage medications. Participants described cases where they had needed to take a more innovative and active role in supporting the I had a gentleman who couldn’t read or write, …and he lived on his own, what we ended up doing with him, with managing his medicines, was taking one of the medicines, [sticky] taping it to a piece of paper and saying this one, and then writing next to it, like, how many times a day to take it, so three dashes …. you learn little ways of, often, you try something, that doesn’t work so you try something else, so it is tricky. (HCP14_Community nurse) In a small number of reported instances, HCPs recognised that their input was needed on a more consistent basis to support medication use. A few HCPs reported telephoning patients to remind them of recent changes to their medications. One HCP narrated an instance with a patient with memory issues who was repeatedly coming to hospital with heart failure symptoms because he was not remembering to take his medication. They subsequently rationalised this to be taken once a day and arranged for a community nurse to visit daily to prompt this administration. However, lack of time to adequately discuss and monitor medicines and related issues was acknowledge to be a limitation. This level of input was unlikely to be routinely available and the HCP who recounted this experience also noted that this was an ‘extreme’ response that could not be support for long. Page 4/10 Page 4/10 Tailored solutions were particularly required in difficult and unusual situations, for example where patients or someone in their household were affected by phobias, addictions, substance abuse or the effects of dementia. One palliative care nurse described how she and her colleagues were working to find constructive and creative ways to manage pain for a patient who was determined to die at home. As the patient, his FCG and their circle of friends had issues with substance misuse, safeguards were put in place to limit the amounts of morphine they could access at any one time. Results We just introduced the patch last week, he was using a lot of the Oramorph, … he’s used the Oramorph less since the patch has gone on. …We always put dates on the bottle and the box. So we can see which bottle he’s still using and which box, and keep an eye on it as well. He knows we do that. (HCP07_Specialist Palliative Care nurse) This HCP also recalled a patient who expressed a fear of needles and refused any injectable medications. In the quote below the HCP described trying to balance respecting the patient’s choice and agency and providing effective care: We can’t use needles. So we are limited in what medication he can have to manage his symptoms anyway. We’ve got patches … And we’ve got buccal things that we can use to manage as much as we can, but obviously, it doesn’t give us the range that we would normally have. (HCP07_Specialist Palliative Care nurse) Recognition, and support for, patient choices about their treatment and care was a strong theme throughout the interviews. A number of HCPs described the considerable effort they were prepared to make to enable these, regardless of whether they considered them to be wise options. Recognition, and support for, patient choices about their treatment and care was a strong theme throughout the considerable effort they were prepared to make to enable these, regardless of whether they considered them to So she’d developed swallowing issues. And so we tried liquid medication, she didn’t like it. … So she went back to taking oral tablets. … And then I can think about an incident, it was interesting, at the hospice, where I got a phone call from the staff nurse saying the carers had said that she’d had a choking episode following taking tablets in the morning, and that, at the hospice, they weren’t going to be prepared to give her tablets any longer. … I said, ‘This isn’t an issue, this lady has capacity to make the decision, she doesn’t want to take liquid medication, she wants to take her tablets, the risks have been explained to her’. (HCP05_Community nurse) There were few references to pharmacists taking on roles to support medicines management or to other HCPs identifying the potential for greater pharmacy integration in the healthcare team. Declarations Ethics approval and consent to participate: Approval was granted in March 2017, by East Midlands, Derby Research Ethics Committee - 17/EM/0091. R&D approvals for each Trust were also acquired. All participants gave written or, in the case of telephone interviews, verbal consent. Consent for publication: All participants gave written or, in the case of telephone interviews, verbal consent to undertake the interviews and for anonymised sections to be used for publication and dissemination. Availability of data and materials: The datasets generated and/or analysed during the current study are not publicly available as they are personal accounts of patients’, families’ and healthcare professional’s experiences. It is essential that we maintain confidentiality and anonymity but sufficiently anonymised parts of the data are available from the corresponding author on reasonable request. Competing interests: The authors declare that there is no conflicts of interest. Funding: This work is supported by the National Institute of Health Research (Health Services & Delivery Research project: 15/70/101). Disclaimer: The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. isclaimer: The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Dep Authors’ contributions: EW, GC and KP were involved in all aspects of data collection, analysis, drafting the article and revising it critically for important intellectual content. KP also designed the study. AL, CA, and CF are part of the study team and have all been involved in the development of the article, revising it critically for intellectual content. All authors have approved the version to be published. Acknowledgements: We would like to acknowledge PPI co-applicant Alan Caswell for his overarching contributions to the study and for contributing to the analysis of the interview data in this paper. Authors’ information: Dr. Eleanor Wilson is a Senior Research Fellow with a background in medical social anthropology. She has considerable experience of research in palliative and end of life care and undertook data collection and analysis for the study. Dr. Glenys Caswell is a sociologist with particularly interest in death and dying. She worked as a Senior Research Fellow on the study, undertaking data collection and analysis. Dr. Asam Latif is a Pharmacist and Senior Research Fellow. Claire Anderson is a Professor of Social Pharmacy. List Of Abbreviations HCP – Healthcare professional FCG – Family caregiver HCP – Healthcare professional FCG – Family caregiver FCG – Family caregiver Limitations A strength of the study is that it represents the views of a wide range of HCPs, including pharmacists, working in diverse community healthcare services and geographic locations. However, the study location in two adjacent English counties means that the findings may not be typical of other settings. It is also important to note that 17/40 participants were considered to be specialists in palliative care and 4/40 condition specific specialists. As such, we would expect them to manage a more complex caseload and be able to implement a wider range of ways to support patients in managing their medications. We recognise that as self-selected participants, they are likely to have an interest and specialist expertise in the management of medications. Interviews may involve accounts in which respondents are likely to be motivated to give a good account of their practice. We are unable to know how this compares with what they actually do in practice. However, data from patients and FCGs in other parts of the study suggest that proactivity by HCPs is far from routine and especially in the last weeks and days of life when families are confronted with real anxieties about the management of medications. Conclusion If we move the discourse forward from one of compliance and adherence and recognise the reasonable, and often practical, difficulties patients and FCG face when managing medications we can look at ways in which HCPs can help them to overcome these issues. Medication reviews, reduction of problematic polypharmacy and the use of dose administration aids such as dosette boxes are some of the established ways in which HCPs can support patients and families in medicines management at the end of life. However, there may be times when HCPs need to ‘think outside the box’ in order to identify and support patients to safely, effectively and independently manage their medications. Discussion This increases risk of adverse events, lack of effective symptom management, and instability of some medications when stored outside their original packs. These types of issues add to the challenge of optimising pain control in the home environment 47, 48. This paper has demonstrated that there is scope for much greater understanding of the reality of patient and FCG experience, the difficulties they face, and the potential for HCPs to engage with innovative and tailored ways of supporting medicines management for seriously ill patients being cared for and dying at home. Our findings further echo those of current literature in that the majority of participants rarely recognised the current or potential contribution of pharmacists as part of the palliative care team 49-54. Discussion These adaptations often made a considerable difference to patients’ ability to manage their medicines effectively. In other instances, HCPs reported having to be quite innovative, and explore a number of routes to help patients overcome the practical problems involved in organising and taking their medicines effectively. While not aimed specifically at palliative care, current UK guidance 18, 39 advocates for professionals to take account of individual patient needs, preferences for treatment, health priorities and lifestyle and recognise that ‘medicines are likely to be just one aspect of a person’s care’ 39. However, to date there has been little work to explore ways of supporting patients and families in medicines management in order to make their lives less stressful 4, 40. The ability to maintain accurate medicine taking could be a critical factor in avoiding unscheduled hospital admissions and determining whether a patient could remain living at home 41-43. This paper seeks to extend beyond the compliance/adherence discourse 44 to recognise the reasonable difficulties patients and families often face in managing complex regimes and how HCPs play a proactive role in helping them overcome these 5. HCPs in this study often reported encouraging the use of simple dose administration aids such as dosette boxes and blister packs. These can be extremely useful and can support patients to remain at home by helping them to manage their own medication. They can also be a useful tool in supporting Home Care Workers to safely prompt and administer medications in patients’ homes 7-9, 45. Conversely, these aids can have negative implications for a person’s independence and agency Page 5/10 Page 5/10 when they disempower patients by removing responsibility for, and understanding of, their own medications 46. They also have practical disadvantages when, as is common in end of life care, medications need to be changed rapidly and frequently 7. This increases risk of adverse events, lack of effective symptom management, and instability of some medications when stored outside their original packs. These types of issues add to the challenge of optimising pain control in the home environment 47, 48. when they disempower patients by removing responsibility for, and understanding of, their own medications 46. They also have practical disadvantages when, as is common in end of life care, medications need to be changed rapidly and frequently 7. Declarations Both are part of the wider study team and have supported the study throughout. Christina Page 6/10 Page 6/10 Faull is a Consultant in Palliative Medicine, working at LOROS Hospice in Leicester, she actively supported recruitment to the study and is part of the wider study team. Kristian Pollock is Professor of Medical Sociology and PI for the study. She designed study and has be involved in all aspects. Faull is a Consultant in Palliative Medicine, working at LOROS Hospice in Leicester, she actively supported recruitment to the study and is part of the wider study team. 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Caring for the Dying Adult: NICE guideline. London: NICE, 2015. http://www.nice.org.uk/guidance/ng31/resources/care-of-dying-adults-in-the-last-days-of-life-1837387324357 41. National Institute for Health and Care Excellence. Caring for the Dying Adult: NICE guideline. London: NICE, 2015. http://www.nice.org.uk/guidance/ng31/resources/care-of-dying-adults-in-the-last-days-of-life-1837387324357 http://www.nice.org.uk/guidance/ng31/resources/care-of-dying-adults-in-the-last-days-of-life-1837387324357 http://www.nice.org.uk/guidance/ng31/resources/care-of-dying-adults-in-the-last-days-of-life-1837387324357 42. Department of Health. End of Life Care Strategy - Promoting high quality care for all adults at end of life. Department of Health, London 2008. http://www.cpa.org.uk/cpa/End_of_Life_Care_Strategy.pdf 43. Gomes B, Calanzani N and Higginson I. Local preferences and place of death in regions within England 2010. Cicely Saunders Institute, 2011. http://www.endoflifecare-intelligence.org.uk/resources/publications/lp_and_place_of_death 43. Gomes B, Calanzani N and Higginson I. Local preferences and place of death in regions within England 2010. Cicely Saunders Institute, 2011. http://www.endoflifecare-intelligence.org.uk/resources/publications/lp_and_place_of_death 4. Brown MT and Bussell JK. Medication Adherence: WHO Cares? Mayo Clinic Proceedings. 2011; 86: 304-14. 45. Duerden M. What is the place for monitored dosage systems? Drug and Therapeutics Bulletin. 2018; 56: 102-6. 5. Duerden M. What is the place for monitored dosage systems? Drug and Therapeutics Bulletin. 2018; 56: 102-6. 6. Nunney J and Raynor D. How are multi-compartment compliance aids used in primary care? The Phamaceutic 47. Gardiner C, Gott M, Ingleton C, Hughes P, Winslow M and Bennett MI. Attitudes of Health Care Professionals to Opioid Prescribing in End-of-Life Care: A Qualitative Focus Group Study. 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References Journal of the American Geriatrics Society. 2007; 55: 590-5. 21. Sheehy-Skeffington B, McLean S, Bramwell M, O'Leary N and O'Gorman A. Caregivers Experiences of Managing Medications for Palliative Care Patients at the End of Life: A Qualitative Study. American Journal of Hospice & Palliative Medicine. 2014; 31: 148-54. 22. Morin L, Vetrano DL, Rizzuto D, Calderón-Larrañaga A, Fastbom J and Johnell K. Choosing Wisely? Measuring the Burden of Medications in Older Adults near the End of Life: Nationwide, Longitudinal Cohort Study. The American Journal of Medicine. 2017; 130: 927-36.e9. 23. May CR, Eton DT, Boehmer K, et al. Rethinking the patient: using Burden of Treatment Theory to understand the changing dynamics of illness. BMC Health Services Research. 2014; 14: 281. 24. Alhusein N, Macaden L, Smith A, et al. 'Has she seen me?': a multiple methods study of the pharmaceutical care needs of older people with sensory impairment in Scotland. BMJ Open. 2018; 8: e023198. 5. Hughes CM. Medication Non-Adherence in the Elderly: How Big is the Problem? [Review]. Drugs & Aging. 2004; 26. Gellad WF, Grenard JL and Marcum ZA. A Systematic Review of Barriers to Medication Adherence in the Elderly: Looking Beyond Cost and Regimen Complexity. The American journal of geriatric pharmacotherapy. 2011; 9: 11-23. Page 7/10 Page 7/10 27. Shin J, Habermann B and Pretzer-Aboff I. Challenges and strategies of medication adherence in Parkinson's disease: A qualitative study. Geriatric Nursing. 2015; 36: 192-6. Looking at medication adherence: An evidence review. British Journal of Community Nursing. 2017; 22: 485-7. 28. Chapman S. Looking at medication adherence: An evidence review. British Journal of Community Nursing. 28. Chapman S. Looking at medication adherence: An evidence review. British Journal of Community Nursing. 2017; 22: 485-7. 29. Pound P, Britten N, Morgan M, et al. Resisting medicines: a synthesis of qualitative studies of medicine taking. Social Science & Medicine. 2005; 61: 133- 55. 30. Fallsberg M. Reflections on medicines and medication: A qualitative analysis among people on long term drug regiments. Sweden, Linkoping University: Swedish Pharmaceutical Press, 1991. 31. Payne S, Turner M, Seamark D, et al. Managing end of life medications at home—accounts of bereaved family carers: a qualitative interview study. BMJ Supportive & Palliative Care. 2015; 5: 181-8. alik D. Palliative care at home: Carers and medication management. Palliative & Supportive Care. 2008; 6: 349-56 2. Anderson BA and Kralik D. Palliative care at home: Carers and medication management. Role Number of participants Specialist palliative care nurses* 15 Community nurses** 12 Consultants (palliative medicine, gerontology) 2 Pharmacists 4 GPs 7 Total40 *Specialist palliative care nurses – those working at a Band 6 or 7 level with additional training in palliative care. These may include: Clinical Nurse Specialists/Hospice at home nurses – these nurses provide advice and support to patients and families as well as having expertise in the management of symptoms. They may be assigned to work in patients’ homes or with care homes. Macmillan Nurses – some specialist palliative care nurses are badged under the brand of ‘Macmillan Cancer Support’, a national cancer charity. These nurses focus on providing advice for the management of complex symptoms and/or psychological distress. **This is an overarching group of nurses working in the community including District nurses, Community Matrons and Clinical Nurse Specialists in non-palliative specialisms such as heart failure and neurological conditions. In the UK, qualified nurses’ start at Band 5 and go up to Band 8. Nurses at Band 6 have advanced training, skills and experience; those at Band 7 have managerial roles in addition. Figure 2 Polypharmacy and deprescribing Table Table 1: Participants by profession Page 8/10 Figure 1 Dose administration aids COREQChecklistBMC.pdf WP1HCPinterviewguidev104.01.2017.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 9/10 COREQChecklistBMC.pdf WP1HCPinterviewguidev104.01.2017.pdf Page 10/10

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Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain – day–night differences in the chromophores and optical properties

Atmospheric chemistry and physics

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Yuquan Gong1,2, Ru-Jin Huang1,2,3, Lu Yang1,2, Ting Wang1, Wei Yuan1,2, Wei Xu1, Wenjuan Cao1, Yang Wang4,5, and Yongjie Li6 1State Key Laboratory of Loess and Quaternary Geology, CAS Center for Excellence in Quaternary Science and Global Change, Institute of Earth Environment, Chinese Academy of Sciences, 710061 Xi’an, China 2University of Chinese Academy of Sciences, Beijing 100049, China 3Institute of Global Environmental Change, Xi’an Jiaotong University, Xi’an 710049, China 4School of Geographical Sciences, Hebei Normal University, Shijiazhuang, China 5State Key Joint Laboratory of Environmental Simulation and Pollution Control, Beijing, China 6Department of Civil and Environmental Engineering, Faculty of Science and Technology, University of Macau, Taipa, Macau SAR 999078, China Correspondence: Ru-Jin Huang ([email protected]) Correspondence: Ru-Jin Huang ([email protected]) Received: 3 January 2023 – Discussion started: 2 March 2023 Revised: 21 July 2023 – Accepted: 16 October 2023 – Published: 14 December 2023 Correspondence: Ru-Jin Huang ([email protected]) Received: 3 January 2023 – Discussion started: 2 March 2023 Revised: 21 July 2023 – Accepted: 16 October 2023 – Published: 14 December 2023 Abstract. Brown carbon (BrC) aerosol is light-absorbing organic carbon that affects radiative forcing and atmo- spheric photochemistry. The BrC chromophoric composition and its linkage to optical properties at the molecular level, however, are still not well characterized. In this study, we investigate the day–night differences in the chro- mophoric composition (38 species) and optical properties of water-soluble and water-insoluble BrC fractions (WS-BrC and WIS-BrC) in aerosol samples collected in Shijiazhuang, one of the most polluted cities in China. We found that the light absorption contribution of WS-BrC to total BrC at 365 nm was higher during the day (62 ± 8 %) than during the night (47 ± 26 %), which is in line with the difference in chromophoric polarity between daytime (more polar nitrated aromatics) and nighttime (more less-polar polycyclic aromatic hydrocar- bons, PAHs). The high polarity and water solubility of BrC in the daytime suggests the enhanced contribution of secondary formation to BrC during the day. There was a decrease in the mass absorption efficiency of BrC from nighttime to daytime (2.88 ± 0.24 vs. 2.58 ± 0.14 for WS-BrC and 1.43 ± 0.83 vs. 1.02 ± 0.49 m2 g C−1 for WIS-BrC, respectively). Large polycyclic aromatic hydrocarbons (PAHs) with four- to six-ring PAHs and nitrophenols contributed to 76.7 % of the total light absorption between 300–420 nm at nighttime, while nitro- catechols and two- to three-ring oxygenated PAHs accounted for 52.6 % of the total light absorption during the day. Measurement report Atmos. Chem. Phys., 23, 15197–15207, 2023 https://doi.org/10.5194/acp-23-15197-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain – day–night differences in the chromophores and optical properties Yuquan Gong1,2, Ru-Jin Huang1,2,3, Lu Yang1,2, Ting Wang1, Wei Yuan1,2, Wei Xu1, Wenjuan Cao1, Yang Wang4,5, and Yongjie Li6 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain 15198 et al., 2018; Kasthuriarachchi et al., 2020; Yuan et al., 2021) and the diurnal variation of WS-BrC in fluorescence and in- organic fractions (Deng et al., 2022; Zhan et al., 2022; Li et al., 2021); however, the research of BrC chemical com- position on day–night differences is scarce. In this study, the optical properties and chemical composition of WS-BrC and WIS-BrC in both daytime and nighttime PM2.5 samples collected in Shijiazhuang, one of the most heavily polluted cities in the Beijing–Tianjin–Hebei region, were analyzed us- ing a high-performance liquid chromatography (HPLC) sys- tem equipped with a photodiode array (PDA) detector and a high-resolution Orbitrap mass spectrometer (HRMS). The PM2.5 samples were collected in Shijiazhuang, one of the most heavily polluted cities in the Beijing–Tianjin–Hebei re- gion. Besides, the relationship between the concentration and light-absorbing contributions of the BrC subgroups was ana- lyzed. The object of this study is to investigate the day–night differences in the optical properties and chromophore com- position of BrC and to explore the effect of primary emis- sions and atmospheric processes on the light absorption and chemical composition of BrC. 2.1 Sample collection Day and night PM2.5 samples were collected on the quartz- fiber filters (8 × 10 in., Whatman, QM-A; filters prebaked at 750 ◦C, over 3 h) through a high-volume air sampler (Hi- Vol PM2.5 sampler, Tisch, velocity of flow ∼1.03 m3 min−1, Cleveland, OH) from 17 January to 13 February 2014. Day- time samples were collected from 08:30 to 18:30 (∼10 h), and nighttime samples are collected from 18:30 to the next day at 08:30 (∼14 h). After collection, the samples were stored in a freezer (−20 ◦C) until analysis. The sampling site was located on the rooftop of a building (∼15 m above ground) in the Institute of Genetics and Developmental Bi- ology, Chinese Academy of Sciences (38.2◦N, 114.3◦E), which is surrounded by a residential–business mixed zone. 1 Introduction Light-absorbing organic carbon aerosols, also termed brown carbon (BrC) aerosol, are ubiquitous in the atmosphere (Iinuma et al., 2010; Yuan et al., 2016; Huang et al., 2021). Growing evidence has shown that BrC can reduce atmo- spheric visibility, affect atmospheric photochemistry, and change the regional and global radiation balance (Kirchstet- ter et al., 2004; Laskin et al., 2015; Hammer et al., 2016). Besides, some components in BrC, such as polycyclic aro- matic hydrocarbons (PAHs), are highly toxic and carcino- genic, which can adversely impact human health (Alcanzare, 2006; Zhang et al., 2009; Huang et al., 2014). The extent of these effects is closely related to the optical properties and chemical composition of BrC, which are still not well under- stood. BrC is often classified into water-soluble (WS-BrC) and water-insoluble (WIS-BrC) fractions because these two frac- tions are largely different in chemical composition and light absorption. For example, abundant nitrophenols were de- tected in WS-BrC, while polycyclic aromatic hydrocarbons (PAHs) were the main component of WIS-BrC (Huang et al., 2018, 2020). The difference in BrC chemical composition is associated with the emission sources. For example, methyl nitrocatechols are specific to biomass burning, while PAHs are mainly emitted by fossil fuel combustion (Kitanovski et al., 2012; Dat and Chang, 2017). Atmospheric oxidation can further complicate the BrC chromophores dynamically, lead- ing to light-absorbing enhancement or bleaching. For exam- ple, Li et al. (2020) reported that the mass absorption effi- ciency (MAE) of some nitroaromatic compounds (e.g., nitro- catechols) from biomass burning can be enhanced by about 2–3 times by oxidation to generate secondary chromophores. Yet, prolonged photo-oxidation reactions (exposure to sun- light for few hours) of these nitroaromatic can generate small fragment molecules (e.g., malonic acid, glyoxylic acid) and rapidly reduce the particle absorption (Hems and Abbatt, 2018; Y. Wang et al., 2019; Li et al., 2020). The complexity in composition and sources, as well as the dynamics in their at- mospheric processing, limits our understanding in BrC chro- mophores and their links to light absorption. Yuquan Gong1,2, Ru-Jin Huang1,2,3, Lu Yang1,2, Ting Wang1, Wei Yuan1,2, Wei Xu1, Wenjuan Cao1, Yang Wang4,5, and Yongjie Li6 The total mass concentrations of the identified chromophores showed larger day–night difference during the low-pollution period (day-to-night ratio of 4.3) than during the high-pollution period (day-to-night ratio of 1.8). The large day–night difference in BrC composition and absorption, therefore, should be considered when estimating the sources, atmospheric processes, and impacts of BrC. Published by Copernicus Publications on behalf of the European Geosciences Union. Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain 15199 sure reliable absorbance measurements (absorbance between 0.2 and 0.8 at 300 nm in this study), the filtrate was diluted with appropriate folds before absorption spectra measure- ments. In this study, the light absorption of WIS-BrC is ob- tained from MS-BrC minus WS-BrC. As shown in Fig. S1, the summed absorbance of WS-BrC and WIS-BrC is very close to the absorbance of MS-BrC (difference less than 5 %). Therefore, the interferences of solvent and pH on the measurement of WIS-BrC should be very limited. The pH of the water extracts was not adjusted because highly diluted water extracts were used to measure the light absorption, and little change in pH was observed for water extracts of differ- ent samples. The light absorption coefficient (Abs) and ab- sorption data were calculated following the equation known as the absorption Ångström exponent, which depends on the types of chromophores in the solution. In this study, AAE was calculated by linear regression of log10 Absλ ver- sus log10λ at 300–400 nm. The MAE values of authentic standards including nitro- phenols, PAHs, and OPAHs (MAES,λ) at a wavelength of λ were calculated by the following equation (Laskin et al., 2015): MAES, λ = Aλ −A700 l × C ln(10), (4) (4) where C (µg mL−1) is the concentration of the standards in the extracts. Absλ = (Aλ −A700) Vl Vb × l × ln(10), (1) 2.3 BrC chemical composition analysis (1) The main chromophores in WS-BrC and WIS-BrC were identified by the HPLC–PDA–HRMS platform (Thermo Electron, Inc.), and the details are presented in our previ- ous study (Huang et al., 2020). Firstly, the filter samples (3.5 ∼48.3 cm2) were ultrasonically extracted with 6 mL of the ultrapure water for 30 min and repeated two times. The extracts were filtered through a PVDF filter (0.45 µm) to re- move insoluble materials. Then the solution was subjected to a solid phase extraction (SPE) cartridge (Oasis HLB, USA) to remove water-soluble inorganic salt ions. On the other hand, the residual filters were dried and the WIS-BrC frac- tions were further extracted two times with 6 mL of methanol for 30 min to extract the WIS-BrC fractions. Afterward, the extracts of WS-BrC and WIS-BrC chromophores were dried with a gentle stream of nitrogen and then redissolved in 150 µL of ultrapure water and methanol. where Absλ (Mm−1) represents the sample absorption coef- ficient at a wavelength of λ, Aλ is the absorbance recorded (random wavelength), and A700 is for explaining baseline drift as the reference during data analysis. To account for baseline drift that may occur during analysis, absorption at all wavelengths below 700 nm is referenced to that at 700 nm, where there is no absorption for BrC extracts. Vl (mL) is the total volume of solvent (water or methanol) used to extract the quartz-fiber filters; Vb (m3) is the volume of the air sam- pled through the filter punch; l (0.94 m) is the optical path length of the UV–VIS spectrophotometer; and ln (10) is the absorption coefficient with base e, which is the natural loga- rithm using the logarithm conversion with the base 10. where Absλ (Mm−1) represents the sample absorption coef- ficient at a wavelength of λ, Aλ is the absorbance recorded (random wavelength), and A700 is for explaining baseline drift as the reference during data analysis. To account for baseline drift that may occur during analysis, absorption at all wavelengths below 700 nm is referenced to that at 700 nm, where there is no absorption for BrC extracts. https://doi.org/10.5194/acp-23-15197-2023 2.3 BrC chemical composition analysis Vl (mL) is the total volume of solvent (water or methanol) used to extract the quartz-fiber filters; Vb (m3) is the volume of the air sam- pled through the filter punch; l (0.94 m) is the optical path length of the UV–VIS spectrophotometer; and ln (10) is the absorption coefficient with base e, which is the natural loga- rithm using the logarithm conversion with the base 10. The mass absorption efficiency (MAE) of the filter extracts at a wavelength of λ can be defined as The BrC factions were analyzed by an HPLC–PDA– HRMS platform (including the Dionex UltiMate system and the high-resolution Q Exactive Plus hybrid quadrupole– Orbitrap mass spectrometer). Here, the extracts were loaded onto an Accucore RP-MS column (Thermo Scientific) by the binary solvent with an aqueous solution containing 0.1 % formic acid and a methanol solution containing 0.1 % formic acid as mobile phases L1 and L2, eluting at a flow rate of 0.3 mL min−1. The process of gradient elution was set as fol- lows: the concentration of L2 was held at 15 % at the begin- ning and then linearly increased to 30 % from 0 to 15 min, linearly increased to 90 % from 15 to 45 min, held at 90 % from 45 to 50 min, and then decreased to 15 % from 50 to 52 min and held there for 60 min. The spectrometer was equipped with an electrospray ionization (ESI) source and details about the operation parameters are similar to those of previous studies (Liu et al., 2016; Huang et al., 2020). Briefly, the mass spectra of different BrC fractions in Shi- jiazhuang were acquired in both negative (ESI (−)) and pos- itive (ESI (+)) modes in the mass range between m/z 100 and 800. Strongly polar aromatic hydrocarbons like nitro- phenol and carboxylic acid are preferentially ionized in ESI (–) mode (Lin et al., 2017). OPAHs and nitrogen heterocyclic MAEλ = Absλ/COM, (2) (2) 2.2 Light absorption measurement In recent years, a growing number of studies have investi- gated the chromophore composition of BrC and found that nitrophenols, low-ring acids/alcohols, PAHs, and carbonyl oxygenated PAHs (OPAHs) were the major chromophores in BrC (Teich et al., 2017; Yuan et al., 2020; Huang et al., 2020). Some chromophores in BrC can be generated from both pri- mary emission and secondary formation. For example, 4- nitrophenol and 4-nitrocatechol can be emitted directly from biomass burning and can also be generated through photo- oxidation reactions (Kitanovski et al., 2012; Yuan et al., 2020). The differences in emission sources or atmospheric oxidation conditions have a significant effect on the chemical composition of BrC chromophores. Previous studies mainly focused on seasonal variations of BrC chromophores (Wang A portion filter (about 0.526 cm2 punch) was taken from collected samples and sonicated for 30 min in 10 mL of ul- trapure water (>18.2 M) or methanol (J. T. Baker, HPLC grade), and then the extracts WS-BrC and methanol-soluble BrC (MS-BrC) were obtained. The extracts were filtered with a 0.45 µm polyvinylidene fluoride (PVDF; water-soluble) or polytetrafluoroethylene (PTFE; water-insoluble) pore sy- ringe filter to remove insoluble substances. The light ab- sorption spectra of the filtrate were tested using a UV–VIS spectrophotometer (Ocean Optics) over the range from 250 to 700 nm, equipped with a liquid waveguide capillary cell (LWCC-3100, World Precision Instruments, Sarasota, FL, USA), following the method of Hecobian et al. (2010). To en- Atmos. Chem. Phys., 23, 15197–15207, 2023 https://doi.org/10.5194/acp-23-15197-2023 3.1 Optical properties of BrC during the day and night For example, the AAE and MAE365 of BrC emitted from biomass burning (AAE ∼7.31 and MAE365 ∼1.01 m2 g C−1, respectively) (Siemens et al., 2022) showed large differences compared to that from vehi- cle emissions (AAE ∼10.5 and MAE365 ∼0.32 m2 g C−1) (Xue et al., 2018). Besides, photochemical oxidation of fresh BrC from coal combustion resulted in considerable changes in AAE and MAE365; e.g., the AAE and MAE365 of fresh coal combustion emission are 7.2 and 0.84 ± 0.54 m2 g C−1, Figure 1. (a) Day–night absorption spectra (Abs, in the wavelength range of 300–500 nm), mass absorption efficiency (MAE, deter- mined at 365 nm), and absorption Ångström exponent (AAE, cal- culated between 300 and 400 nm) of water-soluble/insoluble BrC (WS-/WIS-BrC) in Shijiazhuang. (b) Light-absorbing proportion of WS-BrC and WIS-BrC between 300 and 500 nm. PAHs were quantified in ESI (+) mode, while PAHs were de- tected by PDA spectroscopic analysis due to their super low ionization efficiency in ESI. The absorption spectra of chro- mophores were measured by a PDA detector in the wave- length range of 190–700 nm. The results of this study were corrected using a blank. The elemental composition of individual chromatographic peaks was assigned with the molecular formula calcula- tor in Xcalibur 4.0 software using a mass tolerance of ±3 ppm, and the maximum numbers of atoms for the for- mula calculator were set as 30 12C, 60 1H, 15 16O, 3 14N, 1 32S, and 1 23Na. To eliminate the chemically unreasonable formulas, the identified formulas were constrained by set- ting 0.3 ≤H / C ≤3.0, 0.0 ≤O / C ≤3.0, 0.0 ≤N / C ≤0.5, and 0.0 ≤S / C ≤0.2 in ESI mode and 0.3 ≤H / C ≤3.0, 0.0 ≤O / C ≤3.0, 0.0 ≤N / C ≤1.3, and 0.0 ≤S / C ≤0.8 in ESI+ mode, as suggested in a previous study (Lin et al., 2012). Further, the calculated neutral molecular formulas that did not fit the nitrogen rule were excluded. In total, 20 WS-BrC chromophores (two quinolines, four two- to three-ring OPAHs, four nitrocatechols, six nitrophenols, and four aromatic alcohols and acids) and 18 WIS-BrC chro- mophores (three four-ring OPAHs and 15 PAHs) were iden- tified, and their concentrations were quantified with authen- tic standards (28 species) or surrogates (10 species) (see Ta- ble S1). 3.1 Optical properties of BrC during the day and night Figure 1a shows the average absorption spectra of WS- BrC and WIS-BrC at the wavelength range between 300 and 500 nm during the day and the night. It can be seen that the light absorption of both WS-BrC and WIS-BrC sharply increased toward the short wavelength. The average absorbance of WS-BrC is 46.04 ± 35.92 Mm−1 (at 365 nm) during the day, which is higher than at night (35.68 ± 35.50 Mm−1). However, the light absorption of WIS-BrC at 365 nm is much lower during the night (27.90±24.80 Mm−1) than during the day (40.89 ± 23.42 Mm−1). The day–night differences of light absorption of WS-BrC and WIS-BrC in- dicate the difference in water solubility and polarity of the chromophores. The average AAE of WS-BrC (AAEWS-BrC) and WIS-BrC (AAEWIS-BrC) during the day are 5.10 ± 0.28 and 6.36 ± 0.45, respectively, which are lower than those at night (5.51 ± 0.40 and 6.97 ± 0.80, respectively). Note that during both the day and night the AAEWS-BrC is lower than AAEWIS-BrC, which is different from findings in previous studies (see Table S2). For example, Huang et al. (2020) found that the AAEWS-BrC was higher (8.2 ± 1.0 and 8.2 ± 1.0 in Beijing and Xi’an, respectively) than that of AAEWIS-BrC (5.7 ± 0.2 and 5.4 ± 0.2 in Beijing and Xi’an, respectively). Besides, MAE365 values of WS-BrC (2.88 ± 0.24 and 2.58±0.14 m2 g C−1) are 2.0 and 2.5 times those of WIS-BrC (1.43 ± 0.83 and 1.02 ± 0.49 m2 g C−1) during the day and night, respectively. For example, the MAE365 val- ues of WS-BrC (1.22 ± 0.11 and 1.00 ± 0.18 m2 g C−1) are 0.7 and 0.5 times those of WIS-BrC (1.66±0.48 and 1.82± 1.06 m2 g C−1) in winter for Beijing (Cheng et al., 2016) and Xi’ an (Li et al., 2020), respectively. This result indicates that the chemical composition of BrC in the most polluted city, Shijiazhuang, is different from other urban areas on primary sources and secondary aging process. However, both WS- BrC and WIS-BrC have higher MAE365 and average AAE values during the day than the night. This suggests that the day–night differences of AAE and MAE365 of BrC fractions are likely associated with the different primary emissions and atmospheric aging processes (Cheng et al., 2016; Q. Wang et al., 2019; Wang et al., 2020). Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain tified by PDA spectroscopic analysis due to their super low ionization efficiency in ESI (see Table S1). Figure 1. (a) Day–night absorption spectra (Abs, in the wavelength range of 300–500 nm), mass absorption efficiency (MAE, deter- mined at 365 nm), and absorption Ångström exponent (AAE, cal- culated between 300 and 400 nm) of water-soluble/insoluble BrC (WS-/WIS-BrC) in Shijiazhuang. (b) Light-absorbing proportion of WS-BrC and WIS-BrC between 300 and 500 nm. MAEλ = Absλ/COM, where COM(µg m−3) stands for the concentration of water- soluble organic carbon (WSOC) or methanol-soluble organic carbon (MSOC). The concentrations of WSOC were mea- sured with a total-organic-carbon–total-nitrogen (TOC–TN) analyzer (TOC-L, Shimadzu, Japan). The concentration of organic carbon (OC) was measured by a thermal–optical car- bon analyzer (DRI, model 2001) with the IMPROVE A pro- tocol (Chow et al., 2011). Note that MSOC is usually re- placed with OC because previous studies have shown that methanol has a high extraction efficiency (∼90 %) for OC. But it is difficult to completely extract the OC by methanol (Chen and Bond, 2010; Cheng et al., 2016; Xie et al., 2019). Here, water-insoluble organic carbon (WISOC) is obtained from MSOC minus WSOC. The wavelength dependence for light absorption by chro- mophores in solution can be characterized by a power law equation: Absλ = K · λ−AAE, (3) Absλ = K · λ−AAE, (3) where K is the fitting parameter of the extracts which is con- stant related to the chromophoric concentration, and AAE is Atmos. Chem. Phys., 23, 15197–15207, 2023 https://doi.org/10.5194/acp-23-15197-2023 3.1 Optical properties of BrC during the day and night Therein, the WS-BrC chromophores, benzanthrone (21#) and benzo[b]fluoren-11-one (22#), were quantified by mass spectrometry analysis in either negative or positive ESI mode, while the rest of WIS-BrC chromophores were quan- Atmos. Chem. Phys., 23, 15197–15207, 2023 https://doi.org/10.5194/acp-23-15197-2023 15201 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain which is much higher than those in aged samples (6.4 and 0.14 ± 0.08 m2 g C−1, respectively) (Ni et al., 2021). et al., 2020; Chen et al., 2021). Our previous study has found that the emitted organic aerosols from coal combustion had a clear increase at midnight in Shijiazhuang (Huang et al., 2019; Lin et al., 2020). Thus, the large contribution of ni- trophenols and four- to six-ring PAHs to total mass concen- tration at night may be impacted by emissions from the coal combustion. Figure 1b shows the light absorption contributions of WS- BrC and WIS-BrC to total BrC over the wavelength range of 300–500 nm. It is obvious that the absorption contribution of WS-BrC is increased from 53.8 % at 300 nm to 87.4 % at 500 nm during the day and from 38.4 % to 61.5 % during the night. The higher absorption contributions of WS-BrC at longer wavelengths during the day compared to that of the night may be related to photo-oxidation reaction in the day- time (Y. Wang et al., 2019; Chen et al., 2021). The absorption contribution of WS-BrC accounts for 62 ± 8 % of the total BrC absorption at 365 nm during the day but only 47 ± 8 % during the night. The large difference in BrC light absorp- tion between samples from the day and those from the night observed in this study is comparable with previous studies (Shen et al., 2019; Li et al., 2020) and indicates the signifi- cant day–night difference in chemical composition. To investigate the source of the BrC chromophores, the mass concentrations (these concentrations of chromophores are OC normalized) of the day and night were compared. The day-to-night ratios of identified BrC compounds in mass con- centrations are shown in Fig. 3. It can be seen that the aver- age day-to-night ratios of WS-BrC chromophores are 4.87 for quinolines, 3.49 for two- to three-ring OPAHs, 3.47 for nitrocatechols, 0.48 for nitrophenols, and 2.53 for aromatic alcohols/acids, respectively. 3.2 Composition and absorption contribution of BrC during the day and night In total, 38 major chromophores were quantified in WS- BrC and WIS-BrC with HPLC–PDA–HRMS analysis, and the concentrations of these chromophores are shown in Ta- ble S3. According to the characteristics of the molecular structures and absorption spectra, these chromophores are di- vided into 10 subgroups, including two quinolines, four ni- trocatechols, six nitrophenols, four aromatic alcohols/acids, four two- to three-ring OPAHs, three four-ring OPAHs, two three-ring PAHs, four four-ring PAHs, five five-ring PAHs, and four six-ring PAHs. Detailed information about these chromophores is listed in Table S4. Figure 2 shows the chem- ical composition of the identified BrC components during the day and night. The total concentration of these chromophores during the day (169.8 ng m−3) is similar to that at night (171.8 ng m−3), and the chemical composition of the BrC subgroups is clearly different between the day and night. For example, nitrocatechols, aromatic alcohols/acids, and two- to three-ring OPAHs are the major contributors to the total mass concentration of identified BrC chromophores during the day (accounting for 23.3 %, 22.3 %, and 16.6 %, respectively). These BrC chromophores, however, are the minor compo- nents during the night (accounting for 12.1 %, 15.6 %, and 6.9 %, respectively). This result indicates the enhanced for- mation of these chromophores during the day. On the con- trary, the relative contributions of nitrophenols and four- to six-ring PAHs are much lower during the day (15.3 % and 15.2 %, respectively) than those during the night (35.8 % and 24.0 %, respectively). During the night, 4-nitrophenol (4NP) contributes 24.4 % of the total concentration, followed by 2- methyl-4-nitrophenol, fluoranthene, and chrysene (2M4NP 4.7 %, FLU 4.6 %, CHR 4.6 %, respectively). The higher contributions of nitrophenols and four- to six-ring PAHs at night are likely caused by enhanced primary emissions (Lin The average day-to-night ratio (∼0.48) of nitrophenols is smaller than 1, and this result is similar to previous stud- ies (Yuan et al., 2016; Schnitzler and Abbatt, 2018). Al- though both nitrophenols and nitrocatechols can be emitted from biomass burning, they show largely different day–night variation patterns. The higher concentrations of nitrocate- chols during daytime indicate enhanced secondary forma- tion, which is similar to the results observed in urban Beijing (Cheng et al., 2021). In addition, previous studies found that emissions from residential coal-fired heating are significant sources of nitrophenols (Wang et al., 2018; Lu et al., 2019). 3.1 Optical properties of BrC during the day and night Previous studies have found that quinolines are important products of fossil fuel combustion and were used as tracers of the vehicular exhaust (Banerjee and Zare, 2015; Xue et al., 2018; Lyu et al., 2019). Thus, the higher day-to-night ratio of quinolines may be due to in- creased primary emissions from vehicles during the day. Ni- trophenols and vanillin are typical biomass burning tracers for atmospheric aerosols (Harrison et al., 2005; Scaramboni et al., 2015; Huang et al., 2021). Previous studies have iden- tified secondary formation as an important source of phthalic acid (PA) and methyl-nitrocatechols (Chow et al., 2015; Zhang and Hatakeyama, 2016; Liu et al., 2017). In this study, vanillin, phthalic acid, and three methyl-nitrocatechols (in- cluding 4M5NC, 3M6NC, and 3M5NC) isomers have high day-to-night ratios (4.16, 3.75, and 3.28, respectively). The high day-to-night ratios of these BrC chromophores suggest that biomass burning and secondary formation likely play important roles in the daytime source of BrC. 15202 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain 15202 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain 15202 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain 15202 Figure 2. Mass fraction of the identified BrC chromophores during the day and night (details of the identified BrC chromophores are shown in Table S1). he identified BrC chromophores during the day and night (details of the identified BrC chromophores are show Figure 2. Mass fraction of the identified BrC chromophores during the day and night (details of the identified BrC chromophores are shown in Table S1). cal emissions may be responsible for the majority of four- to six-ring PAHs during the night. et al., 2005; Wang et al., 2020). High absorbance of nitro- phenols at night is closely related to their higher mass frac- tion at night. The absorption characteristics of four- to six- ring PAHs are significantly different from the nitrophenols, and their absorption per unit mass is larger than that of nitro- phenols. The much higher per-unit-mass absorbance of PAHs than the low-ring aromatic hydrocarbons (e.g., aromatic alco- hols/acids) is due to their strongly conjugated systems. It is worth noting that the absorption contributions of some BrC compounds (including quinolines, aromatic alcohols/acids, four-ring OPAHs, three-ring PAHs four subgroups) are much lower than those of the above-mentioned BrC compounds be- cause of their lower mass concentration or light absorption coefficient. Figure S2 shows the light absorption contributions of the BrC subgroups to total BrC subgroups in the wave- length range between 300 and 420 nm (the absorptions above 420 nm are too low to exactly estimate the contributions), ex- hibiting large day–night differences. For example, quinolines show evident absorption below 340 nm (3.6 % at 310 nm dur- ing the day) but negligible contribution above 360 nm. Nitro- phenols exhibit a maximum contribution at about 350 nm, while nitrocatechols show higher absorption in the wave- length range from 360 to 400 nm. For PAHs, the absorption maxima shift to a longer wavelength with the increase in the aromatic rings (e.g., 320 nm for four-ring PAHs and 400 nm for six-ring PAHs). Overall, the combined light absorption contributions of nitrophenols, nitrocatechols, and PAHs are 86.5 % and 80.1 % (averaged between 300 and 420 nm) at night and during the day, respectively. 15202 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain This result is similar to previous studies in which PAHs and nitroaromatic com- pounds were identified as the major chromophores (Huang et al., 2020; Yuan et al., 2020). 3.2 Composition and absorption contribution of BrC during the day and night The higher concentrations of nitrophenols during nighttime, however, suggest that they are mainly emitted from primary emission sources such as residential heating during winter in North China. Compared with the WS-BrC chromophores (the day-to-night ratio >2.53), the day-to-night ratios of the WIS-BrC chromophores approach or are below 1, with av- erage ratios of 1.46 for three-ring PAHs, 1.34 for four-ring OPAHs, 0.74 for four-ring PAHs, 0.91 for five-ring PAHs, and 0.79 for six-ring PAHs. A number of studies showed that coal combustion was the dominant source of PAHs (Wang et al., 2018; Xie et al., 2019; Yuan et al., 2020). Thus, the lo- https://doi.org/10.5194/acp-23-15197-2023 Atmos. Chem. Phys., 23, 15197–15207, 2023 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain 15203 Figure 3. Day-to-night ratios of the concentrations of different BrC chromophores. Figure 3. Day-to-night ratios of the concentrations of different BrC chromophores. Figure 3. Day-to-night ratios of the concentrations of different BrC chromophores. dation. Nitrophenols exhibit a higher mass fractional contri- bution during the night than the day, indicating a significant contribution from primary emissions (Lu et al., 2019; Lin et al., 2020). Besides, previous investigations have shown that NOx concentrations and relative humidity are higher at night in Shijiazhuang, which may have accelerated the formation of nitrophenols in the dark (Yuan et al., 2016; Huang et al., 2019). This result exhibits a clear day–night difference dur- ing the low-pollution period compared to the high-pollution period, which indicates that the low-pollution period is easily influenced by the external environment (e.g., solar radiation and wind speed). high-pollution periods. In the high-pollution period, how- ever, the mass concentration of quinolines is much lower than other BrC chromophores, and there is no evident difference between day and night. The mass fraction of aromatic alco- hols/acids during the day (35.4 %) is much higher than during the night (12.0 %) in the low-pollution period. For the high- pollution period, the mass fraction of aromatic alcohols/acids shows little difference between the day and night. How- ever, their mass concentration during the day (55.5 ng m−3) is higher than that during the night (31.9 ng m−3). Therein, the mass concentration of phthalic acid (a tracer from pho- tochemical oxidation) contributes more than 60 % to the aro- matic alcohols/acids during the day for the low- and high- pollution period (Zhang and Hatakeyama, 2016). This evi- dence may suggest that there is stronger photo-chemical oxi- dation for aromatic alcohols/acids during the day, especially in the low-pollution period. The day–night light absorption contribution of WS-BrC and WIS-BrC chromophores in different pollution periods is shown in Fig. 4b. For the low-pollution period, the light ab- sorption contribution of the 10 BrC subgroups shows a large difference during the day and night. Therein, the WS-BrC chromophores (e.g., quinolines, nitrophenols, and nitrocat- echols) are the main contributor (accounting for ∼75 % at 365 nm) of total identified BrC during the day. While, the WIS-BrC chromophores (e.g., four- to six-ring PAHs) be- come an abundance contributor (accounting for ∼65 % at 365 nm) during the night. https://doi.org/10.5194/acp-23-15197-2023 3.3 Comparisons between the low- and high-pollution period The relative contributions of day–night subgroups of BrC chromophores in light absorption and mass concentration were further investigated for different pollution levels. The sampling campaign was classified into a low-pollution period (PM2.5<150 µg m−3) and a high-pollution period (PM2.5>250 µg m−3). Figure 4a shows the mass fractional contributions of the identified subgroups during these peri- ods, which show an evidently different composition during day and night. For example, the mass fraction of quinolines during the day (∼24.1 %) is much higher than during the night (3.4 %) in the low-pollution period, which may be re- lated to increased vehicle emissions during the day (Rogge et al., 1993; Lyu et al., 2019). Moreover, during the low- pollution period, with good atmospheric dispersion condi- tions during the day, the fractional concentration of BrC is only 56.9 ng m−3, which is much lower than the nights and The light absorption contribution of these BrC subgroups exhibits obvious day–night differences. For example, the ab- sorption contribution of two- to three-ring OPAHs and nitro- catechols at 365 nm increased by ∼2.0 and ∼3.5 times dur- ing the day compared to that during the night (see Fig. S3). This result differs from previous studies (Kampf et al., 2012; Gao et al., 2022), which indicated that light absorption of BrC compounds were enhanced after exposure to photo- oxidation. On the other hand, the absorption contributions of nitrophenols and four- to six-ring PAHs at 365 nm are ∼1.6 times and ∼2.2 times higher at night than during the day, respectively. The day–night difference of light absorption of nitrophenols is comparable with previous studies (Harrison Atmos. Chem. Phys., 23, 15197–15207, 2023 https://doi.org/10.5194/acp-23-15197-2023 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain 15204 Figure 4. Day–night fractional contributions of mass concentrations (a) and light absorption (b) of the 10 BrC subgroups in the low-pollution period and high-pollution period. Here the BrC chromophore is the main chromophore substance that has been identified. In panel (b) WS- BrC is below the dotted line and WIS-BrC is above the dotted line. Figure 4. Day–night fractional contributions of mass concentrations (a) and light absorption (b) of the 10 BrC subgroups in the low-pollution period and high-pollution period. Here the BrC chromophore is the main chromophore substance that has been identified. In panel (b) WS- BrC is below the dotted line and WIS-BrC is above the dotted line. from residential heating) as well as the dynamic develop- ment of planetary boundary layer height. Moreover, these day–night differences are largely affected by the air pollution level, which determines the concentrations of BrC precursors (e.g., aromatic hydrocarbon and phenols) and oxidants (e.g., NOx, NO q 3, and OH), as well as meteorological conditions (e.g., solar irradiation and RH) (Liu et al., 2012; Laskin et al., 2015; Wang et al., 2019). For example, our results found that the day–night difference of BrC fractions is more pro- nounced in chemical composition and light absorption dur- ing the low-pollution period than the high-pollution period. These factors may show different effects on the formation and photobleaching of different types of the identified chro- mophores. However, our current understanding of the forma- tion mechanisms of and influencing factors on these iden- tified chromophores is still incomplete (Huang et al., 2018; Yuan et al., 2020). Therefore, a combination of more labo- ratory and field studies is needed to (1) make a comprehen- sive characterization of the chromophore composition BrC in ambient aerosol and (2) explore thoroughly the formation mechanisms of different types of BrC chromophore. This will significantly enhance our understanding of atmospheric BrC formation mechanisms and therefore improve the accu- racy of the atmospheric effects of BrC in air quality and cli- mate models. or formation mechanisms between the day and night. Our results show a significant day–night difference in mass con- tributions and absorption contributions of BrC components at different pollution levels. This suggests that the variation of BrC chromophores in different pollution periods may be caused by different sources and weather conditions. Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain There is an obvious day–night dif- ference in light absorption in the low-pollution period, which is consistent with the difference in their mass concentration contribution. Different from the low-pollution period, the light absorption contribution of the total WS-BrC and WIS- BrC chromophores showed no significant day–night differ- ences during the high-pollution period. However, the absorp- tion contributions of subgroups in WS-BrC chromophores have a significant day–night difference (e.g., nitrocatechol and nitrophenols) during the high-pollution period, which is due to the changes in the mass contributions. WS-BrC chro- mophores have stronger light absorption during the day and night compared to the WIS-BrC chromophores during the high-pollution period. Specifically, the absorption contribu- tion of nitrocatechols and nitrophenols combined accounts for 66.1 % during the day and 60.7 % at night at 365 nm, re- spectively, which depends on the different emission sources The mass fractional contribution of nitrocatechols is lower during the day than the night in the low-pollution period, while there is obvious secondary formation during the day for the high-pollution period. This likely suggests that the daytime conditions of the high-pollution period are inductive for the generation of nitrocatechols. The mass fractional con- tribution of PAHs during the day is much lower than the night in the low-pollution period. At night, residential coal heat- ing is an important source of PAHs, and therefore the day- time contributions of PAHs are much lower than the night- time ones (Wang et al., 2017; Ni et al., 2021), while there is no day–night difference for PAHs in the high-pollution period, which is related to the stable sources and stagnant weather conditions (Huang et al., 2019; Lin et al., 2020). It is noteworthy that the mass contributions of the nitrophenols (nighttime is 2–3 times more than daytime) and two- to three- ring OPAHs (daytime is ∼2 times more than nighttime) are opposite between the day and night. This demonstrates that they have stable sources compared to other BrC subgroups even during the low-pollution period and high-pollution pe- riod. The higher mass fractional contribution of two- to three- ring OPAHs during the day is related to photochemical oxi- https://doi.org/10.5194/acp-23-15197-2023 Atmos. Chem. Phys., 23, 15197–15207, 2023 Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain Y. Gong et al.: Measurement report: Brown carbon aerosol in po 15205 Data availability. Detailed data can be obtained from https://doi.org/10.5281/zenodo.7690230 (Gong, 2023). Data availability. Detailed data can be obtained from https://doi.org/10.5281/zenodo.7690230 (Gong, 2023). Banerjee, S. and Zare, R. N.: Syntheses of isoquinoline and substi- tuted quinolines in charged microdroplets, Angew. Chem., 127, 15008–15012, 2015. Chen, L.-W. A., Chow, J. C., Wang, X., Cao, J., Mao, J., and Watson, J. G.: Brownness of Organic Aerosol over the United States: Evidence for Seasonal Biomass Burning and Photobleaching Effects, Environ. Sci. Technol., 55, 8561–8572, https://doi.org/10.1021/acs.est.0c08706, 2021. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/acp-23-15197-2023-supplement. Chen, Y. and Bond, T. C.: Light absorption by organic carbon from wood combustion, Atmos. Chem. Phys., 10, 1773–1787, https://doi.org/10.5194/acp-10-1773-2010, 2010. Author contributions. R-JH designed the study. Data analysis was done by YG and R-JH. YG and R-JH interpreted data, prepared the figures, and wrote the manuscript. LY, TW, WY, WX, WC, YW, and YL commented on and discussed the manuscript. Cheng, X., Chen, Q., Li, Y., Huang, G., Liu, Y., Lu, S., Zheng, Y., Qiu, W., Lu, K., Qiu, X., Bianchi, F., Yan, C., Yuan, B., Shao, M., Wang, Z., Canagaratna, M. R., Zhu, T., Wu, Y., and Zeng, L.: Secondary Production of Gaseous Nitrated Phenols in Polluted Urban Environments, Environ. Sci. Technol., 55, 4410- 4419, https://doi.org/10.1021/acs.est.0c07988, 2021. Competing interests. The contact author has declared that none of the authors has any competing interests. Cheng, Y., He, K. B., Du, Z. Y., Engling, G., Liu, J. M., Ma, Y. L., Zheng, M., and Weber, R. J.: The characteristics of brown carbon aerosol during winter in Beijing, Atmos. Environ., 127, 355–364, https://doi.org/10.1016/j.atmosenv.2015.12.035, 2016. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, pub- lished maps, institutional affiliations, or any other geographical rep- resentation in this paper. While Copernicus Publications makes ev- ery effort to include appropriate place names, the final responsibility lies with the authors. Chow, J. C., Watson, J. G., Robles, J., Wang, X., Chen, L.-W. A., Trimble, D. L., Kohl, S. D., Tropp, R. J., and Fung, K. K.: Qual- ity assurance and quality control for thermal/optical analysis of aerosol samples for organic and elemental carbon, Anal. Bioanal. Chem., 401, 3141–3152, 2011. Chow, K. S., Huang, X. H., and Yu, J. Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain Z.: Quantification of ni- troaromatic compounds in atmospheric fine particulate matter in Hong Kong over 3 years: field measurement evidence for sec- ondary formation derived from biomass burning emissions, Env- iron. Chem., 13, 665–673, 2015. Acknowledgements. We are very grateful to the National Natu- ral Science Foundation of China (NSFC) (no. 41925015), the Strate- gic Priority Research Program of the Chinese Academy of Sci- ences (no. XDB40000000), the Key Research Program of Fron- tier Sciences from the Chinese Academy of Sciences (no. ZDBS- LY-DQC001), and the Cross Innovative Team fund from the State Key Laboratory of Loess and Quaternary Geology (no. SKL- LQGTD1801), who supported this study. Dat, N. D. and Chang, M. B.: Review on characteris- tics of PAHs in atmosphere, anthropogenic sources and control technologies, Sci. Total Environ., 609, 682–693, https://doi.org/10.1016/j.scitotenv.2017.07.204, 2017. Deng, J., Ma, H., Wang, X., Zhong, S., Zhang, Z., Zhu, J., Fan, Y., Hu, W., Wu, L., Li, X., Ren, L., Pavuluri, C. M., Pan, X., Sun, Y., Wang, Z., Kawamura, K., and Fu, P.: Measure- ment report: Optical properties and sources of water-soluble brown carbon in Tianjin, North China – insights from organic molecular compositions, Atmos. Chem. Phys., 22, 6449–6470, https://doi.org/10.5194/acp-22-6449-2022, 2022. Financial support. This work was supported by the National Natural Science Foundation of China (NSFC) under grant no. 41925015, the Strategic Priority Research Program of the Chi- nese Academy of Sciences (grant no. XDB40000000), the Key Re- search Program of Frontier Sciences from the Chinese Academy of Sciences (grant no. ZDBS-LY-DQC001), and the Cross Innovative Team fund from the State Key Laboratory of Loess and Quaternary Geology (grant no. SKLLQGTD1801). Gao, Y., Wang, Q., Li, L., Dai, W., Yu, J., Ding, L., Li, J., Xin, B., Ran, W., and Han, Y.: Optical prop- erties of mountain primary and secondary brown carbon aerosols in summertime, Sci. Total Environ., 806, 150570, https://doi.org/10.1016/j.scitotenv.2021.150570, 2022. Gong, Y.: Dataset for “Measurement report: Brown Carbon Aerosol in Polluted Urban Air of North China Plain: Day-night Dif- ferences in the Chromophores and Optical Properties”, Zenodo [data set], https://doi.org/10.5281/zenodo.7690230, 2023. Review statement. This paper was edited by Stefania Gilardoni and reviewed by three anonymous referees. Hammer, M. S., Martin, R. V., van Donkelaar, A., Buchard, V., Torres, O., Ridley, D. A., and Spurr, R. J. Y. Gong et al.: Measurement report: Brown carbon aerosol in polluted urban air of the North China Plain D.: Interpreting the ultraviolet aerosol index observed with the OMI satellite in- strument to understand absorption by organic aerosols: implica- tions for atmospheric oxidation and direct radiative effects, At- mos. Chem. Phys., 16, 2507–2523, https://doi.org/10.5194/acp- 16-2507-2016, 2016. https://doi.org/10.5194/acp-23-15197-2023 4 Conclusions In general, our study shows the large day–night differences in optical properties and chemical composition of the bulk BrC in the urban atmosphere. Therein, WS-BrC is the main light- absorbing contributors during the day, while WIS-BrC is the main light-absorbing compound at night. The polar WS-BrC has higher MAE365 values compared to the less-polar WIS- BrC, mainly due to the different conjugate systems and func- tional groups in the two fractions. Different types of the iden- tified BrC chromophores exhibit unique characteristics of day–night differences, reflecting their particular sources and formation pathways. For example, nitrocatechols and two- to three-ring OPAHs are important contributors to mass con- centration and light absorption during the day, while four- to six-ring PAHs and nitrophenols become the significant con- tributors at night. Day–night differences of BrC chromophores are associ- ated with different sources during the day (mainly secondary formation and vehicle emission) and night (mainly emissions https://doi.org/10.5194/acp-23-15197-2023 Atmos. Chem. Phys., 23, 15197–15207, 2023 References Alcanzare, R. J. C.: Polycyclic aromatic compounds in wood soot extracts from Henan, China, M.Sc. Thesis, The Depart- ment of Chemical Engineering, Louisiana State University and Agricultural and Mechanical College, Baton Rouge, LA, 132, https://doi.org/10.31390/gradschool_theses.2377, 2006. Atmos. Chem. 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General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Digital Object Identifier (DOI): 10.1002/jmri.28499 Published In: Journal of Magnetic Resonance Imaging Edinburgh Research Explorer Citation for published version: Singh, T, Joshi, S, Kershaw, LE, Dweck, MR, Semple, SI & Newby, DE 2022, 'Manganese‐Enhanced Magnetic Resonance Imaging of the Heart', Journal of Magnetic Resonance Imaging. https://doi.org/10.1002/jmri.28499 View this article online at wileyonlinelibrary.com. DOI: 10.1002/jmri.28499 Received May 2, 2022, Accepted for publication Oct 11, 2022. *Address reprint requests to: T.S., University of Edinburgh, Room SU. 305, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK. E-mail: [email protected] GRANT SOURCES: T.S. is supported by the British Heart Foundation (RE/18/5/34216) and Medical Research Council (MR/T-29/53/1). S.J. and M.R.D. are supported by the British Heart Foundation (FS/17/19/32641, RG/16/10/32375, RE/18/5/34216). D.E.N. is supported by the British Heart Foundation (CH/09/002, RG/16/10/32375, RE/18/5/34216) and is the recipient of a Wellcome Trust Senior Investigator Award (WT103782AIA). For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising from this submission. From the 1BHF/University Centre for Cardiovascular Science, University of Edinburgh, UK; 2Edinburgh Heart Centre, Royal Infirmary of Edinburgh, UK; and 3Edinburgh Imaging, University of Edinburgh, UK This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 REVIEW Manganese-Enhanced Magnetic Resonance Imaging of the Heart Trisha Singh, BM,1,2,3* Shruti Joshi, MBBS,1,2,3 Lucy E Kershaw, MSci, PhD,1,3 Marc R Dweck, MBChB, PhD,1,2,3 Scott I Semple, MSc, PhD,1,3 and David E Newby, DM, PhD1,2,3 Manganese-based contrast media were the first in vivo paramagnetic agents to be used in magnetic resonance imaging (MRI). The uniqueness of manganese lies in its biological function as a calcium channel analog, thus behaving as an intracellular con- trast agent. Manganese ions are taken up by voltage-gated calcium channels in viable tissues, such as the liver, pancreas, kid- neys, and heart, in response to active calcium-dependent cellular processes. Manganese-enhanced magnetic resonance imaging (MEMRI) has therefore been used as a surrogate marker for cellular calcium handling and interest in its potential clini- cal applications has recently re-emerged, especially in relation to assessing cellular viability and myocardial function. Calcium homeostasis is central to myocardial contraction and dysfunction of myocardial calcium handling is present in various cardiac pathologies. Recent studies have demonstrated that MEMRI can detect the presence of abnormal myocardial calcium han- dling in patients with myocardial infarction, providing clear demarcation between the infarcted and viable myocardium. Fur- thermore, it can provide more subtle assessments of abnormal myocardial calcium handling in patients with cardiomyopathies and being excluded from areas of nonviable cardiomyocytes and severe fibrosis. As such, MEMRI offers exciting potential to improve cardiac diagnoses and provide a noninvasive measure of myocardial function and contractility. This could be an invaluable tool for the assessment of both ischemic and nonischemic cardiomyopathies as well as providing a measure of func- tional myocardial recovery, an accurate prediction of disease progression and a method of monitoring treatment response. Evidence Level: 5 y Evidence Level: 5 Technical Efficacy: Stage 5 Technical Efficacy: Stage 5 © 2022 The Authors. Journal of Magnetic Resonance Imaging published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine. 1 Journal of Magnetic Resonance Imaging Journal of Magnetic Resonance Imaging extracellular space before returning to the circulation for excretion by the kidneys. Due to its strong paramagnetic prop- erties and subsequent commercial development, gadolinium- based media became the preferred contrast agent, eventually leading to a decline in interest for manganese-based contrast media. This review provides a synopsis of previously published work and discusses the potential future clinical applications of MEMRI of the heart. Nonchelated Forms of Manganese Manganese chloride was one of the earliest manganese-based contrast media to be used in MRI and can be administered orally. Once absorbed, manganese chloride freely dissociates into manganese and chloride ions. The manganese ions are strongly paramagnetic and actively enter myocytes via voltage-gated calcium channels. However, due to competition with calcium, early studies demonstrated acute cardiac com- promise, especially with high doses of manganese.12 As such, MEMRI remained underdeveloped for many years due to initial safety concerns and concomitant advances in gadolinium- based imaging techniques. An important imaging characteristic of manganese ions is that they behave in a similar manner to calcium ions and are actively taken up by L-type voltage-gated calcium chan- nels and sodium–calcium exchangers.10,11 This means that uptake is seen in viable tissues, particularly those with a predominance of calcium channel activity, such as the liver, pancreas, brain, kidney, and heart (Fig. 1). Although also an analog of calcium, gadolinium ions are fully chelated in gadolinium-based contrast media, because of the need to prevent gadolinium ion dissociation and any associated toxicity. As such, gadolinium-based contrast media do not cross the cell membrane and accumulate in the extravascular Historical Background Manganese was the first element used as a contrast agent in MRI.2,3,7 Certain characteristics make it a desirable contrast agent. Like gadolinium, it has paramagnetic properties thereby shortening T1 relaxation of water, enabling increased contrast in tissues where it accumulates and enhancing ana- tomical delineation.2 Manganese is also a naturally occurring trace element in the human body, which is required for sev- eral biochemical processes through its role as a co-factor in the activation of several classes of enzymes.8 Humans obtain most of their manganese requirements through the diet and it is eliminated predominantly through the kidneys and liver.9 These properties contrast with gadolinium, which is toxic in its free unbound form and has no known biological function in humans. Formulations The formulations of manganese-based contrast media define how they behave in vivo. In particular, they determine whether there is any associated toxicity, whether they can act as an intracellular contrast agent or whether they behave as an extracellular contrast agent. J. MAGN. RESON. IMAGING 2022. J. MAGN. RESON. IMAGING 2022. C ardiac magnetic resonance imaging (MRI) has a major role in the diagnosis, evaluation, and tissue characterization of a range of cardiovascular diseases.1 Conventional cardiac mag- netic resonance with gadolinium-based contrast media can be complemented by T1-mapping techniques, which allow the quantitative assessment of myocardial tissue. Furthermore, T1 mapping can risk stratify and provide prognostic value in condi- tions, such as dilated cardiomyopathy or hypertrophic cardiomyopathy.1 providing intracellular contrast of viable tissue on MRI.2,3 Manga- nese was introduced as liver-specific contrast agent to visualize hepatic tumors and has subsequently been applied to preclinical neuronal assessment,4 and pancreatic beta-cell activation.5 The major interest in manganese-enhanced magnetic resonance imag- ing (MEMRI) of the heart lies in its biological function. In 1981, Hunter et al6 proposed that uptake of free manganese ions in the heart can be used to measure calcium uptake because manganese is retained intracellularly, and myocytes can be labeled without reduction in cardiac function by maintaining a very low manga- nese concentration in the perfusate. C a r Manganese, a calcium analog, has paramagnetic properties and can cross intact cell membranes via calcium channels, © 2022 The Authors. Journal of Magnetic Resonance Imaging published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine. 1 Journal of Magnetic Resonance Imaging Partially Chelated Subsequent work has sought to overcome the potential acute toxicity of high-dose manganese ions. For intracellular myocardial imaging, manganese must be freely available for FIGURE 1: Manganese uptake in the body. Manganese-enhanced images (a) at native T1 and post-manganese T1 (30 minutes) demonstrating manganese enhancement in the liver (L), pancreas (P) and kidneys (K). Conversely, little enhancement is seen in skeletal muscle (SM). T1 decay curves (b) demonstrate greatest reduction in T1 in liver (green), followed by pancreas (light blue), kidney (green), heart (red) and skeletal muscle (dark blue). Dotted line represents end of manganese dipyridoxyl diphosphate infusion. Manganese uptake (c) in liver (green), pancreas (light blue), kidney (green), heart (red) and skeletal muscle (dark blue). FIGURE 1: Manganese uptake in the body. Manganese-enhanced images (a) at native T1 and post-manganese T1 (30 minutes) demonstrating manganese enhancement in the liver (L), pancreas (P) and kidneys (K). Conversely, little enhancement is seen in skeletal muscle (SM). T1 decay curves (b) demonstrate greatest reduction in T1 in liver (green), followed by pancreas (light blue), kidney (green), heart (red) and skeletal muscle (dark blue). Dotted line represents end of manganese dipyridoxyl diphosphate infusion. Manganese uptake (c) in liver (green), pancreas (light blue), kidney (green), heart (red) and skeletal muscle (dark blue). 2 Singh et al.: Manganese-Enhanced MRI of the Heart FIGURE 2: Types of manganese-based contrast media. Manganese chloride (a), manganese gluconate (b), manganese dipyridoxyl diphosphate (c) and O-carboxymethyl chitosan manganese-diethylenetriamine pentaacetate are examples of nonchelated and chelated manganese-based contrast agents, respectively. MnCl2 = manganese chloride; EVP 1001-1 = manganese gluconate; MnDPDP = manganese dipyridoxyl diphosphate; CMCS (Mn-DTPA) = O-carboxymethyl chitosan manganese-diethylenetriamine pentaacetate. g g FIGURE 2: Types of manganese-based contrast media. Manganese chloride (a), manganese gluconate (b), manganese dipyridoxyl diphosphate (c) and O-carboxymethyl chitosan manganese-diethylenetriamine pentaacetate are examples of nonchelated and chelated manganese-based contrast agents, respectively. MnCl2 = manganese chloride; EVP 1001-1 = manganese gluconate; MnDPDP = manganese dipyridoxyl diphosphate; CMCS (Mn-DTPA) = O-carboxymethyl chitosan manganese-diethylenetriamine pentaacetate. TABLE 1. Safety Historically, there have been safety concerns regarding the early forms of manganese-based contrast media.26,27 Brurok and Jyunge conducted a series of preclinical studies of the car- diotoxic effects of manganese both during and after exposure of perfused rodent hearts to different concentrations of man- ganese chloride and manganese dipyridoxyl diphosphate.26–29 They found that manganese chloride was 8–10 times more potent and competed too strongly with myocardial calcium uptake, causing myocardial depression, hypotension, and cardiac arrest.27 Onset of cardiac depression was immediate upon introduction of 30 mM manganese into the perfusate and normal function was restored within minutes upon removal of manganese chloride.27 Extensive research has been done in assessing dose con- centration effects of manganese on longitudinal relaxation. Early preclinical studies demonstrated a close correlation between tissue manganese content and intracellular longitudi- nal relaxation (R1).3,16,19 Furthermore, they reported close to a linear correlation between intracellular R1 (1/T1) and ex vivo manganese concentration.20,21 Studies in man have shown significant myocardial shortening of T1 relaxation at 5 μmol per kg bodyweight of manganese dipyridoxyl diphos- phate (current approved dose), which is not linearly propor- tional to the total manganese infused. This contrasts with the marked dose–response seen in liver tissue.22 In humans, 70% of cytosolic calcium is from sarcoplasmic reticulum stores and approximately 30% is from extracellular uptake.11 This, and the higher distribution of calcium channel activity in the liver, pancreas and kidneys, could explain the relatively lower uptake of manganese in the myocardium (Fig. 1). Partially Chelated Manganese-Based Contrast Agents Contrast Agent Clinical Dose (μmol/kg) Approved for Clinical Use Previous or Currently Recruiting Studies Chelated Partial: MnDPDP 5 Yes Manganese-enhanced MRI (MEMRI) of the myocardium (NCT03607669) MEMRI in ischemic, inflammatory and Takotsubo Cardiomyopathy (NCT04623788) The DAPA-MEMRI Trial (NCT04591639) PANCREAS MEMRI (NCT05298735) Full: CMCS (Mn-DTPA) 0.03 No None Nonchelated MnCl2† 5 No None EVP1001-1 1–10 No Clinical Trial of MEMRI to assess peri- infarct Injury (NCT02933034) Efficacy of EVP1001-1 in the Assessment of myocardial viability in patients with cardiovascular disease (NCT01989195) MnDPDP = manganese dipyridoxyl diphosphate; CMCS(Mn-DTPA) = O-carboxymethyl chitosan manganese-diethylenetriamine pen- taacetate; MnCl2† = manganese chloride. TABLE 1. Manganese-Based Contrast Agents Contrast Agent Clinical Dose (μmol/kg) Approved for Clinical Use Previous or Currently Recruiting Studies Chelated Partial: MnDPDP 5 Yes Manganese-enhanced MRI (MEMRI) of the myocardium (NCT03607669) MEMRI in ischemic, inflammatory and Takotsubo Cardiomyopathy (NCT04623788) The DAPA-MEMRI Trial (NCT04591639) PANCREAS MEMRI (NCT05298735) Full: CMCS (Mn-DTPA) 0.03 No None Nonchelated MnCl2† 5 No None EVP1001-1 1–10 No Clinical Trial of MEMRI to assess peri- infarct Injury (NCT02933034) Efficacy of EVP1001-1 in the Assessment of myocardial viability in patients with cardiovascular disease (NCT01989195) MnDPDP = manganese dipyridoxyl diphosphate; CMCS(Mn-DTPA) = O-carboxymethyl chitosan manganese-diethylenetriamine pen- taacetate; MnCl2† = manganese chloride. TABLE 1. Manganese-Based Contrast Agents 3 Journal of Magnetic Resonance Imaging likely due to slow release of manganese ions from dipyridoxyl diphosphate and elimination via hepatobiliary and urinary excre- tion. To date, manganese dipyridoxyl diphosphate (Teslascan; General Electric Healthcare) is the only manganese-based con- trast medium to have been approved for magnetic resonance imaging in humans, with a primary indication for hepatic tumor imaging.24 cardiomyocyte uptake. To date, two different methods have been employed producing distinct clinical-grade agents, both with excellent safety profiles: 1) co-administration with cal- cium and 2) partial chelation (Fig. 2, Table 1). CO-ADMINISTRATION WITH CALCIUM GLUCONATE. To counter the adverse effects of free manganese ions, intrave- nous manganese co-administered with calcium gluconate can result in a short plasma half-life and rapid myocardial uptake with little redistribution.13 This has been developed for clin- ical use as EVP 1001-1 (SeeMore, Eagle Vision Pharmaceu- ticals, Downingtown, USA), and it has reduced the toxic effects of high-dose manganese ions and achieved sufficient T1 relaxation for imaging. However, not much work has been done with this formulation. Fully Chelated Biocompatible macromolecular manganese-based contrast media have been generated using O-carboxymethyl chitosan (CMCS), diethylenetriamine pentaacetate (DTPA), and man- ganese (Mn). According to in vitro studies, CMCS-(Mn- DTPA) exhibits good cellular and blood biocompatibility at doses necessary for MRI. The relaxivity of CMCS-(Mn- DTPA) is approximately 3.5 and 5.5 times higher than that of gadolinium-DTPA and manganese dipyridoxyl diphosphate, respectively.25 However, this is only when it is chelated to DTPA. Similar to gadolinium-DTPA, aqueous CMCS-(Mn- DTPA) is stable enough to prevent the release of any manga- nese ions. Thus, partial chelation of manganese enables it to act as an intracellular agent, whereas full chelation results in a stronger paramagnetic agent but one that images the extracel- lular rather than intracellular compartment and hence cannot estimate calcium influx. MANGANESE DIPYRIDOXYL DIPHOSPHATE. Unlike stron- ger chelates, which are designed not to dissociate, dipyridoxyl diphosphate chelation allows manganese to uncouple and cir- culate as a protein-bound complex.14 The dynamics of myocar- dial uptake of manganese dipyridoxyl diphosphate have been described in detail elsewhere.11 In brief, after intravenous administration in humans, manganese dipyridoxyl diphosphate undergoes dephosphorylation and transmetallation with zinc to release manganese ions into the plasma.14–18 This controlled release of manganese ions facilitates rapid intracellular manga- nese uptake and renal clearance.15,16 Administering manganese in this way successfully mitigates toxicity while retaining crucial paramagnetic and biological properties for adequate intracellu- lar contrast. Safety They found that manganese chloride was 8–10 times more potent and competed too strongly with myocardial calcium uptake, causing myocardial depression, hypotension, and cardiac arrest.27 Onset of cardiac depression was immediate upon introduction of 30 mM manganese into the perfusate and normal function was restored within minutes upon removal of manganese chloride.27 Subsequent preclinical studies utilizing partially chelated manganese dipyridoxyl diphosphate demonstrated that manganese-induced negative inotropy can be avoided by maintaining low extracellular manganese concentrations, and over time, contrast enhancement of the myocardium rapidly grows to detectable levels.30 Partially chelated manganese- based contrast agents have been used in several clinical studies in patients with varying cardiac pathologies and demonstrate an excellent safety profile.12 There have been no reports of cardiac toxicity or changes in heart rate, blood pressure or car- diac contractility during manganese dipyridoxyl diphosphate infusion despite administration to patients with acute Manganese taken up by heart cells is retained for hours and does not redistribute, unlike calcium, which rapidly redis- tributes between intra and extracellular compartments.16 Myocardial manganese uptake from manganese dipyridoxyl diphosphate is slower than from manganese chloride23 and is cleared in 24 h without significant organ retention. This is 4 Singh et al.: Manganese-Enhanced MRI of the Heart myocardial infarction or those with poor left ventricular sys- tolic dysfunction.31–33 Naturally occurring deficiency is rare, but toxicity from over- exposure following environmental or occupational contact is recognized and can result in manganese accumulation in the globus pallidus, manifesting as headaches, gait disturbance and extra-pyramidal symptoms and is referred to as “man- ganism.”42 However, neurotoxicity due to inhalation expo- sure has only been observed in miners,43 smelters,44 and welders45 over the course of years. Furthermore, previous studies have demonstrated a significant correlation between airborne manganese levels and manganism in welders, with estimated exposure dosages of 5–20 kg (containing 0.3–6% Mn) per working day per person.46,47 Although gadolinium-based contrast agents are safe and well tolerated, it is important to remember that early formula- tions were toxic. It should also be remembered that gadolinium has no known physiological function or regulated secretory pathways in humans. In its free state, gadolinium is highly toxic. Safety There are several potential mechanisms for this,34,35 and the most well recognized is gadolinium ions interfering with many calcium ion channel-dependent processes.36 For nearly a decade, there was an association between gadolinium-based contrast media and the development of nephrogenic systemic fibrosis in patients with severe renal impairment.37 This risk was much higher in patients given acyclic gadolinium-based contrast media. As such, these formulations (Gd-DTPA, Gd-DTPA-BMA, and Gd-DTPA-BMEA) have been suspended by the European Medicines Agency and cases of nephrogenic systemic fibrosis have subsequently fallen dramatically. Risk of manganese accumulation with intravenous manganese-based contrast agents is minimal.48 Two factors aid in preventing this. First, total manganese exposure is very low even with repeated contrast doses. Second, manganese is naturally eliminated from the body through several regulatory processes. Furthermore, phase III clinical trials evaluated safety in patients with renal and hepatic impairment and demonstrated no clinical or laboratory evidence suggestive of manganese toxicity48 in any of the 48 patients with liver cir- rhosis, including six subjects with hepatic failure. A more recent study has reported that an intravenous infusion of a standard imaging dose (5 μmol/kg) of manganese dipyridoxyl diphosphate in healthy human volunteers raised signal inten- sity in exocrine glands in the head and neck, in the choroid plexus, and in the anterior pituitary gland but not beyond the intact blood–brain barrier.49 However, there is a lack of long- term safety data with the use of manganese-based contrast agents and future work should focus on establishing this. It was previously widely believed that gadolinium-based contrast agents are rapidly and completely eliminated from the human body. However, gadolinium can accumulate in tissues, such as the brain, bone,38 and kidneys,39 in patients who have received gadolinium-based contrast media despite normal renal function. Furthermore, retention of gadolinium can be higher in those who have repeated exposure.40 Further work is needed to clarify propensity of macrocyclic agents to accumulate in the central nervous system and to establish if this is associated with any clinical sequelae. These safety con- cerns highlight our current dependence on gadolinium and the relative lack of alternatives. Most human exposure to manganese occurs naturally as it is present in nearly all diets, with stable levels maintained by homeostatic mechanisms almost regardless of intake.41 Manganese is an essential mineral required for a range of enzymes central to normal physiological function of the body. Manganese T1 Mapping and Patlak Modeling Manganese causes shortening of T1 relaxation time of water protons due to its paramagnetic properties. MEMRI is there- fore best visualized and quantified using T1-weighted imag- ing. However, T1 mapping allows for quantitative assessment of manganese uptake in cells, because of the linear relation- ship between the longitudinal relaxation rate R1 (1/T1) and manganese concentration.55,56 Many animal studies have con- firmed that manganese-enhanced imaging enhances tissue contrast by manganese uptake and retention in viable cells, resulting in shortening of T1.23,29,30,54,57,58 Skjold et al were the first to demonstrate the effective- ness of manganese in imaging the healthy human myocar- dium.22 They applied T1 mapping to short-axis slices of the left ventricular myocardium in healthy volunteers before and after intravenous infusion of manganese dipyridoxyl diphos- phate (5–15 μmol/kg).22,53,54 T1 values from bloodpool and myocardium measured over time demonstrated a sharp reduc- tion in blood pool T1 during initial infusion phase followed by normalization to baseline by 30 minutes (Fig. 4). Myo- cardial T1 values also demonstrate a rapid initial descent (infusion phase), but this is followed by a plateau phase (34%–46%),22 likely attributable to ongoing intracellular myocardial uptake and accumulation. Myocardial Calcium Handling Myocardial contraction is controlled by excitation– contraction coupling which allows for rapid changes in FIGURE 3: Myocardial calcium homeostasis and manganese uptake. Calcium homeostasis in normal myocardium (a) and manganese uptake via voltage-gated calcium channels (b). Ca2+ = calcium ion; Na+ = sodium ion; H+ = hydrogen ion; Mn2+ = manganese ion; NCX = sodium–calcium exchangers; NHE-1 = sodium hydrogen exchanger; RyR = ryanodine receptors; PLB = phospholamban; SERCA = sarcoplasmic reticulum calcium adenosine triphosphatase. FIGURE 3: Myocardial calcium homeostasis and manganese uptake. Calcium homeostasis in normal myocardium (a) and manganese uptake via voltage-gated calcium channels (b). Ca2+ = calcium ion; Na+ = sodium ion; H+ = hydrogen ion; Mn2+ = manganese ion; NCX = sodium–calcium exchangers; NHE-1 = sodium hydrogen exchanger; RyR = ryanodine receptors; PLB = phospholamban; SERCA = sarcoplasmic reticulum calcium adenosine triphosphatase. 5 Journal of Magnetic Resonance Imaging normal myocardium compared to infarcted regions.53,54 Thus, abnormal myocardium would result in reduced manganese uptake. This led to the idea that manganese could be used as a surrogate marker of calcium handling (Fig. 3). calcium ion concentrations in the sarcoplasmic reticulum leading to contraction (systole) and relaxation (diastole, Fig. 3).50 Calcium homeostasis in the myocardium is essential for this and is controlled by several mechanisms. During sys- tole, calcium ions are actively transported into the sarcoplasmic reticulum via L-type voltage-gated calcium channels. Calcium ions bind to ryanodine receptors resulting in the efflux of an even higher concentration of calcium ions from the sarcoplas- mic reticulum into the cytosol.50 “Calcium induced, calcium release” in turn activates calcium-sensitive contractile proteins (troponin C, troponin NC), which leads to myocardial con- traction. During diastole, sarcoplasmic reticulum calcium aden- osine triphosphatase (SERCA 2a) facilitates calcium entry back into the sarcoplasmic reticulum in addition to their exit into the extracellular space via the sodium–calcium exchanger and mitochondrial uptake.50 The net result is a reduced calcium concentration in the cytosol and myocardial relaxation. Phospholamban, a regulatory protein, promotes calcium efflux, resulting in reduced myocardial contraction. When phosphory- lated, it causes disinhibition of SERCA 2a and subsequent increase in myocardial contraction. Myocardial Manganese Uptake The major interest in manganese lies in its biological functional- ity. In 1970, Ochi51 first demonstrated that manganese ions were taken up by L-type voltage-gated calcium channels in cardiomyocytes.51 Uptake of manganese was 35-fold slower in the resting heart compared to the beating heart. Moreover, addi- tion of agents known to influence calcium uptake by myocytes led to similarly detected alteration in manganese uptake.6,52 Early ex vivo animal studies showed that accumulated manga- nese led to markedly increased longitudinal relaxation rates in Myocardial manganese uptake can be quantified by tracer kinetic modeling. Patlak et al were the first to provide a graphical analysis of unidirectionality of transfer and of the FIGURE 4: Manganese-enhanced magnetic resonance imaging of healthy myocardium. Short-axis T1 mapping in healthy myocardium with manganese dipyridoxyl diphosphate (a). Rapid reduction in T1 is seen in the blood pool (green, b), followed by rapid normalization by 30 minutes. In contrast, the T1 value of myocardium (red) shows steady and sustained reduction throughout the imaging time period (b). Dotted line represents end of manganese dipyridoxyl diphosphate infusion. FIGURE 4: Manganese-enhanced magnetic resonance imaging of healthy myocardium. Short-axis T1 mapping in healthy myocardium with manganese dipyridoxyl diphosphate (a). Rapid reduction in T1 is seen in the blood pool (green, b), followed by rapid normalization by 30 minutes. In contrast, the T1 value of myocardium (red) shows steady and sustained reduction throughout the imaging time period (b). Dotted line represents end of manganese dipyridoxyl diphosphate infusion. 6 Singh et al.: Manganese-Enhanced MRI of the Heart Singh et al.: Manganese-Enhanced MRI of the Heart influx constant, using brain uptake data.59 The multitime approach produces information about the exchange rate of the compartments that rapidly and reversibly exchange with plasma. Furthermore, this can be used to assess the rate con- stant of any type of irreversible process within any organ sys- tem.59 The Patlak two-compartment model has become the commonest modelling approach in cardiac manganese- enhanced imaging. This assumes the influx of manganese ions from a reversible (ve, extracellular and vascular space) into a largely irreversible compartment (vi, cardiomyocyte during the imaging period). Myocardial Manganese Uptake This apparent unidirectional influx con- stant (Ki) for the transfer of manganese from plasma to irre- versible compartments vi, can be measured, using Equation 1: disease.31,33,54 Furthermore, only minor differences in myo- cardial manganese uptake were seen between administration of manganese over 5 or 30 minutes.31,33,54 There are limited data on the repeatability and repro- ducibility of MEMRI, which is essential for its future clinical application. Spath et al performed interobserver reproducibil- ity for healthy volunteers (n = 20) and patients with hyper- trophic (n = 10) and dilated cardiomyopathy (n = 10). This demonstrated no significant difference in native T1 and post- manganese T1 between two independent observers for healthy volunteers (P = 0.89 and 0.97, respectively) and patient cohorts.33 An ongoing sub-study is evaluating intraobserver and interobserver reproductivity and scan– rescan repeatability of manganese-enhanced magnetic T1 mapping and kinetic modeling in healthy volunteers and patients with acute myocardial infarction, hypertrophic and dilated cardiomyopathy (Singh T, unpublished data). Ct tð Þ Ca tð Þ ¼ Ki ðt 0 Ca τð Þdτ Ca tð Þ þve ð1Þ ð1Þ where Ct and Ca are the manganese concentration in myocar- dial tissue and blood pool (arterial input function), respec- tively. This formula is equivalent to the Patlak model59 and describes that if a contrast medium is irreversibly trapped in the tissue within the imaging period, the instantaneous tissue concentration (myocardial T1) divided by the instantaneous arterial concentration (bloodpool T1) plotted against the inte- grated arterial concentration divided by the instantaneous arterial concentration, will result in linearization of the data (Fig. 5).54,59 Flow or Function Contractile abnormalities can arise from either a decrease in calcium availability, such as from sarcoplasmic reticular dysfunction, or a decreased responsiveness of the myofilaments to calcium, such as from proteolysis of troponin I.64 restricted artery and this was likely to represent lower blood flow.13 Similarly, it would be reasonable to assume that reduced manganese uptake seen in patients with acute myo- cardial infarction can be ascribed to reduced flow due to coro- nary artery occlusion.61 As a calcium analog, the influx of manganese into the cardiomyocyte is through voltage-gated calcium channels. As such, the rate of this influx could be altered under conditions that increase or decrease calcium influx into the heart. Cal- cium channel blockers are known to decrease cardiac function by decreasing calcium influx into the heart. Animal models have demonstrated that pretreatment of ex vivo mouse myo- cardium with diltiazem resulted in reduced manganese- induced T1 shortening. This inhibition was similar between all agents with manganese chloride experiencing a maximal mean reduction of 30% in T1 shortening, while EVP1001-1 and manganese dipyridoxyl diphosphate experienced reduc- tions of up to 43% and 32%, respectively.62 Conversely, β-adrenergic agonists, such as dobutamine, are known to increase calcium influx into the heart to increase contractile function. Hu et al52 demonstrated upregulation of myocardial manganese uptake with dobutamine, favoring flow-related mechanism. Improved contrast between normal and ischemic myocardium with greater reduction in remote myocardial T1 during both dipyridamole and dobutamine stress has been reported with EVP1001-1 in both acute and chronic models of ischemia.13 However, this has not been replicated in other studies. Further work is required to establish the true impact of flow vs. function. ACUTE MYOCARDIAL INFARCTION. Animal models have assessed quantification of myocardial infarction with MEMRI.61,65,66 In a rat myocardial infarction model, manga- nese enhancement was present in the normal myocardium but absent in the infarcted tissue.67 In an ischemia reperfusion model, manganese dipyridoxyl diphosphate enhancement on in vivo and ex vivo MRI correlated well but was smaller than the areas on triphenyl tetrazolium chloride staining. This suggests that MEMRI detects both infarcted and stunned myocardium.65,66 A recent murine study compared manganese- enhanced and gadolinium-enhanced T1 mapping measurements of myocardial infarction size at 1 hour, 1 day and 14 days after arterial occlusion. Flow or Function Manganese uptake in the heart is determined by both the amount of manganese being delivered to the myocardium (blood flow) and the amount that is taken up into the inter- cellular space (function), instead of passing through the circu- lation. This is crucial in interpreting patterns of manganese uptake according to differences in regional function and it is not a simple question since flow and function are highly interconnected, particular in patients with ischemic heart dis- ease. The heart does not retain all the manganese that enters its circulation. Instead, the uptake of manganese is deter- mined by competitive uptake by calcium channels, which can be approximated by a rate constant (influx from reversible to irreversible compartment).54,59 However, manganese uptake (Ki) is an amalgamated measure that does not distinguish between flow and transfer into the cells. Early animal studies applied a two-compartment model to MEMRI of the retina and successfully estimated apparent retinal transfer constant Ki.60 Years later, Skjold was the first to apply tracer kinetic modelling to R1 curves, a unidirec- tional influx constant for manganese (Ki), was measured as an index of myocardial calcium channel activity in healthy vol- unteers.54 This demonstrated that the use of unidirectional manganese influx rates may be a valuable research tool for in vivo studies of myocyte functioning in myocardial The evidence for flow and function is sparse and contra- dictory. Preclinical studies reported relative low uptake of manganese observed in myocardium served by a partially FIGURE 5: Patlak modeling. Patlak formulation—schematic of (a) model compartments and transfer constant Ki, describing passage from reversible to irreversible compartment (b) data analysis. Cm(t) = myocardial manganese concentration; Cb(t) = blood manganese concentration. FIGURE 5: Patlak modeling. Patlak formulation—schematic of (a) model compartments and transfer constant Ki, describing passage from reversible to irreversible compartment (b) data analysis. Cm(t) = myocardial manganese concentration; Cb(t) = blood manganese concentration. 7 Journal of Magnetic Resonance Imaging cardiomyocytes. This region is important because, in the acute phase, cells may demonstrate myocardial stunning. This refers to persistent myocardial contractile dysfunction tran- siently induced by acute ischemia despite reperfusion and absence of irreversible damage. It is clear that stunned myo- cardium is characterized by abnormalities in excitation– contraction coupling.64 However, despite intense research efforts, there remains some ongoing controversy as to the exact nature of these abnormalities. Flow or Function The authors reported that MEMRI provides an early biomarker on final infarct size after permanent coronary occlusion.68 More recently, serial MEMRI has been used to investi- gate the time course of changes in patients with acute myo- cardial infarction.32 Regions with transmural infarction demonstrated partial recovery of T1 values similar to that of bloodpool (Fig. 6). The lack of manganese uptake here likely represents absence of myocardial calcium handling in the infarct area secondary to myocardial necrosis and absent myocyte viability. Unlike late-gadolinium enhancement, where contrast agent accumulates in areas of myocardial necrosis, manganese uptake occurs in viable tissue, therefore absence or reduction of manganese uptake suggests absent or impaired calcium handling, thereby behaving as an inverse of gadolinium imaging (Fig. 6). Late-gadolinium enhancement is the gold standard for visualization and quantification of myocardial infarction size. Cardiac Applications With increasing interest in MEMRI over the last decade, this technique has been translated to assess myocardial calcium handling in early clinical studies. Myocardial Infarction Mean T1 decay times in bloodpool (green), infarct (red), peri-infarct (orange) and remote region (blue) in patients with acute myocardial infarction (d). Mean myocardial manganese uptake (Ki- mL/min/100 g of tissue) defined by Patlak modeling in patients with acute myocardial infarction and healthy volunteers (e). compared with 18F-FDG PET, suggesting both cellular cal- cium and glucose uptake are robust and concordant markers of myocardial viability.62 infarcted myocardium.32 After 3 months, myocardial manga- nese uptake in the peri-infarct region was similar to that of the remote regions. This suggests that the lack of manganese uptake in the peri-infarct area may represent myocardial stunning. Due to radiation exposure and lack of 18F-FDG PET availability, gadolinium became invaluable in identifying scarred nonviable myocardium. However, viability in gadolinium-enhanced imaging is simply inferred and cannot be measured directly or quantitatively. Although data for chronic ischemia are limited, manganese uptake in the peri- infarct region at 1 month does appear to be greater than infarct zone in animal models.70 This implies that MEMRI can enhance myocardium within injured but not infarcted myocardium and could be an important biomarker of viability. ESTABLISHED MYOCARDIAL INFARCTION. Beyond infarct quantification, manganese-enhanced magnetic resonance has the potential to assess myocardial viability with potential application to myocardium with chronic myocardial contrac- tile dysfunction secondary to ischemia: the so-called “hiber- nating” myocardium. Such “hibernating” cardiomyocytes remain viable and often restoration of blood flow will lead to some degree of improvement in left ventricular ejection frac- tion. Thus, identifying myocardium with potential for improvement in contractility is vital to determine the appro- priateness of coronary revascularization. Skjold et al53 were the first to describe MEMRI in patients with prior established myocardial infarction. They described the ability of manganese-enhanced magnetic reso- nance to characterize and to quantify viable myocardium directly (Fig. 6). This confirmed the potential to detect viable myocardium, which could prove an invaluable tool in patient selection for revascularization therapies. Moreover, it holds particular promise as a biomarker of treatment efficacy for interventional strategies targeting ischemia-reperfusion injury. 18F-Fluorodeoxyglucose positron emission tomography (18F-FDG PET) is the gold standard for assessing myocardial viability69 offering the greatest sensitivity for viable myocar- dium and comparable specificity to other imaging modalities. Although imaging in this way directly assesses viability through metabolic functionality of tissues (a mechanistically similar method of myocardial viability assessment to MEMRI), its use is often limited by availability and expertise. Myocardial Infarction MYOCARDIAL CALCIUM HANDLING IN MYOCARDIAL INFARCTION. After a period of ischemia and hypoxia, myocardial cells within the infarct region demonstrate major metabolic abnormalities, resulting in myocardial calcium mishandling. Intracellular hydrogen ion aggregation causes a low intracellular pH, and intracellular sodium increases through sodium/hydrogen exchange.63 Excessive intracellular sodium will promote sodium excretion and calcium intake by sodium–calcium exchange, which increases intracellular cal- cium levels, leading to calcium overload. This can cause a series of irreversible cell injury responses, such as cardiac con- tractile dysfunction and apoptosis. Furthermore, when blood flow and oxygen supply to the cardiac tissue returns to nor- mal intracellular calcium overload is further aggravated.63 Spath et al report that MEMRI was more sensitive than late-gadolinium enhancement in detecting dysfunctional myocardium (dysfunctional fraction 40.5  11.9 vs. infarct size 34.9  13.9%; P = 0.02).32 This is likely due to the latter being nonspecific and overestimating infarct territory due to acute edema.32,62 MEMRI tracked more closely with abnormal wall motion than late-gadolinium imaging (mean r2 = 0.72 vs. 0.55, P < 0.0001)32. A step-wise reduction was seen in manganese uptake across remote, peri-infarct and The peri-infarct zone is an area of heterogeneous myo- cardial scar containing fibrotic tissue intermingled with viable 8 8 Singh et al.: Manganese-Enhanced MRI of the Heart FIGURE 6: Manganese-enhanced magnetic resonance imaging in acute myocardial Infarction. Short-axis views of gadolinium- enhanced, native and 30-minute postmanganese T1 map images in patients with acute anteroseptal (a), anterior (b), and inferior c) myocardial infarction. Gadolinium-enhanced images demonstrate the presence of late gadolinium in the anteroseptal, anterior and inferior walls respectively. Conversely, manganese-enhanced images demonstrate reduced manganese uptake (abnormal calcium handling, green) in the anteroseptal, anterior and inferior walls. Mean T1 decay times in bloodpool (green), infarct (red), peri-infarct (orange) and remote region (blue) in patients with acute myocardial infarction (d). Mean myocardial manganese uptake Ki- mL/min/100 g of tissue) defined by Patlak modeling in patients with acute myocardial infarction and healthy volunteers (e). FIGURE 6: Manganese-enhanced magnetic resonance imaging in acute myocardial Infarction. Short-axis views of gadolinium- enhanced, native and 30-minute postmanganese T1 map images in patients with acute anteroseptal (a), anterior (b), and inferior (c) myocardial infarction. Gadolinium-enhanced images demonstrate the presence of late gadolinium in the anteroseptal, anterior and inferior walls respectively. Conversely, manganese-enhanced images demonstrate reduced manganese uptake (abnormal calcium handling, green) in the anteroseptal, anterior and inferior walls. Myocardial Infarction This, combined with the properties of manganese, led to the investigation of MEMRI to assess and quantify myocardial viability directly. Preclinical studies have validated direct quantification of myocardial viability using MEMRI Heart Failure MYOCARDIAL CALCIUM HANDLING IN HEART FAILURE. Due to the fundamental role of calcium in excitation– contraction coupling, dysfunctional myocardial calcium 9 15222586, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jmri.28499 by Test, Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA Journal of Magnetic Resonance Imaging handling is central to the pathophysiology of the failing myo- cardium. The amount of calcium entering the cytoplasm and its rate of removal are major factors in determining rate, intensity and duration of myocardial contraction.71 limits calcium extrusion via forward mode sodium–calcium exchange during diastole. The reduced rate of calcium removal reduces the rate of recovery and is associated with marked delay in myocardial relaxation. Calcium overload con- tributes to arrhythmias and diastolic dysfunction (Fig. 7).75 Several mechanisms contribute to disrupted calcium handling in systolic dysfunction. First, release of calcium from the sarcoplasmic reticulum is reduced which in turn impairs calcium-induced calcium release.72 Second, phosphorylation of L-type voltage-gated calcium channels is increased during heart failure, which results in calcium leaking from the sarco- plasmic reticulum.72 Third, there is a substantial reduction in SERCA2a expression in patients with ischemic cardiomyopa- thy and to a lesser degree with dilated cardiomyopathy.72 On the other hand, phospholamban (PLN) expression and phos- phorylation were relatively unchanged.72 This not only reduces intracellular uptake of calcium via SERCA2a but also increases PLN: SERCA ratio resulting in predominately cal- cium efflux and inhibition of myocardial contraction.72 Finally, although ryanodine receptor expression remains unchanged in the failing myocardium, its function is calcium dependent and therefore affected by intracellular calcium con- centration. Together these factors result in reduced calcium release from the sarcoplasmic reticulum (Fig. 7). DILATED CARDIOMYOPATHY. Dilated cardiomyopathy is the commonest form of cardiomyopathy and is characterized by left ventricular dilatation and dysfunction in the absence of abnormal loading or ischemia. Dysfunctional calcium han- dling is a central feature of left ventricular dysfunction in patients with dilated cardiomyopathy, with alterations in cal- cium handling proteins leading to reduced myocardial con- tractile function.76 As such, the ability to detect and to monitor calcium handling noninvasively has great potential to be used as a marker for risk stratification, disease progression and response to therapy. Gadolinium-enhanced MRI is used to identify fibrosis and the degree of fibrosis is a predictor of all-cause mortality. Heart Failure Similarly native T1 can detect diffuse subclinical fibrosis77 and is an important predictor of ventricular arrhythmias. There are no studies that assess MEMRI in animal models of dilated cardiomyopathy. Myocardial calcium handling is important in cardiac diastology. To achieve relaxation, cytosolic calcium must be sequestered, mainly to the sarcoplasmic reticulum by SERCA2.73 Diastolic calcium is increased in human heart failure, a condition that is likely related, at least in part, to defects in cytosolic calcium removal.72 Elevated intracellular sodium and altered sodium channel properties are present in failing myocardium of humans.74 Changes in intracellular sodium may have a large impact on calcium homeostasis.74 Small increases in sodium, increases calcium influx via reverse-mode sodium–calcium exchange during systole and A small pilot study investigating MEMRI in patients with dilated cardiomyopathy33 demonstrated reduced myo- cardial manganese uptake, suggestive of dysfunctional myo- cardial calcium handling (Fig. 8). Furthermore, MEMRI correlated with left ventricular ejection fraction in patients with dilated cardiomyopathy, suggesting it has potential to track and to quantify more subtle gradations of myocardial dysfunction. The cellular mechanisms underlying this have yet to be explored in detail, but it points to important poten- tial of MEMRI for noninvasive detection of calcium dys- regulation. However, this is not specific to nonischemic FIGURE 7: Calcium dysfunction in the failing myocardium. Several factors cause reduced calcium ion (Ca2+) release from the sarcoplasmic reticulum resulting in systolic heart failure (a). Diastolic heart failure results from reduced rate of Ca2+ removal causing delay in myocardial relaxation (b). Ca2+ = calcium ion; Na+ = sodium ion; H+ = hydrogen ion; Mn2+ = manganese ion; NCX = sodium–calcium exchangers; NHE-1 = sodium hydrogen exchanger; RyR = ryanodine receptors; PLB = phospholamban; SERCA = sarcoplasmic reticulum calcium adenosine triphosphatase. FIGURE 7: Calcium dysfunction in the failing myocardium. Several factors cause reduced calcium ion (Ca2+) release from the sarcoplasmic reticulum resulting in systolic heart failure (a). Diastolic heart failure results from reduced rate of Ca2+ removal causing delay in myocardial relaxation (b). Ca2+ = calcium ion; Na+ = sodium ion; H+ = hydrogen ion; Mn2+ = manganese ion; NCX = sodium–calcium exchangers; NHE-1 = sodium hydrogen exchanger; RyR = ryanodine receptors; PLB = phospholamban; SERCA = sarcoplasmic reticulum calcium adenosine triphosphatase. 10 Singh et al.: Manganese-Enhanced MRI of the Heart FIGURE 8: Manganese-enhanced magnetic resonance imaging in ischemic and nonischemic Cardiomyopathy. Potential Clinical Applications and Future Directions could play a role in the assessment of reversible causes of car- diomyopathies, which show transient myocardial dysfunction. could play a role in the assessment of reversible causes of car- diomyopathies, which show transient myocardial dysfunction. The potential clinical applications of MEMRI are numerous and include diagnosis, risk stratification, and management of a range of patients with cardiac disease. This technique has particular implications for diagnosis, especially for those with an uncertain or subclinical cardiomyopathy. This could include a range of cardiomyopathies including genetic causes of cardiomyopathy with variable penetrance or athlete’s heart. Athlete’s heart is a term referring to a constellation of electri- cal, functional, and structural remodeling that accompanies regular athletic training. This is an important physiological adaption, which helps the physical performance of athletes. What was initially thought to be a benign adaptation to endurance training, we now know that 20% of elite athletes demonstrate residual cardiac chamber enlargement despite detraining.84 This raises the question of whether athlete’s heart is truly benign and how best to identify those at risk. Thus, the ability to diagnose an underlying cardiomyopathy prior to gross left ventricular dysfunction, will allow for early detection and prompt treatment initiation, which may have an impact on outcomes. However, this has yet to be established for MEMRI. CORONAVIRUS DISEASE 2019. During the coronavirus disease 2019 (COVID-19) pandemic, there was major concern surrounding the extent of cardiac involvement and its consequences in patients with COVID-19. Myo- cardial injury was commonly seen in patients hospitalized with COVID-19 and correlated with disease severity and poor clinical outcomes.82 Initial studies also reported abnormal cardiac imaging findings83 although many patients had concomitant cardiovascular risk factors and comorbidities, and there was an absence of appropriate comparator control subjects. Indeed, cardiovascular risk factors and comorbidities accounted for many of the observed abnormalities, and subse- quent studies demonstrated that there was no excess left ven- tricular dysfunction between co-morbidity matched control subjects and those who had been hospitalized with, and recovered from, COVID-19.31 However, patients did have a higher prevalence of right ventricular dysfunction, likely attributable to recent severe viral pneumonia and consequent pulmonary hypertension. The absence of persistent COVID- 19-induced myocardial dysfunction was also confirmed MEMRI31 (Fig. 9). Left ventricular cardiac function and myocardial manganese uptake in patients hospitalized with COVID-19 were similar to that of age, sex and co-morbidity matched control subjects. Heart Failure Short-axis views of gadolinium-enhanced images (a), native T1 (b) and 30-minute post-manganese T1 maps (c) in patients with dilated and hypertrophic cardiomyopathy. Singh et al.: Manganese-Enhanced MRI of the Heart FIGURE 8: Manganese-enhanced magnetic resonance imaging in ischemic and nonischemic Cardiomyopathy. Short-axis views of gadolinium-enhanced images (a), native T1 (b) and 30-minute post-manganese T1 maps (c) in patients with dilated and hypertrophic cardiomyopathy. cardiomyopathies and could potentially be used to assess a wider range of cardiomyopathy phenotypes, although this has yet to be established. of hypertrophy was achieved using an infusion of isoprotere- nol. Following manganese dipyridoxyl diphosphate adminis- tration, there was clear reductions in myocardial T1 relaxivity and reduced manganese uptake in both the septum and the free-wall of the left ventricle, mimicking hypertensive heart disease.81 HYPERTROPHIC CARDIOMYOPATHY. Hypertrophic cardio- myopathy is a primary myocardial disorder characterized by myocyte remodeling, disorganization of sarcomeric proteins, impaired energy metabolism and altered cardiac contractility. Genetic mutations in patients with hypertrophic cardiomyop- athy result in increased mitochondrial activity and altered myofilament calcium sensitivity involving both calcium- dependent and independent processes,78 resulting in increased left ventricular wall thickness with associated diastolic and systolic dysfunction. Furthermore, dysfunctional calcium han- dling appears to precede alterations in metabolic activity and is associated with an increased risk of arrhythmogenesis.78 To date there has only been one study assessing MEMRI in patients with hypertrophic cardiomyopathy. This demonstrated reduced myocardial manganese uptake in areas of hypertrophy, suggesting abnormal myocardial calcium han- dling (Fig. 8). All patients had normal left ventricular func- tion, suggesting that abnormal myocardial calcium handling was predominantly due to diastolic impairment. Diastolic dysfunction in hypertrophic cardiomyopathy is multifactorial and includes prolonged and disordered ventricular relaxation, decreased chamber compliance and abnormal calcium cycling. Myocardial fibrosis is directly linked to the increased risk of ventricular tachyarrhythmia and sudden cardiac death.79 Native T1 is often abnormal, indicating early fibrosis and has shown potential in adding to current risk stratifica- tion.80 There are no animal models of hypertrophic cardio- myopathy, but MEMRI has been evaluated in a murine model of myocardial hypertrophy. Pharmacological induction Interestingly, manganese uptake was reduced in areas of hypertrophy and virtually absent in areas of severe fibrosis, demonstrating a pattern of uptake similar to that of bloo- dpool. Heart Failure This suggests that MEMRI can distinguish between dysfunctional and nonviable cardiomyocytes as seen in patients with transmural acute myocardial infarction.32,61 This ability to differentiate and track viable myocardium 11 Journal of Magnetic Resonance Imaging Potential Clinical Applications and Future Directions This may suggest that MEMRI may therefore have the potential to detect subclinical myocar- dial dysfunction. The ability to detect altered calcium handling over time and quantify cellular myocardial function directly may trans- form our ability to assess myocardial function, enabling early detection and prognostication. This may be an invaluable non-invasive method of monitoring disease progression in various nonischemic cardiomyopathies. With optimization, this technique has potential to allow individualization of heart failure treatment, assessment of treatment efficacy and FIGURE 9: Cardiac magnetic resonance features of patients hospitalized with coronavirus disease-19. Magnetic resonance imaging findings in patients recovering from COVID-19 infection compared to age-, sex-, and co-morbidity-matched volunteers. MEMRI = manganese-enhanced magnetic resonance imaging; LV = left ventricle; RV = right ventricle. FIGURE 9: Cardiac magnetic resonance features of patients hospitalized with coronavirus disease-19. Magnetic resonance imaging findings in patients recovering from COVID-19 infection compared to age-, sex-, and co-morbidity-matched volunteers. MEMRI = manganese-enhanced magnetic resonance imaging; LV = left ventricle; RV = right ventricle. 12 Singh et al.: Manganese-Enhanced MRI of the Heart targeting optimal therapy to those most likely to benefit. A randomized controlled trial has confirmed that 40% of patients that have recovered from dilated cardiomyopathy (resolution of left ventricular ejection fraction) will “relapse” following discontinuation of heart failure medication.85 The ability to detect subclinical myocardial calcium mishandling in this population could potentially identify which individuals should continue with their heart failure treatment. Such applications are conceptual and speculative but MEMRI does now provide the opportunity to explore such approaches in future studies and potentially provide a more targeted or per- sonalized approach to the treatment and management of our patients with cardiomyopathy. fully understood and may involve improvements in cardiac energetics.89 With optimization, this technique has potential to track treatment response noninvasively. The benefits could include individualization of heart failure treatment and targeting optimal therapy to those most likely to bene- fit. Furthermore, this can be potentially be used to assess a wider range of cardiomyopathy phenotypes. MEMRI has been recently assessed in patients with takotsubo syndrome (NCT04623788). It was long thought that there is complete recovery of cardiac function in patients with takotsubo syndrome. Potential Clinical Applications and Future Directions We now know that not only do patients demonstrate ongoing symptoms with abnormalities of cardiac energetics, but they are also at higher risk of mor- bidity and mortality, similar to those with acute myocardial infarction.90 Singh et al demonstrate profound abnormality in myocardial manganese uptake in the acute phase of takotsubo syndrome, which persists during convalescence, despite restoration of left ventricular function (Singh et al, unpublished data). This is the first description of abnormal myocardial calcium handling in takotsubo syndrome. Furthermore, persistent myocardial calcium mishandling may suggest patients develop a long-term “heart failure” like syndrome, which could explain ongoing symptoms adverse long-term outcomes. There remains considerable interest in the development of therapies to improve the recovery of the cardiac function following myocardial infarction and coronary revasculariza- tion. MEMRI can identify viable myocardium within the peri-infarct region and therefore presents a biomarker measure of novel treatment interventions to reduce infarct size. In the field of myocardial stem cell therapy, manganese uptake can be used as a measure of successful delivery and function of implanted stem cells as demonstrated following human amni- otic mesenchymal stem cell delivery in a porcine model of acute myocardial infarction.86 It would be of interest to see whether MEMRI can detect myocardial calcium handling in reversible, focal cardiac pathologies and, more importantly, whether this as well as conventional measures of cardiac function recover during convalescence. An ongoing study is investigating the role of this technique in patients with acute myocarditis (NCT04623788). One in 20 patients with acute myocarditis will go on to develop heart failure91 and earlier detection of abnormal myocardial calcium handling may help guide earlier treatment to prevent gross left ventricular systolic dysfunction and thereby improve outcomes. In the past, we have been unable to quantify cellular myocardial function. With MEMRI, we are now able to assess intracellular myocardial calcium handling. The presence of late-gadolinium enhancement has been proven to carry a poor prognosis in different cardiac conditions such as ische- mic cardiomyopathy, dilated cardiomyopathy and hypertro- phic cardiomyopathy.79,87 However, to date, there has been no assessment of the prognosis of patients with dysfunctional myocardial calcium handling and it will be important to establish whether abnormal myocardial manganese uptake is associated with adverse outcomes and can provide useful prognostic information. Ongoing Studies Beyond the myocardium, manganese-based contrast media have the potential to provide functional assessment in other organs, such as the kidneys, pancreas, and brain. Renal, pancre- atic, and brain tissue have substantial manganese uptake (Fig. 2), and this could provide a noninvasive measure of cellu- lar calcium handling prior to the onset of renal failure, pancre- atic insufficiency or disease monitoring in neurodegenerative conditions. Patients with diabetes mellitus are at high risk of developing heart failure and often present with breathlessness, despite normal gross cardiac function.88 The use of manganese- enhanced magnetic resonance is being assessed in the detection of preclinical cardiac dysfunction in patients with diabetes mellitus with normal left and right ventricular func- tion. Early detection of altered calcium handling in at-risk cardiomyopathy may enable initiation of preventative or disease-modifying therapy earlier than previously possible, which has potential to improve long-term clinical outcomes. A current study is investigating the role of sodium-glucose transporter 2 (SGLT-2) inhibitor therapy in the treatment of heart failure in patients with and without diabetes mellitus with the use of MEMRI (NCT04591639). The mechanism in which SGLT-2 inhibitors exert beneficial outcomes is not KIDNEYS. L-type calcium channels are expressed and are of significance in the renal cortical preglomerular vessels, juxtamedullary efferent arterioles, and outer medullary vasa recta. As such, manganese uptake and retention into the renal cortex via calcium channels in viable renal issue has been demonstrated and long recognized.3 Currently, renal function is assessed indirectly, using serum markers such as, creatinine, 13 Journal of Magnetic Resonance Imaging urea, and estimated glomerular filtration rate. However, this is not suitable to detect early stages of disease and is fre- quently inaccurate when compared to reference methods. This is likely related to nonfunctional factors, including unmeasured muscle mass and tubular secretion of creati- nine.92 There is an urgent need to develop a noninvasive technique to assess renal function and viability. biological mechanisms of manganese developed in conjunc- tion with advances in its uses for imaging purposes, beginning in the early 1980s when its accumulation, permeability and calcium channel competition in nerve terminals was discov- ered. Synaptic manganese is taken up by the postsynaptic neuron through any of a number of potential calcium- permeable channels or receptors and is repackaged for further transport propagation. Ongoing Studies Jiang et al have implemented a T1-mapping method to obtain quantitative information both dynamically and over a range of manganese chloride concentrations in a murine model of unilateral renal artery stenosis.93 They detected decreased cellular viability and discerned the regional responses to renal artery stenosis. Early human clinical stud- ies94 have described visual uptake of manganese by renal tis- sue, but dedicated renal imaging using MEMRI has yet to be investigated. There were significant issues with manganese-based brain imaging due to the fact that manganese cannot cross an intact blood–brain barrier. Early studies involved administer- ing manganese chloride via a peripheral intravenous injection and reported imaging enhancement. However, this initial application of manganese-enhanced imaging was limited by the need of a chemical or mechanical disruption in the blood–brain barrier. Systemic administration using higher doses was utilized which provided sufficient enhancement, but was complicated by toxicity and required much longer acquisition times.4 Subsequent studies have utilized different delivery techniques, both systemically (fractionated and con- tinuous infusions)97 and locally.98 PANCREAS. Type 1 diabetes is characterized by autoimmune loss of pancreatic beta cell mass leading to metabolic dys- regulation, requiring lifelong insulin therapy. The degree of beta-cell dysfunction at this time often exceeds the percentage beta cell loss, suggesting additional functional impairment in insulin secretion in these patients. The beta-cell deficit in type 1 diabetes mellitus provides a rationale for novel therapeutic strategies aimed at restoring or at least preventing further loss of beta cell mass. In the last decade, MEMRI has been used to detect declining pancreatic beta-cell function in diabetes mellitus. Traditional gadolinium-based contrast media are ineffective at imaging pancreatic beta cells due to their ten- dency to accumulate in the extracellular space. As a calcium analog, manganese is taken up into pancreatic beta cells dur- ing insulin secretion via voltage gated calcium channels, serv- ing as an intracellular contrast agent that represents active insulin secretion.5 Animal models of neurodegenerative diseases with manganese-enhanced imaging mainly focus on anatomy and cytoarchitecture. Following intravenous administration of manganese, contrast in the brain is achieved 24 hours later. Furthermore, reduced manganese uptake was seen in the hip- pocampus and the amygdala in models of Alzheimer’s disease.99 In humans, MEMRI of the brain has been limited. Suto et al100 are the first to use manganese in patients with multi- ple sclerosis in whom there was evidence of blood–brain bar- rier disruption. Ongoing Studies This demonstrated enhancement of active lesions with a temporal and spatial profile distinct from that of gadolinium. Interestingly, chronic lesions did not enhance. The mechanism of manganese accumulation is not yet fully understood. Further study is needed to understand the mech- anism of contrast enhancement as well as cell uptake. Mouse models of type 1 diabetes mellitus demonstrated a decrease in manganese uptake in T-cell receptor transgenic nonobese diabetic mice, with normal uptake in non-diabetic mice. This mirrored loss of beta-cell function, which was con- firmed by pancreatic insulin measurements and histology.5 Several other preclinical studies have shown potential for non- invasive detection of changes in functional beta-cell mass.95 References 18. Schmidt PP, Toft KG, Skotland T, Andersson K. Stability and trans- metallation of the magnetic resonance contrast agent MnDPDP mea- sured by EPR. J Biol Inorg Chem 2002;7(3):241-248. https://doi.org/ 10.1007/s007750100290. 1. Dass S, Suttie JJ, Piechnik SK, et al. Myocardial tissue characterization using magnetic resonance noncontrast t1 mapping in hypertrophic and dilated cardiomyopathy. Circ Cardiovasc Imaging 2012;5(6):726- 733. https://doi.org/10.1161/CIRCIMAGING.112.976738. 1. Dass S, Suttie JJ, Piechnik SK, et al. Myocardial tissue characterization using magnetic resonance noncontrast t1 mapping in hypertrophic and dilated cardiomyopathy. Circ Cardiovasc Imaging 2012;5(6):726- 733. https://doi.org/10.1161/CIRCIMAGING.112.976738. 19. Scholz TD. Reduction in myocardial high energy phosphate spin lattice relaxation times using manganese. NMR Biomed 1994;7(3): 137-140. https://doi.org/10.1002/nbm.1940070306. 2. Lauterbur PCDM, Rudin AM. Augmentation of tissue water proton spin-lattice relaxation rates by in vivo addition of paramagnetic ions. New York, NY: Academic Press; 1978. 20. Nordhoy W, Anthonsen HW, Bruvold M, et al. Intracellular manganese ions provide strong T1 relaxation in rat myocardium. Magn Reson Med 2004;52(3):506-514. https://doi.org/10.1002/mrm.20199. 3. Kang YS, Gore JC. Studies of tissue NMR relaxation enhancement by manganese dose and time dependences. Invest Radiol 1984;19(5): 399-407. https://doi.org/10.1097/00004424-198409000-00012. 3. Kang YS, Gore JC. Studies of tissue NMR relaxation enhancement by manganese dose and time dependences. Invest Radiol 1984;19(5): 399-407. https://doi.org/10.1097/00004424-198409000-00012. 3. Kang YS, Gore JC. Studies of tissue NMR relaxation enhancement by manganese dose and time dependences. Invest Radiol 1984;19(5): 399-407. https://doi.org/10.1097/00004424-198409000-00012. 21. Southon TE, Grant D, Bjornerud A, et al. NMR relaxation studies with MnDPDP. Acta Radiol 1997;38(4 Pt 2):708-716. 4. Lin YJ, Koretsky AP. Manganese ion enhances T1-weighted MRI dur- ing brain activation: An approach to direct imaging of brain function. Magn Reson Med 1997;38(3):378-388. https://doi.org/10.1002/mrm. 1910380305. 22. Skjold A, Vangberg TR, Kristoffersen A, Haraldseth O, Jynge P, Larsson HB. 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Next Steps S hd Since withdrawal from the European market in 2012 by the marketing-authorization holder, no manganese contrast medium has been clinically available. The reasons for this are multifactorial but the decision was principally driven by the lack of large-scale commercial interest, despite promising clin- ical pilot data. It is important to highlight that no safety con- cern caused its withdrawal, simply the lack of clinical demand in hepatobiliary imaging. Furthermore, early clinical studies have established that manganese is safe in various cardiac conditions.31–33 There are currently no available preparations of manganese-based contrast media for widespread clinical use and has never been licensed for cardiac applications. This continues to limit the use of MEMRI in research and clinical settings. However, the formulation of manganese dipyridoxyl The proof-of-concept studies have demonstrated that MEMRI can distinguish between patients with type 2 diabetes mellitus and normoglycemic control subjects.96 An ongoing study (NCT05298735) is investigating the role of MEMRI of the pancreas in patients with type 1 and type 2 diabetes mellitus. This could provide a noninvasive measure of pancre- atic beta-cell function, which holds promise in monitoring of disease progression and the assessment of treatment response. BRAIN. Lin and Koretsky were the first to demonstrate that manganese contrast can be used as a noninvasive direct mea- surement of neuronal function.4 Understanding of the 14 Singh et al.: Manganese-Enhanced MRI of the Heart Am J Physiol Gastrointest Liver Physiol 2022;322(1):G79-g92. https:// doi.org/10.1152/ajpgi.00299.2021. diphosphate for clinical use is clearly feasible, as evidenced by its current use in clinical studies (Table 1). The provision and availability of manganese-based contrast media is likely to change with the re-emergence of commercially available prep- arations anticipated in the near future. Am J Physiol Gastrointest Liver Physiol 2022;322(1):G79-g92. https:// doi.org/10.1152/ajpgi.00299.2021. 10. 10. Spath NB, Thompson G, Baker AH, Dweck MR, Newby DE, Semple SIK. Manganese-enhanced MRI of the myocardium. Heart 2019;105(22): 1695-1700. https://doi.org/10.1136/heartjnl-2019-315227. 11. Wendland MF. Applications of manganese-enhanced magnetic reso- nance imaging (MEMRI) to imaging of the heart. NMR Biomed 2004; 17(8):581-594. https://doi.org/10.1002/nbm.943. The next step in the development of MEMRI of the heart would be to use this technique in larger clinical pro- grams and multicenter trials. This will allow for further assess- ment of its use across a wider population, different T1-mapping sequences and magnetic resonance scanners, as well as providing best evidence of effectiveness. 12. 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KETIDAKTEPATAN PENJATUHAN PIDANA PENJARA TERHADAP PENYALAHGUNA NARKOTIKA

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Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Kata kunci: Rehabilitasi; Pidana Penjara; Penyalahguna Narkotika Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Abstrak Penelitian ini bertujuan untuk menganalisis esensi keberadaan UU Narkotika dan ratio legis Pasal 103 UU Narkotika. Secara faktual, terdapat beda tafsir dan beda pendapat antara penegak hukum tentang penerapan UU Narkotika. Hal ini terbukti pada putusan pengadilan, yang menjatuhkan putusan pidana penjara dan rehablitasi, walaupun pidana penjara lebih besar jumlahnya. Beda tafsir antara penegak hukum mengakibatkan belum tercapainya tujuan diberlakukannya UU Narkotika berkaitan dengan penyalahguna narkotika. Metode penelitian yang digunakan dalam artikel ini adalah penelitian normatif dengan menggunakan pendekatan perundang- undangan (Statute Approach). Analisis terhadap rumusan masalah dilakukan secara preskriptif dengan menggunakan penafsiran Gramatikal dan penafsiran Sistematis. Ketentuan hukum yang dianalisis adalah UU Nomor 35 Tahun 2009 Tentang Narkotika dan Surat Edaran Mahkamah Agung Nomor 4 Tahun 2010 tentang Penempatan Penyalahgunaan. Kesimpulan dari artikel ini menunjukkan bahwa Pasal 103 UU Narkotika mengandung unsur ratio legis yang tepat untuk dijadikan acuan bagi aparat penegak hukum untuk memberikan atau menjatuhkan hukuman rehabilitasi terhadap penyalahguna narkotika. Fakta lain menunjukkan bahwa penjatuhan pidana penjara terhadap penyalahguna narkotikaternyata kurang efisien karena tidak mampu menimalisasi jumlah penyalahguna narkotika. Penjatuhan pidana penjara terhadap penyalahguna narkotika dinilai tidak tepat. 177 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Keywords: Rehabilitation; Imprisonment; Narcotics Abuser Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 1 Anang Iskandar ,Pengguna Narkoba Wajib di Rehabilitasi Bukan di Penjara, kompasiana.com,31 Maret 2014 pukul 18:53, diakses 31 Maret 2020 pukul 18:53. Abstract This article aims to analyze the essence of the existence of the Narcotics Law and legis ratio Article 103 of Narcotics Law. Factually, there are different interpretations and differences of opinion between law enforcement agencies regarding the application of Narcotics Law. This is evident in the court's decision, which handed down a sentence of imprisonment and rehabilitation, even though the prison sentence was greater in number. The different interpretations between law enforcers result in not achieving the goal of the enactment of the Narcotics Law relating to narcotics abusers. The research method used in this article is a normative study using the statute approach. Analysis of the formulation of the problem is done prescriptive using Grammatical interpretation and Systematic interpretation. The legal provisions analyzed were Law Number 35 of 2009 concerning Narcotics and Supreme Court Circular Letter Number 4 of 2010 concerning Placement of Abuse. The conclusion of this article shows that Article 103 of the Narcotics Law contains the right ‘ratio legis’ element to be used as a reference for law enforcement officials to give or impose rehabilitation sentences on narcotics abusers. Other facts show that the imprisonment of narcotics abusers is less efficient because it is unable to minimize the number of narcotics abusers. The imprisonment of narcotics abusers is considered inappropriate. 178 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 2Anang Iskandar,Hakim Wajib Memvonis Rehabilitasi bagi Penyalah Guna Narkotika, OpiniIlustrasi PA Ilustrasi PA 6 November 2017 diakses 5 Juli 2020 pukul 08 30 WIB p , , p 3 Anton Sudanto, Penerapan Hukum Pidana Narkotika Di Indonesia, Jurnal Hukum : Anang Iskandar,Hakim Wajib Memvonis Rehabilitasi bagi Penyalah Guna Narkotika, OpiniIlustrasi---PA Ilustrasi---PA, 6 November 2017, diakses 5 Juli 2020 pukul 08.30 WIB. 3 Adil Vol 7 No 1, Universitas YARSI, 2017 Jakarta, hlm, 138-161. https://dx.doi.org/10.33476/ajl.v8i1.457 p p 3 Anton Sudanto, Penerapan Hukum Pidana Narkotika Di Indonesia, Jurnal Hukum : Adil Vol 7 No 1, Universitas YARSI, 2017 Jakarta, hlm, 138-161. 3 Anton Sudanto, Penerapan Hukum Pidana Narkotika Di Indonesia, Jurnal Hukum : Adil Vol 7 No 1, Universitas YARSI, 2017 Jakarta, hlm, 138-161. https://dx.doi.org/10.33476/ajl.v8i1.457 p 3 Anton Sudanto, Penerapan Hukum Pidana Narkotika Di I Adil Vol 7 No 1, Universitas YARSI, 2017 Jakarta, hlm, 138-161. 2Anang Iskandar,Hakim Wajib Memvonis Rehabilitasi bagi Penyalah Guna Narkotik OpiniIlustrasi---PA Ilustrasi---PA, 6 November 2017, diakses 5 Juli 2020 pukul 08.30 WIB. A. Latar Belakang Diterbitkannya Undang-Undang Nomor 35 Tahun 2009 tentang Narkotika yang selanjutnya disebut UU Narkotika merupakan salah satu langkah pemerintah guna melawan jumlah penyalahgunaan narkotika yang semakin mengkhawatirkan di Indonesia. Tujuannya yaitu mendukung kepentingan ilmu kesehatan dan pengetahuan dengan menjamin ketersediaan narkotika, mencegah penggunaan narkotika yang tidak sesuai aturan (penyalahgunaan narkotika), dan memberantas peredaran gelap narkotika. Sekitar 4 juta orang Indonesia telah mengonsumsi narkoba, narkoba yang dikonsumsi merupakan narkoba jenis baru berbentuk sintetis ataupun illegal. Penegakan hukum terhadap kasus pidana narkotika telah dilakukan secara maksimal oleh aparat penegak hukum dan telah banyak yang mendapat kekuatan hukum tetap (putusan) di pengadilan. Adanya penegakan hukum ini diharapkan dapat menjadi pencegah maraknya kasus narkoba, tetapi hal yang terjadi malah sebaliknya kasus narkoba menjadi semakin meningkat menjangkit jutaan orang Indonesia. Penanganan kasus penyalahgunaan narkoba di Indonesia kebanyakan diberikan sanksi badan ataupun denda, namun di sisi lain rehabilitasi menjadi pilihan hakim dalam memutus kasus penyalah guna narkotika. Contohnya pada kasus penyalahguna narkoba yang dilakukan artis Ridho Rhoma, Iwa K dan Ello yang mendapat hukuman untuk direhabilitasi. Hal ini tentu menjadi aneh ketika kasus yang sama memiliki putusan yang berbeda-beda, padahal dasar dalam memutuskan kasus narkoba atau narkotika hakim telah berpedoman pada Undang- undang ‘khusus’ yaitu UU Narkotika. Namun di sisi lain seperti yang disampaikan oleh Anang Iskandar, secara fakta hakim memiliki kewenagan ekstra, kewenangan ekstra yang dimaksud yakni, 179 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 179 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 80-8842 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 , , , , //dx.doi.org/10.33476/ajl.v8i1.457 Istri Mas Candra, Perlindungan Hukum Terhadap Penyalahgunaan Narkotika Dengan Berlakunya Undang-Undang Nomor 35 Tahun 2009 Tentang Narkotika, Jurnal Magister Hukum Universitas Udayana Denpasar Vol 1 No 1, 2012, hlm 20. 4 Hafied Ali Gani, Rehabilitasi Sebagai Upaya Depenalisasi Bagi Pecandu Narkotika, Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 j y g g 5 Istri Mas Candra, Perlindungan Hukum Terhadap Penyalahgunaan Narkotika Dengan Berlakunya Undang-Undang Nomor 35 Tahun 2009 Tentang Narkotika, Jurnal Magister Hukum Universitas Udayana Denpasar Vol 1 No 1, 2012, hlm 20. Jurnal Hukum Mei 2015, Universitas Brawijaya Malang, 2015, Malang, hlm 1-20 4 Hafied Ali Gani, Rehabilitasi Sebagai Upaya Depenalisasi Bagi Pecandu Narkotika, Jurnal Hukum Mei 2015, Universitas Brawijaya Malang, 2015, Malang, hlm 1-20 5 Istri Mas Candra, Perlindungan Hukum Terhadap Penyalahgunaan Narkotika Dengan Berlakunya Undang-Undang Nomor 35 Tahun 2009 Tentang Narkotika, Jurnal Magister Hukum Universitas Udayana Denpasar Vol 1 No 1, 2012, hlm 20. DOI: https://doi.org/10.24843/JMHU.2012.v01.i01.p01 Universitas Udayana Denpasar Vol 1 No 1, 2012, hlm 20 DOI: https://doi.org/10.24843/JMHU.2012.v01.i01.p01 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana hakim dapat menjatuhkan hukuman sesuai Pasal 10 Kitab Undang-undang Hukum Pidana (KUHP) dan menyatakan tidak bersalah dan membebaskan. Kewenangan ekstra di sini bersifat fakultatif sehingga hal ini menjadi dasar hakim memberikan saknsi badan ataupun denda terhadap penyalah guna narkotika. Tentunya juga dengan pemberian sanksi badan terhadap penyalah guna narkotika menyebabkann keuangan negara tidak stabil karena penjara digunakan terhadap yang bukan peruntuknya.2 Penelitian ini terkait dengan penelitian Anton Sudanto (2017) yang mengangkat bagaimana penerapan hukum pidana dan pengaturannya mengenai tindak pidana narkoba di Indonesia. Dalam penelitiannya tersebut dijelaskan bahwak konsep dari hukum pidana untuk narkotika itu sendiri mencakup tindakan krimininal, hukum pidana dan non-pidana (penal). Tindakan kriminal merupakan ilmu penanggulangan kejahatan yang dapat dilakukan dengan memadukan penerapan sarana pidana dan pencegahan tanpa menggunakan sarana pidana. Tindakan Hukum pidana adalah upaya penanggulangan kejahatan dengan menggunakan sarana pidana. Sedangkan terkait tindakan non pidana adalah tindakan pencegahan sebelum terjadinya kejahatan.3 Sedangkan penelitian oleh Hafied Ali Gani (2015) tersebut menulis konsep yang hampir mirip dengan tulisan penelitian ini yaitu mengenai hukuman rehabilitasi bagi pecandu narkotika, yang dasar pembahasannya juga berlandaskan Pasal 103 UU Narkotika mengenai kewenangan hakim untuk menjatuhkan hukuman rehabilitasi bagi pecandu narkotika yang dapat terbukti atau tidak terbukti. Penelitian tersebut menjelaskan bahwa rehabilitasi dapat dijadikan upaya depenalisasi, rehabalitasi dianggap maatregel atau sebagai sanksi tindakan, disebut juga sebagai sanksi yang ada dalam undang-undang narkotika, artikel ini juga membahasa mengenai sanksi pidana bagi penyalahguna narkotika. Hafied Ali juga 180 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 180 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 8842 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 U ve s tas Udaya a e pasa Vo No , 0 , 0. DOI: https://doi.org/10.24843/JMHU.2012.v01.i01.p01 j y g g 5 Istri Mas Candra, Perlindungan Hukum Terhadap Penyalahgunaan Narkotika Denga B l k U d U d N 35 T h 2009 T t N k tik J l M i t H k 6 Roni Gunawan Raja Gukguk, Nyoman Serikat Putra Jaya, Tindak Pidana Narkotika Sebagai Transnasional Organized Crime, Jurnal Pembangunan Hukum Indonesia Vol 1, No 3, Fakultas Hukum Universitas Diponegoro, 2019, Semarang, hlm 337-351. https://doi.org/10.14710/jphi.v1i3.337-351 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Hal yang berbeda lagi diangkat Roni Gunawan Raja Gukguk dan Nyoman Serikat Putra Jaya (2019) yang lebih membahas mengenai perkembangan kejahatan narkotika sebagai salah satu kejahatan transnasional, bagaimana upaya pemerintah dalam memberantas peredaran narkoba, dan bagaimana efek dari penyalahgunaan narkotika pada saat ini sangat meresahkan semua umat manusia, karena pada saat ini narkotika adalah sebuah momok bagi seluruh bangsa pada umumnya dan bangsaIndonesia pada khususnya.6 Penelitian ini lebih menekankan bagaimana pemerintah Indonesia dalam memerangi atau memberantas kejahatan narkotika. Berdasarkan uraian diatas, penelitian ini ditulis dengan tujuan agar dapat menjawab isu hukum yang ditulis dan menjawab permasalahan yang belum diangaky pada penelitian sebelumnya seperti menganalisis unsur-unsur yang terkandung dalam Pasal 103 UU Narkotika sehingga Pasal 103 UU Narkotika benar-benar dapat dijadikan acuan oleh aparat hukum dalam menjatuhkan hukuman terhadap penyalahguna narkotika. Sehingga aparat hukum bisa secara seragam untuk menjatuhkan hukuman rehabilitasi bagi penyalahguna narkotika yang terbukti ataupun tidak terbukti bersalah menggunakan narkoba, dan hukuman pidana penjara dapat dinilai tidak tepat sebagai hukuman yang dapat diterapkan pada penyalahguna narkotika serta tidak ada lagi pembedaan pemberian hukuman terhadap penyalahguna narkotika. Tujuan dari penelitian adalah untuk menganalisis esensi keberadaan UU Narkotika dan ratio legis Pasal 103 UU Narkotika. Secara faktual, terdapat beda tafsir dan beda pendapat antara penegak hukum tentang penerapan UU Narkotika. p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana berpendapat bahwa rehabilitasi dapat melindungi masyarakat dan mewujudkan efektivitas dalam menyelesaikan permasalahan narkotika.4 Meskipun memiliki konsep pemikiran yang hampir sama mengenai rehabilitasi yang didasarkan pada Pasal 103 UU Narkotika, artikel ini memiliki perbedaan yang sangat signifikan, pembahasan dalam artikel ini lebih khusus membahas mengenai Ratio Legis pada Pasal 103 UU Narkotika sehingga Pasal 103 dapat dikatakan sesuai dengan tujuan keberadaan UU Narkotika. Selain itu problematika hukum yang terjadi dalam artikel ini yaitu adanya beda tafsir dan beda pendapat antara penegak hukum atas penerapan berlakunya UU Narkotika. Sehingga tepat tidaknya penjatuhan pidana terhadap penyalah guna narkotika perlu dianalisis. Adapun penelitian Istri Mas Candra (2012) memiliki persamaan isu hukum yang dijadikan latar belakang yakni pemberian sanksi yang berberda terhadap penyalahgunaan narkotika yang dianggap sebagai korban diri sendiri. Penelitian tersebut khusus membahas putusan hakim terhadap penyalahgunaan narkotika dengan berlakunya UU Narkotika berpacu pada Pasal 103 UU Narkotika, dalam artikel tersebut menjawab relevansi perliindungan hukum melalui rehabilitasi terhadap korban penyalahgunaan narkotika, bahwa rehabilitasi tidak lepas dari ide yang mendasari perlindungan hukum terhadap penyalahgunaan narkotika sesuai dengan Pasal 55, Pasal 56, Pasal 103 dan Pasal 127. 5 Penelitian ini hanya mengkaji relevansi hukuman rehabilitasi terhadap penyalahgunaan narkotika yang berdasarkan pasal 55, 56, 103 dan Pasal 127 UU Narkotika. Pembahasannya penelitian ini lebih bervariasi karena tidak hanya sekedar membahas mengenai hukuman rehabilitasi terhadap penyalahgunaan narkotika, melainkan membahas mengenai unsur-unsur yang terkandung dalam Pasal 103 UU Narkotika sehingga pasal ini benar-benar dapat dijadikan acuan untuk memberikan hukuman rehabilitasi terhadap penyalahgunaan narkotika. 181 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 2. Apakah tepat penjatuhan pidana penjara terhadap penyalah guna narkotika? C. Metode Penelitian Penelitian ini merupakan penelitian hukum normatif. Penelitian ini menggunakan pendekatan perundang-undangan (Statute Approach). Data yang dalam penelitian ini adalah data sekunder, yakni menggunakan bahan hukum primer dan bahan hukum sekunder. Bahan hukum primer merupakan produk hukum seperti peraturan perundang-undangan. Sementara bahan hukum sekunder terdiri dari jurnal dan buku yang relevan untuk dianalisis sesuai dengan isu yang dijadikan masalah dalam penelitian ini. Data yang terkumpul disusun, diolah dan dianalisis menggunakan analisis prespektif dengan perbandingan substansi, sehingga akan menjawab isu atau masalah dalam penelitian ini. 7 Parasian Simanungkalit, Model Pemidanaan Yang Ideal Bagi Korban Pengguna Narkoba Di Indonesia, Jurnal Yustisia Vol 1 No 3, Fakultas Hukum Universitas Sebelas Maret, 2012, Solo, hlm 2. DOI: https://doi.org/10.20961/yustisia.v1i3.10090 8Supriyadi Widodo,dkk, Memperkuat Revisi Undang-Undang Narkotika Usulan Masyarakat Sipil Institute for Criminal Justice Reform 2017 hlm 10 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 2. Apakah tepat penjatuhan pidana penjara terhadap penyalah guna narkotika? C. Metode Penelitian p g y 8Supriyadi Widodo,dkk, Memperkuat Revisi Undang-Undang Narkotika Usulan Masyarakat Sipil, Institute for Criminal Justice Reform,2017, hlm 10. B. Perumusan Masalah Berdasarkan latar belakang yang telah diuraikan maka yang menjadi rumusan masalah dalam penelitian ini adalah : 1. Apa yang menjadi ratio legis Pasal 103 UU Narkotika? 1. Apa yang menjadi ratio legis Pasal 103 UU Narkotika? 182 182 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 a. Ratio Legis Pasal Kasus Penyalahguna Narkotika Narkoba merupakan masalah global, ratusan juta orang yang tercatat dalam United Nation on Drugs and Crime (UNDC) telah menggunakan zat terlarang tersebut7. Dikenal sejak zaman kolonial Belanda,jenis narkotika yang digunakan yaitu candu dengan jenis mentah, masak, obat, resi, jitjing (ampas candu), morpin dan ganja. Saat itu kolonial Belanda mengeluarkan pengaturan melalui eogonine staatblat Tahun 1927 No 278 kemudian diperbaharui dengan staatblat No. 635 Tahun 1927.8 Sejak kemerdekaan Indonesia 17 Agustus 1945, pemerintah Indonesia telah membuat aturan untuk melindungi dan mengatasi peredaran gelap narkotika. Indonesia terus melakukan kebijakan terhadap penanganan kasus Narkotika termasuk dengan mendukung pengesahan dua perjanjian internasional. Pertama perjanjian tentang narkotika dan yang kedua psikotropika, Indonesia mengatur narkotika dan psikotropika kedalam dua aturan yang berbeda yakni, Undang- 183 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 2 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Undang Nomor 5 Tahun 1997 tentang Psikotropika, dan pengaturan mengenai narkotika Undang-undang Nomor 22 Tahun 1997, Indonesia membentuk lembaga koordinasi dalam mengambil langkah kebijakan nasional di bidang narkotika. Badan Koordinasi Narkotika Nasional sebagai lembaga koordinasi menjadi benteng utama dalam memberantas penyalahgunaan narkotika, yang kemudian berdasarkan keputisan Presiden Rl Nomor l7 Tahun 2002 berubah nama menjadi Badan Narkotika Nasional (BNN).9 Permasalahan peredaran narkoba merupkan permasalahan yang mudah cepat berkembang,dengan demikian pemerintah Indonesia terus melakukan pembaharuan terhadap regulasi narkotika agar sesuai dengan perkembangan zaman. Maka lahirlah Undang-undang Nomor 35 Tahun 2009 Tentang Narkotika. Pengguna narkotika dijadikan subyek utama dalam UU Narkotika. Pengguna narkotika dapat disebut sebagai pecandu, pecandu adalah orang yang menggunakan atau menyalahgunakan narkotika dalam keadaan ketergantungan pada narkotika. Selain itu, UU Narkotika juga menyatakan bahwa peecandu narkotika secara fisik maupun psikis wajib diberikan rehabilitasi medis maupun sosial.10 Pada dasarnya undang-undang narkotika menganut konsep strict liability mengandung unsur pertanggungjawaban mutlak. Artinya setiap orang yang memenuhi unsur-unsur pidana pada undang-undang narkotika dapat dipertanggung jawabkan secara mutlak. Sesuai tujuannya undang-undang narkotika sebagai yang tertuang dalam Pasal 4 UU Narkotika. Pasal tersebut bermakna bahwa keberadaan UU Narkotika dijadikan wadah utama untuk menyelamatkan Indonesia dari maraknya penyalahguna narkotika yakni dengan menjamin, dan mencegah penyebaran penyalahguna narkotika. Penyalahguna narkotika diatur dalam Pasal 1 Angka 15 UU Narkotika, penyalahguna adalah orang yang menggunakan narkotika tanpa hak atau melawan hukum. Secara terlampir penggolongan jenis narkotika juga dijelaskan dalam undang-undang narkotika, dimana narkotika digolongkan kedalam tiga jenis yakni 9 Ibid 10Ibid Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 184 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 8842 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 11 Surat Edaran Mahkamah Agung Nomor 4 Tahun 2010 tentang Penempatan Penyalahgunaan Angka 3 huruf a Penyalahgunaan, Angka 3 huruf a. 11 Surat Edaran Mahkamah Agung Nomor 4 Tahun 2010 tentang Penempatan Penyalahgunaan, Angka 3 huruf a. Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana menjatuhkan hukuman rehabilitasi terhadap terdakwa hakim wajib menjelaskan secara tegas dan jelas dimana tempat pelaksanaan rehabilitasi dalam amar putusannya11. Sehingga Ratio Legis dari Pasal 103 Undang-undang Narkotika adalah hakim wajib memutuskan dan menetapkan hukuman rehabilitasi bagi penyalahguna narkotika. Karena keberadaan Pasal 103 UU Narkotika mengandung unsur yang sesuai dengan tujuan diterbitkannya Undang-undang Narkotika untuk mewujudkan cita-cita bangsa Indonesia agar terbebas dari maraknya penyalahgunaan narkotika. Sehingga Ratio Legis dari Pasal 103 Undang-undang Narkotika adalah hakim wajib memutuskan dan menetapkan hukuman rehabilitasi bagi penyalahguna narkotika. Karena keberadaan Pasal 103 UU Narkotika mengandung unsur yang sesuai dengan tujuan diterbitkannya Undang-undang Narkotika untuk mewujudkan cita-cita bangsa Indonesia agar terbebas dari maraknya penyalahgunaan narkotika. Rehabilitasi merupakan salah satu cara pemberantasan atau pencegahan peredaran narkotika dapat dilakukan dengan mudah ketika para penyalahguna narkotika telah sembuh dari sakitnya karena secara otomatis ketika penyalahguna narkotika telah sembuh dari sakitnya, maka mereka akan berhenti membeli obat-obatan terlarang tersebut kepada bandar, sehingga rantai peredaran narkotika tersebut dapat terputus dan teratasi. Maka diharapkan aparat penegak hukum menjadi kompak atau seragam dalam menangani kasus penyalahguna narkotika khususnya Hakim mampu menjadikan Pasal 103 Undang-undang sebagai ratio legis dalam memutus perkara penyalahguna narkotika. b. Penjatuhan Pidana Terhadap Penyalah Guna Narkotika Adalah Tidak b. Penjatuhan Pidana Terhadap Penyalah Guna Narkotika Adalah Tidak Tepat p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana golongan I,II, dan III. Narkotika Golongan I tidak diperbolehkan digunakan untuk kepentingan pelayanan kesehatan. Golongan I hanya dapat digunakan untuk kepentingan pengembangan ilmu pengetahuan dan teknologi. Penggunaan narkotika golongan I juga perlu mendapatkan persetujuan Menteri atas rekomendasi Kepala Badan Pengawas Obat dan Makanan dengan batas jumlah tertentu. Penggunaan narkotika Golongan I jenis ganja yang digunakan untuk diri sendiri diberikan sanksi pidana penjara paling lama 4 Tahunsesuai yang tertuang dalam Pasal 127 ayat (1) huruf a UU Narkotika. Dalam menyelesaikan atau memutus pidana narkotika terkait penyalahgunaan ganja, hakim wajib memberikan rehabilitasi medis dan sosial. Hal yang menarik pada diskresi yang dimiliki hakim untuk memutus pidana terkait Penyalahgunaan ganja yakni pada Pasal 127 ayat (2) jo. Pasal 103 ayat (1) UU Narkotika, pasal tersebut mengandung makna bahwa hakim memiliki hak untuk menentukan hukuman secara alternatif artinya :  Pertama, hakim dapat menjadikan hukuman rehabilitasi sebagai vonis akhir (putusan tetap) terhadap penyalahguna narkotika yang terbukti bersalah dengan memerintahkan yang bersangkutan menjalani pengobatan dan/atau perawatan melalui rehabilitasi .  Kedua, hakim dapat menetapkan bahwa rehabilitasi bukan merupakan putusan akhir (vonis) bagi penylahguna narkotika yang tidak terbukti bersalah. Artinya, meskipun yang bersangkutan tidak terbukti bersalah mereka tetap wajib menjalankan rehabilitasi sebagai bentuk penekanan terhadap penyalahguna narkotika yang tidak terbukti bersalah untuk tetap melakukan perawatan dan pengobatan. Maka dapat disimpulkan point penting dari keberadaan Pasal ini adalah pentingnya rehabilitasi bagi penyalah guna narkotika baik bersalah maupun tidak bersalah. Berdasarkan uraian diatas, Pasal 103 UU Narkotika secara implisit menegaskan bahwa keberadaan UU Narkotika ini telah memberikan paradigma baru terhadap makna dari pecandu narkotika sendiri. Pecandu narkotika tidak selalu menjadi pelaku utama dari sebuah tindak pidana, melainkan juga sebagai korban dari perbuatannya sendiri dalam menyalahgunakan narkotika. Selain keberadaan UU Narkotika, adanya Surat Edaran Mahkamah Agung (SEMA) Nomor 4 Tahun 2010 tentang Penempatan Penyalahgunaan Narkotika juga mendukung adanya pemberian rehabilitasi terhadap penyalahguna narkotika. Hal ini biasanya juga dijadikan acuan oleh hakim dalam menjatuhkan hukuman rehabilitasi. Dalam 185 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 12 Maudy Pritha Amanda dkk. Penyalahgunaan Narkoba Dikalangan Remaja (Adolescent Substance Abuce), Jurnal Prosiding Penelitian dan PPM, Vol 4 No 2, FISIP Universitas Padjajaran Bandung, 2017, Bandung, hlm 341. https://doi.org/10.24198/jppm.v4i2.14392 1 13 Anang Iskandar, Pengguna Narkoba Wajib di Rehabilitasi Bukan di Penjara, kompasiana.com,31 Maret 2014 pukul 18:53,Diperbarui: 31 Maret 2014 pukul 18:53. 14 Oksidelfa Yanto, Jurnal Hukum dan Peradilan ,Peranan Hakim Dalam Pemberantasan Tindak Pidana Narkoba Melalui Putusan Yang Berkeadilan, Volume 6 Nomor 2, Juli 2017 hlm 259 – 278. DOI: http://dx.doi.org/10.25216/jhp.6.2.2017.259-278 15 Sarwirini dan Riza, Rehabilitation of Narcotics Addicts as the Rights to Health, Atlantis Press, Advances in Social Science, Education and Humanities Research (ASSEHR), Vol 131. https://dx.doi.org/10.2991/iclgg-17.2018.34 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Tepat Narcose (narcosis) atau secara bahasa disebut Narkoba berarti dapat memberikan efek bius sehingga penggunanya dengan mudah tertidur atau terbius. Sedangkan menurut Kamus Besar Bahasa Indonesia, narkoba adalah obat yang dapat merangsang rasa kantuk, menenangkan syaraf dan dapat menghilangkan rasa sakit. Pengertian narkoba juga dijelaskan pada Pasal 1 UU Narkotika, dalam pasal tersebut narkoba dijelaskan sebagai obat atau zat berasal dari tanaman/bukan tanaman, sintetis atau semisintetis yang dapat menimbulkan efek ketergantungan, 186 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 842 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 12 Maudy Pritha Amanda dkk. Penyalahgunaan Narkoba Dikalangan Remaja (Adolescent Substance Abuce), Jurnal Prosiding Penelitian dan PPM, Vol 4 No 2, FISIP Universitas Padjajaran Bandung, 2017, Bandung, hlm 341. https://doi.org/10.24198/jppm.v4i2.14392 1 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Sehingga dapat disimpulkan bahwa narkoba atau narkotika pada dasarnya merupakan salah satu jenis obat oral yang dapat digunakan oleh medis dengan takaran tertentu karena dapat membantu mengurangi bahkan menghilangkan rasa nyeri dan menenangkan syaraf.12 Tapi adapun takaran yang harus dipatuhi dalam penggunaannya sehingga obat jenis narkoba ini tidak memberikan efek ketergantungan (kecanduan) dan tidak disalah gunakan keberedaannya. Seiring perkembangan di era modern saat ini, penyalahgunaan narkoba semakin meluas. Penduduk Indonesia dengan jumlah populasi kurang lebih 250 juta, 4 juta dari jumlah tersebut merupakan anak bangsa yang terjerumus dalam penyalahgunaan narkoba.13 Faktor terjadinya penyalahgunaan narkoba ada dua diantaranya : 1. Diri sendiri, rasa ingin tahu yang sangat besar merupakn pemicu utama seorang penyalah guna narkoba menggunakan narkoba tanpa memikirkan efek yang akan timbul dikemudian hari 2. Lingkungan sosial, faktor lingkungan sosial ini meliputi lingkungan keluarga,sekolah ataupun pergaulan dan lain-lain. Penyalahgunaan narkoba ini didasarkan oleh rasa ingggin coba-coba yang kemudian berkelanjut karena dipenuhi sarana dan prasarana14. “Narcotics crime is classified as crime without victim in the perspective of criminology” dalam ilmu krimonologi kejahatan narkotika merupakan kejahatan yang tidak merugikan korban.15 Pernyataan ini muncul terhadap kasus narkotika yang menjadi penyalahguna saja karena pada dasarnya seorang penyalah guna 187 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 842 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana narkotika merupakan seorang yang harus disembuhkan dari penyakit ketergantungan.16 Penggunaan narkotika bagi sendiri merupakan kejahatan tanpa korban, namun berbeda dengan kasus narkotika sebagai pengedar tentu saja tindakan sebagai pengedar narkoba merugikan korban karena pengedar akan mengajak orang lain untuk menggnakan narkotika sehingga menjadi pecandu. Pengaturan tentang narkotika diatur oleh UU Narkotika, dimana tujuan dari adanya undang-undang tersebut secara spesifik yaitu menyelamatkan masyarakat Indonesia dari penyalahgunaan narkotika dengan cara mencegah dan melindungi serta menjamin upaya rehabilitasi medis dan rehabilitasi sosial bagi penyalah guna dan pecandu. 16 Iwan Joko Prasetyo, R. Ayu Erni Jusnita, and Sanhari Prawiradiredja, Therapeutic Communication Narcotics in Rehabilitation Institution “Rumah Kita” Surabaya, Advances in Social Science, Education and Humanities Research, Vol 165. , , 17 www.medcom.id, Iwa K Miliki Tiga Linting Ganja Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 , , 17 www.medcom.id, Iwa K Miliki Tiga Linting Ganja 18 Hamidah Abdurrachman dkk, Disparitas Putusan Hakim dalam Kasus Narkoba, Jurnal Pandecta Vol.7 No 2, Fakultas Hukum Universitas Negeri Semarang, 2012, Semarang, hlm 217. http://dx.doi.org/10.15294/pandecta.v7i2.2388 19 Junaidi, Penerapan Pasal 34,103, dan 127 Ayat (2) dan (3) Undang-Undang No 35 Tahun 2009 Tentang Narkotika Dalam Penyelesaian Perkara Di Pengadilan Negeri Terhadap Penyalahgunaan Narkotika Bagi Diri Sendiri, Jurnal Binamulia Hukum. Vol 8 No 2, Fakultas Hukum Universitas Krisnadwipayana, 2019 Jakarta, hlm 201. https://doi.org/10.37893/jbh.v8i2.84 20 Tatas Nur Arifin, Implementasi Rehabilitasi, Pecandu Narkotika Dalam Undang-undang Republik Indonesia Nomor 35 Tahun 2009 Tentang Narkotika Sebagai Upaya Non Penal Badan Narkotika Nasional, Jurnal Hukum Universitas Brawijaya Malang, 2013, hlm.14. Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Yang menjadi persoalan adalah penyalah guna ataupun pecandu saat ini sering dianggap sebagai subjek kriminal atau pelaku kejahatan bukan sebagai prespektif sebagai korban, contohnya penyalahguna narkotika yang dilakukan oleh kalangan artist seperti, Iwa Kusuma atau yang lebih akrab dikenal Iwa K yang tersandung kasus penyalah guna narkotika. Dimana saat itu Iwa K sebagai terdakwa diketahui memiliki tiga linting ganja, dalam surat tuntutan jaksa pada tanggal 20 September 2017 disampaikan bahwa terdakwa Iwa K terbukti melanggar ketentuan Pasal 127 UU Narkotika. Dalam tuntutan hingga putusannya hakim memutuskan Iwa K dijatuhkan hukuman untuk menjalani rehabilitasi selama 8 bulan. Hal ini sesuai dengan alasan tuntunan Jaksa Penuntut Umum (JPU) bahwa Iwa K hanyalah pecandu narkoba yang harus disembuhkan, sehingga perlu rehabilitasi di Rumah Sakit Ketergantungan Obat atau RSKO.17 Contoh lainnya datang dari seorang terdakwa yang juga tersandung kasus penyalah guna narkotika yakni Tomy Febriansyah. Salah satu penghuni Rumah Tahanan Bangkalan yang masuk penjara akibat penyalah gunaan narkoba. Berdrasarkan putusaan Pengadilan Negeri Bangkalan dengan nomor register perkara 363/Pid.sus/2019/PN.Bkl terdakwa dijatuhkan hukuman penjara selama 1 tahun dikurangi selama masa penangkapan. Putusan ini berdasarkan tuntutan jaksa 188 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 13 November 2019 yang menuntut terdakwa karena terbukti secara sah melanggar ketentuan Pasal 127 UU Narkotika dengan memiliki narkotika jenis sabu seberat 0,32 gram. Menjadi menarik jika dilihat dari uraian kasus diatas, bahwa dua terdakwa yang sama-sama terbukti sebagai penyalah guna narkotika tetapi malah dijatuhkan jenis hukuman yang berbeda, dapat dikatakan aparat penegak hukum telah memiliki beda tafsir terhadap penerapan undang-undang narkotika.18 Faktanya kedua kasus tersebut sama-sama melanggar ketentuan Pasal 127 UU Narkotika, Pasal 127 UU Narkotika biasanya dijadikan dasar oleh Jaksa Penuntut Umum dalam merumuskan tuntutan bagi penyalah guna narkotika. Dalam Pasal 127 UU Narkotika Ayat (1) dijelaskan penyalahguna narkotika untuk diri sendiri golongan I dijatuhkan sanksi pidana penjara paling lama 4 (empat) tahun, penyalah guna narkotika golongan II untuk diri sendiri dijatuhkan sanksi pidana penjara paling lama 2 (dua) tahun, dan penyalah guna narkotika golongan III untuk diri sendiri dijatuhkan pidana penjara paling lama 1 (satu) tahun. Pasal 127 Ayat (2) UU Narkotika bermakna hakim wajib memperhatikan unsur dalam Pasal 54, 55, dan 103 UU Narkotika dalam memustus perkara penyalahgunaan narkotika,19 dimana dalam Pasal 54 sendiri menjelaskan pecandu narkotika dan penyalahguna narkotika wajib menjalani rehabilitasi medis dan rehabilitasi sosial.20 Pasal 55 berisi penjelasan bahwa orang tua/wali penyalahguna narkotika yang masih dibawah umur diwajibkan untuk melapor kepada pusat kesehatan dan lembaga rehabilitasi, sedangkan apabila penyalahguna telah cukup umur maka diwajibkan untuk melaporkan diri sendiri atau diwakilkan oleh keluarganya ke pusat kesehatan dan lemabaga rehabiltasi. Sedangkan Pasal 103 189 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana sendiri secara singkat menjelaskan bahwa hakim dapat memutus untuk memerintahkan penyalahguna narkotika yang terbukti bersalah untuk menjalani rehabiltasi dan dapat menetapkan penyalahguna narkotika yang tidak terbukti bersalah untuk menjalani pengobatan melalui rehabilitasi. Sehingga bisa disimpukan berdasarkan point penting yang tertuang pada tiga Pasal tadi wajib diperhatikan oleh hakim dalam menangani kasus narkotika agar penerapan undang- undang narkotika dapat diterapkan secara tepat dan benar. p y 22 Op.cit Hafied Ali Gani, hlm7. 21 Supriyadi Widodo dkk,op.cit, hlm 20 21 Supriyadi Widodo dkk,op.cit, hlm 20 22 Op.cit Hafied Ali Gani, hlm7. 21 Supriyadi Widodo dkk,op.cit, hlm 20 22 Op.cit Hafied Ali Gani, hlm7. 21 Supriyadi Widodo dkk,op.cit, hlm 20 22 Op cit Hafied Ali Gani hlm7 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Pada Pasal 127 Ayat (3) UU Narkotika dijelaskan kembali secara tegas dan jelas bahwa penyalahguna narkotika yang terbukti atau tidak terbukti sebagai korban penyalah guna tetap menjalani rehabilitasi medis dan rehabilitasi sosial sehingga rehabilitasi terhadap penyalahguna narkotika bersifat wajib. Salah satu faktor adanya beda tafsir antara penegak hukum atau penerapan yang tidak sesuai tentang undang-undang narkotika disebabkan karena undang- undang tentang narkotika belum mengatur perihal gramatur, jumlah atau berat narkotika yang ditemukan di tangan pengguna sebagai barang bukti sering menjadi permasalahan bagi aparat penegak hukum untuk menentukan apakah orang tersebut dari awal dapat ditetapkan sebagai penyalahguna, pecandu ataupun pengguna yang harus diproses atau tidak.21 Rehabilitasi dikenal sebagai proses pengobatan untuk menyembuhkan pecandu narkotika dari ketergantungan, rehabilitasi terhadap pecandu narkotika merupakan bentuk perlindungan sosial yang tujuannya agar pecandu narkotika dapat tertib sosial dan tidak lagi melakukan penyalahgunaan narkotika saat ia kembali ke lingkungan masyarakat. Berdasarkan Undang-Undang Nomor 35 Tahun 2009 tentang Narkotika terdapat 2 (dua) jenis rehabilitasi, yaitu rehabilitasi medis dan rehabilitasi sosial. Pasal 1 angka 16 Undang-Undang Nomor 35 Tahun 2009 tentang Narkotika.22 Rehabilitasi medis merupakan proses menghentikan penyalahgunaan narkotika yang dilakukan dirumah dan dibawah pantuan dokter, sedangkan 190 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 1-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana rehabilitasi sosial dilakukan secara terpadu baik secara fisik,mental, maupun sosial.23 Kegiatan rehabilitasi yang diberikan dapat berupa pembekalan keahlian, atau keberanian dan bekal rohani agar ketika pecandu narkotika kembali ke lingkungan masyarakat dia dapat melindungi dirinya dan tidak memiliki keinginan untuk mengonsumsi narkoba lagi.24 Selain itu dasar penerapan pemidanaan dengan rehabilitasi pada UU Narkotika keberadaan SEMA (Surat Edaran Mahkamah Agung) No.04 Tahun 2010 Tentang penempatan penyalahgunaan dan Pecandu Narkotika Kedalam Lembaga Rehabilitasi Medis dan Sosial mendukung secara jelas pelaksanaan rehabilitasi terhadap penyalahguna narkotika.25 Dalam UU Narkotika memberikan kewenangan terhadap penegak hukum khususnya Hakim untuk merehabilitasi penyalahguna narkotika. Sesuai Pasal 103 UU Narkotika yang menyatakan “Hakim menjatuhkan hukuman rehabilitasi terhadap penyalah guna yang terbukti bersalah, dan menetapkan untuk menjalani rehabilitasi terhadap penyala guna yang tidak terbukti bersalah”. 23 Yohanes Christ, Pemenuhan Hak Bagi Penyalahguna Narkotika Di Yogyakarta, Jurnal Hukum Universitas Atma Jaya Yogyakarkata, 2015, Yogyakarta, hlm.7 y gy gy 24 Alfajriyah, Eddy Rifai, Diah Gusmiati Pelaksanaan Rehabilitasi Sebagai Upaya Penanggulangan Tindak Pidana Narkotika (Studi loka Rehabilitasi Kalianda), Jurnal Ponale Vol 5 No 6, Fakultas Hukum Universitas Lampung, 2017, Lampung, hlm 13. Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 , p g, , p g, 25Andri Winjaya Laksana, Tinjauan Hukum Pemidanaan Terhadap Pelaku Penyalahguna Narkotika Dengan Sistem Rehabilitasi, Jurnal Pembaharuan Hukum, Vol 2 No.1, Fakultas Hukum Universitas Islam Sultan Agung, 2017, Semarang, hlm 10. DOI: http://dx.doi.org/10.26532/jph.v2i1.1417 26Firman Floranta Adonara, Prinsip Kebebasan Hakim dalam Memutus Perkara Sebagai Amanat Konstitusi, Jurnal Konstitusi.Vol 12 No 2, Mahkamah Konstitusi RI, 2015, Jakarta, hal 2. http://dx.doi.org/10.31078/jk1222 27Anang Iskandar,op.cit 23 Yohanes Christ, Pemenuhan Hak Bagi Penyalahguna Narkotika Di Yogyakarta, Jurnal Hukum Universitas Atma Jaya Yogyakarkata, 2015, Yogyakarta, hlm.7 24 Alfajriyah, Eddy Rifai, Diah Gusmiati Pelaksanaan Rehabilitasi Sebagai Upaya Penanggulangan Tindak Pidana Narkotika (Studi loka Rehabilitasi Kalianda), Jurnal Ponale Vol 5 No 6, Fakultas Hukum Universitas Lampung, 2017, Lampung, hlm 13. 25Andri Winjaya Laksana, Tinjauan Hukum Pemidanaan Terhadap Pelaku Penyalahguna Narkotika Dengan Sistem Rehabilitasi, Jurnal Pembaharuan Hukum, Vol 2 No.1, Fakultas Hukum Universitas Islam Sultan Agung, 2017, Semarang, hlm 10. DOI: http://dx.doi.org/10.26532/jph.v2i1.1417 26Firman Floranta Adonara, Prinsip Kebebasan Hakim dalam Memutus Perkara Sebagai Amanat Konstitusi, Jurnal Konstitusi.Vol 12 No 2, Mahkamah Konstitusi RI, 2015, Jakarta, hal 2. http://dx.doi.org/10.31078/jk1222 27Anang Iskandar,op.cit , p g, , p g, 25Andri Winjaya Laksana, Tinjauan Hukum Pemidanaan Terhadap Pelaku Penyalahguna Narkotika Dengan Sistem Rehabilitasi, Jurnal Pembaharuan Hukum, Vol 2 No.1, Fakultas Hukum Universitas Islam Sultan Agung, 2017, Semarang, hlm 10. Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 28 Ibid p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Dengan demikian adanya undang-undang narkotika yang bersifat ‘khusus’ ini aparat penegak hukum khususnya hakim dituntut untuk mampu merefleksikan isi pasal sesuai yang tertuang dalam undang-undang tersebut.26 Karena undang-undang UU Narkotika menganut “double track system” yang artinya pemidanaan bagi penyalah guna yang digunakan untuk dirinya sendiri diberikan hukuman rehabilitasi sedangkan bagi pengedarnya diberikan hukuman penjara hingga pidana mati. Hal ini berlaku untuk seluruh lembaga pengadilan di Indonesia.27 Selain uraian diatas, dapat 191 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana 42 Ketidaktepatan Penjatuhan Pidana Penjara Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 p y g Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana disimpulkan bahwa hakim dituntut mampu menerapkan regulasi UU Narkotika sesuai dengan tujuannya yaitu ‘melindungi ,menyelamatkan, dan menjamin rehbailitasi’ bagi penyalahguna narkoba. Hal ini juga diperjelas pada Pasal 54 UU Narkotika yang mengandung makna penyalahguna dan pecandu narkotika wajib menjalankan rehabilitasi, sehingga bisa disimpulkan seharusnya penyalahguna dan pecandu narkotika berhak direhabilitasi (disembuhkan) bukan untuk dikirim ke dalam penjara. Regulasi narkotika ini diterbitkan sebagai alat untuk menyembuhkan penyakit penyalah guna narkotika dari sakit ketergantungannya. Menurut menteri kesehatan rehabilitasi sosial dan rehabilitasi medis merupakan hal yang ampuh untuk mengatasi banyaknya penyalah guna narkoba. Berdasarkan Pasal 103 ayat 2 UU Narkotika sebagai bentuk konvensi narkotika yang sudah dilakukan amandemen, rehabilitasi sama halnya dengan hukuman penjara. Penjara dianggap sebagai wadah penyebaran penyalahgunaan narkotika yang sistemis dan tidak mampu menyembuhkan pecandu narkotika, dibandingkan rehabilitasi yang dianggap lebih bermanfat bagi penyalahguna, keluarga, bangsa, dan negara daripada hukuman penjara.28 Sehingga penjatuhan pidana bagi penyalah guna narkotika bukanlah tindakan yang dapat dibenarkan karena dinilai tidak tepat, keberadaan Pasal 103 UU Narkotika. Karena Undang-undang Narkotika senidri diterbitkan secara ‘khusus’ dan menganut “double track system” berarti wajib bukan bersifat fakultatif untuk dipatuhi oleh aparat penegak hukum khususnya hakim untuk menerapkan hukuman rehabilitasi dan sebagainya sesuai dengan yang tertuang dalam undang-undang. Selain itu dengan adanya peraturan pelaksana dapat memperkuat tercapainya esensi yang terkandung dalam undang-undang narkotika. Artian lain tujuan melindungi, menyelamatkan dan menjamin rehabilitasi penyalahguna narkotika dapat dicapai. 28 Ibid 192 Ketidaktepatan Penjatuhan Pidana Penjara p-ISSN : 2541-2345 , e-ISSN : 2580-8842 III. PENUTUP Ratio legis dari Pasal 103 Undang-undang Narkotika adalah hakim wajib memutuskan dan menetapkan hukuman rehabilitasi bagi penyalahguna narkotika. Karena keberadaan Pasal 103 UU Narkotika mengandung unsur yang sesuai dengan tujuan diterbitkannya Undang-undang Narkotika yakni, mencegah, melindungi, dan menyelamatkan bangsa Indonesia dari penyalahgunaan narkotika. Penjatuhan pidana terhadap penyalahguna narkotika dinilai tidak tepat karena banyaknya fakta- fakta atau kasus terkait penyalah guna narkotika yang mengalami pembedaan penanganan kasus yaitu ada sebagian kasus penyalah guna narkotika yang dijatuhkan hukuman penjara namun ada juga yang mendapat hukuman untuk rehabilitasi. Maka rehabilitasi medis dan sosial perlu diperhatikan untuk mencegah, menyelamatkan, dan menjamin rehabilitasi penyalah guna narkoba dapat tercapai. Sehingga penjatuhan hukuman rehabilitasi bagi penyalah guna narkoba dapat serentak dilakukan bukan malah menjatuhkan hukuman penjara. Karena penjatuhan hukuman penjara tehadap penyalah guna narkoba bukan solusi yang tepat, terlebih lagi dapat merugikan keuangan negara karena menggunakan penjara bukan pada peruntukannya. Selain itu agar aparat penegak hukum dapat lebih jeli dan tepat dalam melaksanakan kewenangannya. Buku Supriyadi Widodo,dkk. Memperkuat Revisi Undang-undang Narkotika Usulan Masyarakat Sipil. Jakarta Selatan : Institute for Criminal Justice Reform, 2017. Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Jurnal Alfajriyah, Eddy Rifai, Diah Gusmiati Pelaksanaan Rehabilitasi Sebagai Upaya Penanggulangan Tindak Pidana Narkotika (Studi Lokal Rehabilitasi Kalianda), Jurnal Ponale Vol 5 No 6, Fakultas Hukum Universitas Lampung, 2017, Lampung. Andri Winjaya Laksana, Tinjauan Hukum Pemidanaan Terhadap Pelaku Penyalahguna Narkotika Dengan Sistem Rehabilitasi, Jurnal Pembaharuan 193 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Hukum, Vol 2 No.1, Fakultas Hukum Universitas Islam Sultan Agung, 2017, Semarang, hlm 10. DOI: http://dx.doi.org/10.26532/jph.v2i1.1417 Hukum, Vol 2 No.1, Fakultas Hukum Universitas Islam Sultan Agung, 2017, Semarang, hlm 10. DOI: http://dx.doi.org/10.26532/jph.v2i1.1417 Anton Sudanto, Penerapan Hukum Pidana Narkotika Di Indonesia, Jurnal Hukum : Adil Vol 7 No 1, Universitas YARSI, 2017 Jakarta. https://dx.doi.org/10.33476/ajl.v8i1.457 Anton Sudanto, Penerapan Hukum Pidana Narkotika Di Indonesia, Jurnal Hukum : Adil Vol 7 No 1, Universitas YARSI, 2017 Jakarta. https://dx.doi.org/10.33476/ajl.v8i1.457 Firman Floranta Adonara, Prinsip Kebebasan Hakim dalam Memutus Perkara Sebagai Amanat Konstitusi, Jurnal Konstitusi.Vol 12 No 2, Makhkamah Konstitusi RI, 2015, Jakarta, http://dx.doi.org/10.31078/jk1222 Hafied Ali Gani, Rehabilitasi Sebagai Upaya Depenalisasi Bagi Pecandu Narkotika, Jurnal Hukum Mei 2015, Universitas Brawijaya Malang, 2015, Malang. Hamidah Abdurrachman dkk, Disparitas Putusan Hakim dalam Kasus Narkoba, Jurnal Pandecta Vol.7 No 2, Fakultas Hukum Universitas Negeri Semarang, 2012, Semarang. http://dx.doi.org/10.15294/pandecta.v7i2.2388 Istri Mas Candra, Perlindungan Hukum Terhadap Penyalahgunaan Narkotika Dengan Berlakunya Undang-Undang Nomor 35 Tahun 2009 Tentang Narkotika, Jurnal Magister Hukum Universitas Udayana Denpasar Vol 1 No 1, 2012. Denpasar. DOI: https://doi.org/10.24843/JMHU.2012.v01.i01.p01 Iwan Joko Prasetyo, R. Ayu Erni Jusnita, and Sanhari Prawiradiredja, Therapeutic Communication Narcotics in Rehabilitation Institution “Rumah Kita” Surabaya, Advances in Social Science, Education and Humanities Research, Vol 165. Junaidi, Penerapan Pasal 34,103, dan 127 Ayat (2) dan (3) Undang-Undang Nomor 35 Tahun 2009 Tentang Narkotika Dalam Penyelesaian Perkara Di Pengadilan Negeri Terhadap Penyalahgunaan Narkotika Bagi Diri Sendiri, Jurnal Binamulia Hukum. Vol.8 No 2, Fakultas Hukum Universitas Krisnadwipayana, 2019 Jakarta. https://doi.org/10.37893/jbh.v8i2.84 Maudy Pritha Amanda dkk. Penyalahgunaan Narkoba Dikalangan Remaja (Adolescent Substance Abuce), Jurnal Prosiding Penelitian dan PPM, Vol 4 No 2, Fisip Universitas Padjajaran Bandung, 2017, Bandung. https://doi.org/10.24198/jppm.v4i2.14392 Parasian Simanungkalit, Model Pemidanaan Yang Ideal Bagi Korban Pengguna Narkoba Di Indonesia, Jurnal Yustisia Vol 1 No. 3, Fakultas Hukum Universitas Sebelas Maret, 2012, Solo. Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Jurnal DOI:https://doi.org/10.20961/yustisia.v1i3.10090 Roni Gunawan Raja Gukguk, Nyoman Serikat Putra Jaya, Tindak Pidana Narkotika Sebagai Transnasional Organized Crime, Jurnal Pembangunan Hukum Indonesia Vol 1, No 3, Fakultas Hukum Universitas Diponegoro, 2019, Semarang. https://doi.org/10.14710/jphi.v1i3.337-351 Oksidelfa Yanto, Peranan Hakim Dalam Pemberantasan Tindak Pidana Narkoba Melalui Putusan Yang Berkeadilan Jurnal Hukum dan Peradilan Vol 6 Nomor 2, Mahkamah Agung RI, 2015, Jakarta. DOI: http://dx.doi.org/10.25216/jhp.6.2.2017.259-278 194 Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana Ketidaktepatan Penjatuhan Pidana Penjara Terhadap Penyalahguna Narkotika Deni Setya Bagus Yuherawan, Baiq Salimatul Rosdiana p-ISSN : 2541-2345 , e-ISSN : 2580-8842 Sarwirini dan Riza, Rehabilitation of Narcotics Addicts as the Rights to Health, Atlantis Press, Advances in Social Science, Education and Humanities Research (ASSEHR), Vol 131. https://dx.doi.org/10.2991/iclgg-17.2018.34 Sarwirini dan Riza, Rehabilitation of Narcotics Addicts as the Rights to Health, Atlantis Press, Advances in Social Science, Education and Humanities Research (ASSEHR), Vol 131. https://dx.doi.org/10.2991/iclgg-17.2018.34 Tatas Nur Arifin, Implementasi Rehabilitasi Pecandu Narkotika Dalam Undang- undang Republik Indonesia Nomor 35 Tahun 2009 Tentang Narkotika Sebagai Upaya Non Penal Badan Narkotika Nasional, Jurnal Hukum Universitas Brawijaya Malang, 2013, Malang. Yohanes Christ, Pemenuhan Hak Bagi Penyalahguna Narkotika Di Yogyakarta. Jurnal Hukum Universitas Atma Jaya Yogyakarkata, 2015, Universitas Atma Jaya, Yogyakarta. Perundang-undangan Undang-Undang Nomor 35 Tahun 2009 Tentang Narkotika Undang-Undang Nomor 35 Tahun 2009 Tentang Narkotika Surat Edaran Mahkamah Agung Nomor 4 Tahun 2010 tentang Penempatan Penyalahgunaan Jurnal Ius Constituendum | Volume 5 Nomor 2 Oktober 2020 Anang Iskandar.Pengguna Narkoba Wajib di Rehabilitasi Bukan di Penjara. kompasiana.com. Anang Iskandar.Pengguna Narkoba Wajib di Rehabilitasi Bukan di Penjara. kompasiana.com. p Anang Iskandar.Hakim Wajib Memvonis Rehabilitasi bagi Penyalah Guna Narkotika.OpiniIlustrasi---PA www.medcom.id, Iwa K Miliki Tiga Linting Ganja Anang Iskandar.Hakim Wajib Memvonis Rehabilitasi bagi Penyalah Guna Narkotika.OpiniIlustrasi---PA d id I K Miliki Ti Li i G j Anang Iskandar.Hakim Wajib Memvonis Rehabilitasi bagi Penyalah Guna Narkotika.OpiniIlustrasi---PA www.medcom.id, Iwa K Miliki Tiga Linting Ganja Anang Iskandar.Hakim Wajib Memvonis Rehabilitasi bagi Penyalah Guna Narkotika.OpiniIlustrasi---PA www.medcom.id, Iwa K Miliki Tiga Linting Ganja 195

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Development of an Off‐Grid Solar‐Powered Autonomous Chemical Mini‐Plant for Producing Fine Chemicals

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Development of an Off-Grid Solar-Powered Autonomous Chemical Mini-Plant for Producing Fine Chemicals Tom M. Masson+,[a, b] Stefan D. A. Zondag+,[a] Koen P. L. Kuijpers,[b, c] Dario Michael G. Debije,[e] and Timothy Noël*[a, b] Tom M. Masson+,[a, b] Stefan D. A. Zondag+,[a] K Michael G. Debije,[e] and Timothy Noël*[a, b] and passing clouds, a responsive control system was designed that rapidly adapts the flow rate of the reagents to the light received by the reaction channels. Supplying the plant with solar panels, integrated into the module by placing it behind the LSC to utilize the transmitted fraction of the solar irradiation, allowed this setup to be self-sufficient and fully operational off-grid. Such a system can shine in isolated environments and in a distributed manufacturing world, allowing to decentralize the production of fine chemicals. and passing clouds, a responsive control system was designed that rapidly adapts the flow rate of the reagents to the light received by the reaction channels. Supplying the plant with solar panels, integrated into the module by placing it behind the LSC to utilize the transmitted fraction of the solar irradiation, allowed this setup to be self-sufficient and fully operational off-grid. Such a system can shine in isolated environments and in a distributed manufacturing world, allowing to decentralize the production of fine chemicals. Photochemistry using inexhaustible solar energy is an eco- friendly way to produce fine chemicals outside the typical laboratory or chemical plant environment. However, variations in solar irradiation conditions and the need for an external energy source to power electronic components limits the accessibility of this approach. In this work, a chemical solar- driven “mini-plant” centred around a scaled-up luminescent solar concentrator photomicroreactor (LSC-PM) was built. To account for the variations in solar irradiance at ground level Research Article doi.org/10.1002/cssc.202102011 ChemSusChem Introduction ting to those explorations by creating synthesis systems that are fully independent and that can harvest energy directly from the environment.[2,3] From Christopher Columbus to Neil Armstrong, humankind has always been drawn toward the exploration of new territories. Today, our robots are reaching the border of Mars and will keep on extending further.[1] In those isolated and hostile lands, it becomes fundamental to have self-sustaining systems to provide energy, food and medicine. These systems must cope with environmental fluctuations and be energetically independ- ent. As chemists and chemical engineers, we aim at contribu- The sun is a source of energy accessible to the entire planet, and serves as an ideal solution to power chemistry at isolated locations.[4,5] In this regard, the recent improvements in the field of photoredox processes have greatly expanded the photo- chemical toolbox allowing harvesting of visible light wave- lengths and enabling complex and previously elusive chemical bond transformations.[6–8] These tools can be progressively combined with solar chemistry, but fluctuating solar conditions often limit the attractiveness and reliable applicability of this field. To cope with these fluctuations and to ensure reliable production processes, direct control over the reaction and process parameters must be maintained. [a] T. M. Masson,+ S. D. A. Zondag,+ Prof. Dr. T. Noël Flow Chemistry Group, van't Hoff Institute for Molecular Sciences (HIMS), Universiteit van Amsterdam (UvA), Science Park 904, 1098 XH Amsterdam (The Netherlands) E-mail: [email protected] [b] T. M. Masson,+ Dr. K. P. L. Kuijpers, Dr. D. Cambié, Prof. Dr. T. Noël Department of Chemical Engineering and Chemistry, Sustainable Process Engineering, Micro Flow Chemistry & Synthetic Methodology, Eindhoven University of Technology Het Kranenveld, Bldg 14 – Helix, 5600 MB Eindhoven (The Netherlands) [c] Dr. K. P. L. Kuijpers Current address: Technology & Engineering, Janssen R&D Turnhoutseweg 30, 2340 Beerse (Belgium) [d] Dr. D. Cambié Current address: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces Am Mühlenberg 1, 14476 Potsdam (Germany) [e] Dr. M. G. Debije Department of Chemical Engineering and Chemistry, Stimuli-responsive Functional Materials & Devices, Eindhoven University of Technology Groene Loper 3, Bldg 14 – Helix, 5600 MB Eindhoven (The Netherlands) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under https://doi.org/10.1002/cssc.202102011 © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH. © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH Introduction This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Micro/milli-flow technology presents itself as the better option to harvest solar light owing to the large exchange surface area and the possibilities of scaling.[8–12] As a recent invention from our laboratory, the combination of flow chemistry with inexpensive luminescent solar concentrators[13–16] (LSCs) allows for enhanced, solar-driven photochemical reactions.[17,18] Herein, sunlight energy is collected, converted, concentrated and directed towards the reaction channels, maximizing the number of photons reaching the reaction mixture. However, we realized that a significant fraction of the solar spectrum remained unused (i.e., >600 nm).[18] We sur- mised that these photons (up to 1100 nm)[19] otherwise escaping the reactor can be collected with photovoltaic cells (PV) to produce electricity.[20] This observation inspired us to make the first steps towards an off-grid solar-driven mini-plant by integrating an LSC-PM and a solar panel for energy production. The solar energy that is not used for chemical production can then be productively utilized to drive pumps, mass flow controllers, sensors and a regulation system that maintains constant chemical conversion during fluctuating irradiation.[21] Consequently, this mini-plant provides a steady [b] T. M. Masson,+ Dr. K. P. L. Kuijpers, Dr. D. Cambié, Prof. Dr. T. Noël Department of Chemical Engineering and Chemistry, Sustainable Process Engineering, Micro Flow Chemistry & Synthetic Methodology, Eindhoven University of Technology Het Kranenveld, Bldg 14 – Helix, 5600 MB Eindhoven (The Netherlands) [b] T. M. Masson,+ Dr. K. P. L. Kuijpers, Dr. D. Cambié, Prof. Dr. T. Noël Department of Chemical Engineering and Chemistry, Sustainable Process Engineering, Micro Flow Chemistry & Synthetic Methodology, Eindhoven University of Technology Het Kranenveld, Bldg 14 – Helix, 5600 MB Eindhoven (The Netherlands) g y [e] Dr. M. G. Debije Department of Chemical Engineering and Chemistry, Stimuli-responsive Functional Materials & Devices, Eindhoven University of Technology Groene Loper 3, Bldg 14 – Helix, 5600 MB Eindhoven (The Netherlands) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under https://doi.org/10.1002/cssc.202102011 © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Results and Discussion The oxidation of l-methionine to the corresponding sulfoxide (Figure 1B), previously already used for LSC-PM applications,[17] was chosen as a benchmark reaction, since having water as a solvent is easily scalable and safe to operate outdoors. Interestingly, methionine sulfoxide has many biochemical applications,[25] including in studies of cell ageing and oxidative stress, in peptide sciences,[26] and in material sciences and organic synthesis.[27] For example, it can increase the perme- ability of oxygen in water through a polypeptide film, crucial for contact lenses.[28,29] Traditionally, the oxidation of sulfides is done with peroxides, but this method often suffers from over- oxidation to the corresponding sulfones.[30] Using singlet oxy- gen as a green oxidant solves this selectivity issue, but the reaction conditions are not convenient to scale due to limitations associated with mass transfer and light attenuation.[31] To overcome these challenges, the use of micro- flow chemistry presents itself as an easy strategy to perform this photocatalytic, singlet oxygen mediated reaction.[32] Meth- ylene Blue (MB) is used as photocatalyst, as it is not only able to produce singlet oxygen under low photon energy excitation, but also matches perfectly with the wavelength emitted by the LR305 luminophore in the LSC-PM (see Figure 1C). Introduction ChemSusChem published by Wiley-VCH GmbH ChemSusChem 2021, 14, 5417–5423 5417 Research Article doi.org/10.1002/cssc.202102011 ChemSusChem flow of desired chemicals in a self-sufficient manner without external energy supply, save for the sun. Through modelling and simulations, the optimal orientation of the reactor could be determined depending on the global location. With this data and the capability of the LSC-PM to utilize both direct and diffuse light, solar tracking becomes unnecessary. Notably, this easy-to-build setup can be readily adapted to perform a large variety of photochemical transformations under controlled conditions.[17] both oxygen and reagents to match the current incident light intensity following an experimentally established conversion correlation, maintaining constant production quality. The electrical equipment of the mini-plant is powered by solar panels charging a battery, where the battery acts as a power buffer when the solar irradiation fluctuates. The battery can deliver 5 V DC to supply the control system and 230 V AC to supply the MFC and the pump (see Figure 1A). Design of the solar-driven mini-plant The reactor is based on a 15 mL LSC-PM (470×470×8 mm3) module. This reactor is doped with the commercial fluorescent dye Lumogen F Red 305 (BASF, ‘LR305’),[22] which combines high fluorescent yields[23] and excellent photostability[24] to bring the luminescent properties to the polymer. The assembly of the panel is described in the Supporting Information, and adapted from a previously reported method by our group.[17] The concentrated sunlight is guided towards the reaction channels and used to perform the photochemical transformations. The reactor is fed by an HPLC pump (Knauer Azura P4.1S) that introduces the reaction mixture inside the reactor channels. The incoming liquid flow is merged with an oxygen flow controlled by a mass flow controller (Bronkhorst, O2, max. 25 mLmin1, ‘MFC’). To cope with fluctuating irradiation conditions, which are inevitable when using the sun as light source (e.g., passing clouds, day/ night cycles), a control system including a light sensor is attached to the edge of the LSC-PM.[17] The edge emission (EE) from the LSC lightguide is monitored in real-time, and the data is used to adjust the pump-driven flow rates of Figure 1. Working principle of the system. (A) Scheme of the solar mini-plant showing the main components: the LSC-PM, the external solar panel, the reactant feeds, MFC and the flow control system. (B) Oxidation of l-methionine to l-methionine sulfoxide using Methylene Blue (MB) as photocatalyst: 0.1 m l- methionine, 1 mol% MB in H2O. (C) Wavelength conversion scheme LSC-PM and Methylene Blue. The absorption (red area) and emission (green area) of the LR305 dye compared to the absorption spectrum of the MB photocatalyst (blue area). Superimposed on the spectra is the AM 1.5 solar spectrum. Reprinted with permission of Ref. [18]. Copyright 2016 John Wiley and Sons. Figure 1. Working principle of the system. (A) Scheme of the solar mini-plant showing the main components: the LSC-PM, the external solar panel, the reactant feeds, MFC and the flow control system. (B) Oxidation of l-methionine to l-methionine sulfoxide using Methylene Blue (MB) as photocatalyst: 0.1 m l- methionine, 1 mol% MB in H2O. (C) Wavelength conversion scheme LSC-PM and Methylene Blue. The absorption (red area) and emission (green area) of the LR305 dye compared to the absorption spectrum of the MB photocatalyst (blue area). Superimposed on the spectra is the AM 1.5 solar spectrum. Reprinted with permission of Ref. [18]. Copyright 2016 John Wiley and Sons. Energy supply using photovoltaic cells beneath the LSC-PM is more than 10 times greater than the edge surface area (0.221 m2 vs. 0.015 m2). Hence, for these reasons, the second option was preferred. By placing a silicon PV panel at the back side of the LSC-PM, solar photons with wavelengths that are not absorbed by the LSC-PM can be collected by the PV and used to power all the peripheral equipment. In this way, maximum use is made of the solar spectrum. For PV arrays with no solar tracking technology, an important parameter to maximize the yearly productivity is the tilt angle, whose dependency with the site latitude is well established.[33,34] For LSC-PM systems, little is known on how the different tilt angles can affect the reactor productivity. The design considerations commonly adopted for PVs cannot be directly translated to the LSC-PM system as the latter is also capable of harvesting diffuse irradiance.[35] A ray-tracing Monte- Carlo algorithm implemented in Python (PvTrace) was used to determine the fate of each photon reaching the LSC.[36] This model was adapted to determine the best fixed tilt angle for the LSC-PM located in Eindhoven (The Netherlands) (see Figure 2A). A 40° angle for LSC-PM was determined optimal to maximize the yearly overall number of photons reaching the panel (see Supporting Information). We aimed to validate this rationale experimentally by placing a nominal 18 V solar panel below the LSC-PM and measuring the outdoor energy production levels under a 40° angle approximately. Under sunny conditions, the PV can supply up to 13 W, while, when large clouds pass by, the power production drops to about 4 W. Next, to estimate if a stand-alone mini-plant is possible with a single PV below the LSC-PM, the energy consumption of the pumping system is measured with a power meter (Brennenstuhl PM231E). Of all the electric components, the HPLC pump is the major power consumer in the mini-plant and the demand varies with the required flow rate. The power consumption of the system varies between 12–14 W (see Table 1 and Supporting Information for calculations). From these measurements, it is apparent that a single solar panel (0.38×0.51 m2) below the reactor is not sufficient to supply a fully autonomous plant of energy. Design of the solar-driven mini-plant ChemSusChem 2021, 14, 5417–5423 www.chemsuschem.org © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH 5418 www.chemsuschem.org Research Article doi.org/10.1002/cssc.202102011 ChemSusChem Figure 2. (A) Simulation of the tilt angle impact on yearly productivity of the LSC-PM mini-plant. (B) Photographs of the sky conditions at time of the experiment. (C) Experimental setup. Figure 2. (A) Simulation of the tilt angle impact on yearly productivity of the LSC-PM mini-plant. (B) Photographs of the sky conditions at time of the experiment. (C) Experimental setup. ChemSusChem 2021, 14, 5417–5423 www.chemsuschem.org Outdoor experiment using the off-grid solar-powered autonomous chemical mini-plant Next, an outdoor experiment was conducted using the 0.47× 0.47 m2 LSC-PM reactor in combination with a 0.38×0.51 m2 PV cell behind the reactor under an intermittently cloudy sky (see Figure 2B) in Eindhoven, The Netherlands, from 3 pm to 5 pm on July 15th, 2020 (see Supporting Information for a full technical description). Using the Arduino-based self-regulating protocol, flow rates of HPLC and MFC were continuously adjusted by the control system to maintain constant conver- sion. Two additional light sensors were used to record both the direct incident light power and the edge emission of the LSC- PM at the outdoor location (see Figure 2C). The reaction was conducted for 80 min, with samples taken every 5 minutes and analyzed by HPLC (see experimental section). Reduction in luminosity was observed for certain periods during data collection, corresponding to passing clouds (Fig- ure 2B). Despite the fluctuations of irradiation impinging on our reactor, the constant conversion proves the efficiency of the controlling system to autonomously and instantaneously regu- late fast changes in light intensity (Figure 3A). The liquid flow rate varied between 2.2 mLmin1 and 0.95 mLmin1 to ensure that the reaction mixture is receiving sufficient irradiation to reach the targeted conversion. The throughput of the system can experimentally reach up to 17 mmolh1 of L-methionine sulfoxide under strong direct irradiation conditions (i.e., 60 klux). Under very low irradiation (i.e., 10 klux), the system maintained a throughput of 3 mmolh1. In both scenarios, a high conversion and selectivity for the target compound was ensured. It should be further noted that such high productivities would not be feasible without the light concentrating effect of the LSC-PM that directs even the diffuse light fraction to the reaction channels. As a consequence, more photons can be harvested compared to transparent reactors, especially at the most challenging reaction conditions, that is, Using the same principle, simulations can be used to predict the productivity of the device around the world at different latitudes. Targeting a lower conversion would make the process more photon-efficient but less relevant to produce clean chemicals (Figure 4A and ESI). Since the final goal of this mini- plant is to be capable of implementation off-grid at any location, it is valuable to be able to evaluate the efficiency of a setup anywhere on the globe (or beyond). Simulations and productivity By simulating the productivity of the reactor in Eindhoven using Monte Carlo ray-tracing simulations (see Supporting Informa- tion), the model can be experimentally validated. For this, the days are assumed to be cloudless throughout the entire year. The total number of photons reaching the reaction mixture during an entire year is depicted in Figure 3B. In comparison to a non-tilted reactor (Figure 3C), the productivity is more stable throughout the year for the 40° tilted reactor, but with a lower peak productivity in summer. The enhanced productivity due to the addition of the luminescent dye can be seen when comparing the results shown in Figure 3D, where the doped reactor exhibits nearly quadrupled photon absorption as previously reported.[18] With this system, a maximum photon absorption of 2.8 molday1 can be attained. While our experi- ment uses photons for chemical conversion, a biphasic mixture is established with oxygen slugs. Since a 1:1 volumetric gas-to- liquid ratio is required to perform the reaction, the photons reaching the gas will not be absorbed by the photocatalyst to perform the oxidation. Applying the same simulation to the time of the experiment, taking the gaseous volume fraction and the quantum yield of methylene blue[38] (0.39) into account, a maximum productivity of around 55 mmolh1 is predicted. The experiment resulted in a productivity of 17 mmolh1. The difference between the two productivities can be explained by the high conversion target of the reaction. In the experiment, more than 99% conversion is maintained consistently since conversion was prioritized over productivity. This target was selected as it would be associated with lower and less energy- demanding downstream purification costs, such as the in-line catalyst removal by activated charcoal.[17] Energy supply using photovoltaic cells However, positioning the solar panel besides the reactor provides enough power (19 W under sunny conditions and 6 W during cloudy days) and could meet the power demands of the system (Table 1). Yet, by separating the PV panel from the reactor, the entire surface covered would be doubled while producing the same quantity of high-value chemicals. To cope with this issue, another option would be to increase the surface area of the plant as the peripheral electronic components, including pumps, would remain the Regarding the positioning of the PV cell, two options were investigated. Attaching tailor-made PV cells to the edges of the panel as deployed on standard LSC systems was considered as a first option.[37] This design can be favourable since the LSC-PM concentrates the photons towards the edges of the lightguide. However, the need for tailored PV cells increases the price for such a device. Furthermore, the small surface area covered by the PV cells, added to the fact that the LSC-PM reaction channels are meant to gather most of the photons, reduces the attractiveness of this design. The second location investigated places the PV cell directly beneath the reactor. It should be noted that the solar cell and the luminescent dye do not use the same wavelengths and are thus compatible. A PV cell below the reactor could thus process wavelengths between 600– 1100 nm, while the more energetic wavelengths are used to catalyse chemical reactions. Moreover, the surface area available Table 1. Outdoor energy measurements of a solar panel at a 40° angle placed either beneath or next to the LSC-PM at varying edge emission of the LSC-PM (EE). Condition EE [klux] Punder LSC-PM [W] Pnext to LSC-PM [W] Pconsumed [W] Sunny 40–44 13 19 14 Very cloudy 16–20 4 6 12 19 © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH Table 1. Outdoor energy measurements of a solar panel at a 40° angle placed either beneath or next to the LSC-PM at varying edge emission of the LSC-PM (EE). © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH 5419 Research Article doi.org/10.1002/cssc.202102011 ChemSusChem cloudy weather with high fractions of diffuse light (See Simulations and productivity section). same. Consequently, by increasing the size of the reactor and the PV cell underneath, the produced power would surpass the power consumed, as shown in Table 2. Energy supply using photovoltaic cells In addition, despite the drop in power produced at very cloudy weather, the surplus of energy harvested during sunny moments can be stored in a battery and can, subsequently, act as a buffer to supply this shortfall (sunny conditions are required for approximately 25% of the time for truly autonomous operation at very cloudy weather). © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH Outdoor experiment using the off-grid solar-powered autonomous chemical mini-plant Based on the spectral and irradiance data of a location (using pvlib[39]), we can determine both the optimal tilt angle and give indication of the eventual productivity. An adequate location does not necessarily have to be strongly irradiated by the sun to be suitable, as is normally required for standard silicon PV cells. Thanks to efficient diffuse light collection by the LSC, northern European countries could still use the LSC-PM device for outdoor chemical production, for example. Table 2. Extrapolated power consumed and power produced for a 1 m2 LSC-PM system with a 1 m2 solar panel mounted directly underneath the reactor. Condition EE [klux] Punder LSC-PM [W] Pconsumed [W] Sunny 40 63 39 Very cloudy 16 21 29 5 ChemSusChem 2021, 14, 5417–5423 www.chemsuschem.org Table 2. Extrapolated power consumed and power produced for a 1 m2 LSC-PM system with a 1 m2 solar panel mounted directly underneath the reactor. Yearly productivity is compared at the optimal angle for the four locations with varying latitudes depicted in Figure 4B; North Cape, Eindhoven, Almeria and Townsville, using a 0.22 m2 LSC-PM reactor. Here, North Cape was chosen because it is the northernmost location in mainland Europe, Eindhoven was Condition EE [klux] Punder LSC-PM [W] Pconsumed [W] Sunny 40 63 39 Very cloudy 16 21 29 5420 © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH ChemSusChem Research Article doi.org/10.1002/cssc.202102011 Research Article doi.org/10.1002/cssc.202102011 ChemSusChem Figure 3. (A) Measurement of direct luminosity (blue area) and edge emission (green area) for 80 min under cloudy conditions 15th of July at 3 pm, and the conversion of L-methionine to L-methionine sulfoxide (red dots). Yearly number of photons absorbed by the mini-plant in Eindhoven in 2020: (B) in normal operating conditions, (C) with no tilt angle and (D) without luminescent dye. 3. (A) Measurement of direct luminosity (blue area) and edge emission (green area) for 80 min under cloudy conditions 15th f L thi i t L thi i lf id ( d d t ) Y l b f h t b b d b th i i l t i Ei dh Figure 3. (A) Measurement of direct luminosity (blue area) and edge emission (green area) for 80 min under cloudy conditions 15th of July at 3 pm, and the conversion of L-methionine to L-methionine sulfoxide (red dots). ChemSusChem 2021, 14, 5417–5423 www.chemsuschem.org © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH Outdoor experiment using the off-grid solar-powered autonomous chemical mini-plant Yearly number of photons absorbed by the mini-plant in Eindhoven in 2020: (B) in normal operating conditions, (C) with no tilt angle and (D) without luminescent dye. This demonstrates that the solar-powered mini-plant can be deployed and practically used for chemicals production at almost any location where solar energy can be harvested. chosen to be able to compare our simulations to the outdoor experimental results. In Almería the largest European solar concentrating test and research center, Plataforma Solar de Almería, is located, and Townsville was chosen as a sunny location in the Southern Hemisphere. As expected, higher productivities can be reached in regions closer to the equator, like Townsville (up to 955 mol of absorbed photons per year) or Almería (up to 899 mol of absorbed photons per year). However, the mini-plant is still capable of reliably producing chemicals from March until November in remote environments like North Cape (up to 527 mol of absorbed photons per year). Applying this easy-to-build system to a known industrial photochemical process, such as Dragoco’s rose oxide synthesis,[40,41] can justify its use against other options. The key photooxidation can be performed under solar irradiation[42] and would benefit from our LSC-PM technology. The yearly production of this fragrance molecule is 60–100 tons (390– 650 kmol).[43] Thus, by locating the mini-plant in Townsville, a yearly productivity of 955 mol could be reached. To reach a 5421 ChemSusChem 2021, 14, 5417–5423 www.chemsuschem.org © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH Research Article doi.org/10.1002/cssc.202102011 ChemSusChem Figure 4. Simulations around the world. (A) Geographical positions of the different locations simulated. Open-source map from uMap. (B) Simulations of the daily number of photons received in 2020 by the mini-plant (0.22 m2) with optimized tilt angles in North Cape (Norway), Eindhoven (The Netherlands), Almería (Spain), and Townsville (Australia). Figure 4. Simulations around the world. (A) Geographical positions of the different locations simulated. Open-source map from uMap. (B) Simulations of the daily number of photons received in 2020 by the mini-plant (0.22 m2) with optimized tilt angles in North Cape (Norway), Eindhoven (The Netherlands), Almería (Spain), and Townsville (Australia). © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH Conclusion productivity of 650 kmolyear1 of rose oxide, approximately 150 m2 of coverage would be needed. Actual industrial solar setups would require around 1900 m2 of space deploying parabolic mirrors costing up to 196 Em2;[43] in comparison, the LSC panels required to build the reactor are sold for 99 Em2. Since two LSCs are needed per reactor (see Supporting Information), the price of the light concentrating material reaches 198 Em2. With a similar cost and a smaller surface area required to produce the same quantity of product, the LSC-PM mini-plant is actually a promising alternative with more flexible deployment options to industrial photochemical plants. Moreover, as the entire plant is run on solar energy, no energy cost is present in the Operating Expenditures (OPEX), making it a sustainable strategy for future chemical production. productivity of 650 kmolyear1 of rose oxide, approximately 150 m2 of coverage would be needed. Actual industrial solar setups would require around 1900 m2 of space deploying parabolic mirrors costing up to 196 Em2;[43] in comparison, the LSC panels required to build the reactor are sold for 99 Em2. Since two LSCs are needed per reactor (see Supporting Information), the price of the light concentrating material reaches 198 Em2. With a similar cost and a smaller surface area required to produce the same quantity of product, the LSC-PM mini-plant is actually a promising alternative with more flexible deployment options to industrial photochemical plants. Moreover, as the entire plant is run on solar energy, no energy cost is present in the Operating Expenditures (OPEX), making it a sustainable strategy for future chemical production. Herein, we describe the development of an off-grid, solar- powered, autonomous chemical mini-plant for producing fine chemicals under fluctuating solar light irradiation. The reactor consists of a scaled-up LSC-PM, which converts direct and diffuse sunlight energy to a wavelength range that matches the absorption spectrum of the photocatalyst and, subsequently, guides this fluorescent light towards embedded reaction channels to drive photochemical transformations. To maintain constant conversion, a calibrated control system assures almost instantaneous adjustments in the chemical feed. This guaran- tees that the mini-plant can even be implemented in a rapid weather-changing environment. To operate the system off-grid, photovoltaic cells were integrated underneath the reactor to 5422 ChemSusChem 2021, 14, 5417–5423 www.chemsuschem.org ChemSusChem 2021, 14, 5417–5423 © 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH Research Article doi.org/10.1002/cssc.202102011 ChemSusChem deploy the system in an energy-neutral manner. Conclusion Consequently, this mini-plant can perform photochemical reactions autono- mously, even in the absence of a power grid. Furthermore, using our ray-tracing model, we can predict the optimal array orientation and estimate the productivity of this photochemical mini-plant based on the local solar spectral data. [13] M. G. Debije, P. P. C. Verbunt, Adv. Energy Mater. 2012, 2, 12–35 [14] B. McKenna, R. C. Evans, Adv. Mater. 2017, 29, 1606491. [15] I. Papakonstantinou, M. Portnoi, M. G. Debije, Adv. Energy Mater. 2021, 11, 2002883. [16] M. G. Debije, R. C. Evans, G. Griffini, Energy Environ. Sci. 2021, 14, 293– 301. [17] D. Cambié, J. 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https://europepmc.org/articles/pmc7680442?pdf=render

English

Exosome mediated delivery of functional nucleic acid nanoparticles (NANPs)

Nanomedicine

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Abstract RNAi-based technologies have shown biomedical potential; however, safe and efficient delivery of RNA remains a barrier for their broader clinical applications. Nucleic acid nanoparticles (NANPs) programmed to self-assemble and organize multiple therapeutic nucleic acids (TNAs) also became attractive candidates for diverse therapeutic options. Various synthetic nanocarriers are used to deliver TNAs and NANPs, but their clinical translation is limited due to immunotoxicity. Exosomes are cell-derived nanovesicles involved in cellular communication. Due to their ability to deliver biomolecules, exosomes are a novel delivery choice. In this study, we explored the exosomemediated delivery of NANPs designed to target GFP. We assessed the intracellular uptake, gene silencing efficiency, and immunostimulation of exosomes loaded with NANPs. We also confirmed that interdependent RNA/DNA fibers upon recognition of each other after delivery, can conditionally activate NF-kB decoys and prevent pro-inflammatory cytokines. Our study overcomes challenges in TNA delivery and demonstrates future studies in drug delivery systems. Author Manuscript Published in final edited form as: Published in final edited form as: Nanomedicine. 2020 November ; 30: 102285. doi:10.1016/j.nano.2020.102285. Exosome mediated delivery of functional nucleic acid nanoparticles (NANPs) Senny Nordmeiera,*, Weina Keb,*, Kirill A. Afoninb, Victoria Portnoya aSystem Biosciences (SBI), 2438 Embarcadero Way, Palo Alto, CA 94303, USA bNanoscale Science Program, Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC, USA. Senny Nordmeiera,*, Weina Keb,*, Kirill A. Afoninb, Victoria Portnoya aSystem Biosciences (SBI), 2438 Embarcadero Way, Palo Alto, CA 94303, USA bNanoscale Science Program, Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC, USA. Author Manuscript Correspondence: Victoria Portnoy [email protected] +1-408-813-9595 and Kirill A. Afonin [email protected], +1-704-687-0685. * Correspondence: Victoria Portnoy [email protected] +1-408-813-9595 and Kirill A. Afonin [email protected], +1-704-687-0685. *-Equal contribution CRediT author statement Senny Nordmeier: investigation, project administration, writing-original draft Weina Ke: resources, writing-original draft Kirill Afonin: visualization, writing-reviewing and editing Victoria Portnoy: supervision, writing-reviewing and editing Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. CONFLICT OF INTERESTS The authors report no conflicts of interest in this work. HHS Public Access Author manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Author Manuscript INTRODUCTION Nucleic acid nanoparticles (NANPs) are modular nanoscaffolds composed of multiple oligonucleotides programmed to self-assemble into precise 3D structures with well-defined properties (1–4). Rationally designed NANPs can be further decorated with cocktails of therapeutic nucleic acids (TNAs), which may differ in composition, secondary structure, and mechanism of action, allowing for synchronized targeting of multiple cellular pathways. This structural versatility, and the ability to finely control the NANPs’ sizes, shapes, composition, multivalences, and therapeutic payloads, makes this technology an attractive option for biomedical applications (5–7). For example, a previously tested combinatorial RNAi strategy of NANPs functionalized with six different Dicer Substrate (DS) RNAs (8) were effective in simultaneous targeting six distinct parts of the HIV-1 genome (1, 9). These NANPs functionalized with DS RNAs relied on the assistance from the intracellular enzyme Dicer that initiated the nuclease-assisted release of siRNAs from the NANPs. Moreover, by simply extending either the 5’- or/and 3′- ends of each strand of the NANP’s composition with its unique functionality, NANPs can be formulated to precisely package not only different TNAs but also fluorophores, targeting agents, small-molecule drugs, proteins, or other therapeutic cargoes (10–12). Author Manuscript Auth Author Manuscript A sophisticated approach to the conditional intracellular activation of RNAi was introduced through interdependent RNA/DNA NANPs in which the RNA (and/or DNA) functionalities were split into two inactive NANPs (13–15). Driven by sequence complementarity, the inactive NANPs could recognize each other inside the cells and release the activated functionalities upon isothermal reassociation (2, 9, 16). Using the same approach, RNA/DNA fibers were recently designed for enhanced control of deliverable functionalities (e.g., DS RNAs targeting mutated BRAFV600E in melanoma cells) along with higher stability against enzymatic degradation in human blood and tunable rates of intracellular reassociation (17). Another advantage of this user-friendly approach: the DNA part of the RNA/DNA fibers contained split NF-kB (nuclear factor kappa-light-chain enhancer of activated B cells) decoy sequences, which activate upon intracellular fiber reassociation and restrain the immunostimulatory response. NF-kB is expressed in most mammalian cells and remains sequestered in an inactive state in the cytoplasm with the inhibitory protein IκB. Activation of NF-kB is initiated by a variety of stimuli, resulting in signal-induced degradation of IκB proteins by proteasomes. Subsequently, the NF-κB complex can enter the nucleus and initiate gene expression of pro-inflammatory cytokines. Keywords y Extracellular vesicles; exosomes; RNA nanotechnology; NANPs; immunostimulation Graphical abstract Graphical abstract illustrates the experimental design of the current work. Exosomes were isolated, packaged with functionalized NANPs and characterized in in vitro and cell culture experiments, demonstrating a novel TNA delivery system. Author Manuscript diT author statement ny Nordmeier: investigation, project administration, writing-original draft Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The authors report no conflicts of interest in this work. Keywords Extracellular vesicles; exosomes; RNA nanotechnology; NANPs; immunostimulation dmeier et al. Page 2 Nordmeier et al. Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. INTRODUCTION NF-κB decoys (18– 20) mimic the κB consensus sequence and upon the decoys binding to NF-κB translocate into the nucleus is prevented. Author Manuscript Activation of NF-kB is initiated by a variety of stimuli, resulting in signal-induced degradation of IκB proteins by proteasomes. Subsequently, the NF-κB complex can enter the nucleus and initiate gene expression of pro-inflammatory cytokines. NF-κB decoys (18– 20) mimic the κB consensus sequence and upon the decoys binding to NF-κB translocate into the nucleus is prevented. Page 3 Nordmeier et al. Since all NANPs are composed of negatively charged, hydrophilic biopolymers, they cannot easily penetrate through biological membranes and mainly rely on the use of different synthetic carriers for efficient cell internalization (17, 21–25). Over the last few decades, extensive research efforts have been undertaken to improve the stability, efficacy, and specificity of TNA delivery systems, such as liposomes (26), micelles (27), nanoparticles (28), hydrogels (29), and viruses (30). In spite of these attempts, most of these systems suffer from immunogenicity, cytotoxicity, rapid blood clearance, and poor biodistribution (31), hindering further clinical translation of therapeutic NANP platforms. Therefore, an urgent need to identify new endogenous sources for potential delivery systems that avoids the complications associated with synthetic materials is vital. Since all NANPs are composed of negatively charged, hydrophilic biopolymers, they cannot easily penetrate through biological membranes and mainly rely on the use of different synthetic carriers for efficient cell internalization (17, 21–25). Over the last few decades, extensive research efforts have been undertaken to improve the stability, efficacy, and specificity of TNA delivery systems, such as liposomes (26), micelles (27), nanoparticles (28), hydrogels (29), and viruses (30). In spite of these attempts, most of these systems suffer from immunogenicity, cytotoxicity, rapid blood clearance, and poor biodistribution (31), hindering further clinical translation of therapeutic NANP platforms. Therefore, an urgent need to identify new endogenous sources for potential delivery systems that avoids the complications associated with synthetic materials is vital. Author Manuscript Author Author Manuscript Extracellular vehicles (EVs) are membrane enclosed vesicles ranging from 30 nm to 1,000 nm (or even larger) in size and secreted by cells into the extracellular space (32, 33). EVs comprise of exosomes and microvesicles, which are differentiated by their biogenesis, content, size, release pathways, and function (34). Exosomes have been widely investigated as emerging novel delivery vehicles for biological cargos. Nanomedicine. Author manuscript; available in PMC 2021 November 01. INTRODUCTION These structures range from 30 nm to 150 nm in size (35) and are released into most biological fluids. Biological molecules, such as proteins, lipids, and nucleic acids, undergo transport via exosomes from the cell of origin to recipient cells (36). Exosomes form by inward budding of the cell membrane, a process which generates early endosomes. After the endosomes mature into multivesicular bodies (MVB), they fuse with the cell membrane and exit from the cell; alternatively, they can fuse with a lysosome for degradation. (Fig. 1A) (37). Reflective of the mechanism of biogenesis, exosomes are considered to be the “mini versions” of their parental cells in terms of the specially sorted “cargo” they carry (38). The putative function of exosomes is to perform intercellular communication and trigger physiological responses. Interaction between exosomes and recipient cells is promoted via receptor-mediated endocytosis (39), micropinocytosis (40), or membrane fusion (41). Once internalized, the exosomal contents are released into the cytosol directly or through back-fusion with the endosomal membrane, eliciting the effect on the recipient cell (42). Such features of the exosomes highlight their potential as drug delivery systems to alter gene expression by delivering therapeutic genetic materials. Several studies have investigated their performance as therapeutic vehicles for exogenous genetic material delivery as well as their post-delivery functionality. The first published study showed that exosomes could efficiently deliver exogenous siRNAs to the brains of mice with successful knockdown of mRNA and BACE1 protein with negligible immune response (43). Later, a myriad of studies reported using exosomes to deliver siRNAs and knockdown targeted genes in different diseases. For example, exosome- mediated delivery has shown promise in silencing specific genes, including VEGF, EGFR, AKT, MAPK, and KRAS (44); inhibiting luciferase expression (45); treating hepatitis C infection; causing massive cell death of recipient cells by RAD51 gene suppression (46); and survivin siRNA suppression for the treatment of bladder cancer (47). Recently, exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry shRNAs or siRNAs against the KrasG12D mutation known for triggering pancreatic cancer growth in multiple mouse models (48). METHODS Author Manuscript INTRODUCTION The use of exosomes as delivery vehicles demonstrates a plethora of advantages, including biocompatibility, efficacy, stability, and membrane permeation capability; as well as diminished toxicity; low immunogenicity; low Author Manuscript exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry shRNAs or siRNAs against the KrasG12D mutation known for triggering pancreatic cancer growth in multiple mouse models (48). The use of exosomes as delivery vehicles demonstrates a plethora of advantages, including biocompatibility, efficacy, stability, and membrane permeation capability; as well as diminished toxicity; low immunogenicity; low exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry shRNAs or siRNAs against the KrasG12D mutation known for triggering pancreatic cancer growth in multiple mouse models (48). The use of exosomes as delivery vehicles Page 4 Nordmeier et al. Page 4 off-target effect; and the ability to cross the blood-brain-barrier (31, 49–53). As a result, exosomes can significantly outperform other delivery methods. Author Manuscript In this current work, we investigated the use of exosomes as carriers for delivering functional NANPs of different shapes, sizes, and compositions, among which were globular RNA cubes, planar RNA rings, and linear RNA/DNA fibers. We assessed, in vitro, the stability of exosome-loaded NANPs, in addition to their intracellular uptake, silencing efficiency, and immunostimulatory activity. Cell culture MDA-MB-231-GFP, MDA-MB-231 and HUVEC cells were used in this study. All oligonucleotides were purchased from Integrated DNA Technologies (IDTDNA.com, Coralville, Iowa, USA). All NANP compositions used in this project are listed in the Supplementary Material Table 1. Nanomedicine. Author manuscript; available in PMC 2021 November 01. Assemblies of RNA/DNA fibers, RNA cubes and RNA rings targeting GFP and analysis using native-PAGE All individual strands were purified by an 8 M urea polyacrylamide gel (8% acrylamide, 19:1), gel extracted, eluted and dissolved in endotoxin-free water. All individual RNA strands for cube and rings were synthesized via in vitro run-off transcription (IVT) assay, purified by an 8 M urea polyacrylamide gel (8% acrylamide, 19:1), gel extracted, eluted and dissolved in endotoxin-free water. RNA/DNA fibers, RNA cubes, and RNA rings were assembled in one-pot by combining individual monomers at equimolar concentrations in an assembly buffer. All NANPs were analyzed on 8% non-denaturing native polyacrylamide gel (19:1 for fibers, 37.5:1 for rings and cubes). Author Manuscript Atomic force microscopy (AFM) imaging of NANP A freshly cleaved mica surface modified with APS (1-(3-aminopropyl) silatrane) was used for AFM imaging, which was performed on the MultiMode AFM NanoScope IV system (Bruker Instruments, Santa Barbara, CA, USA) in tapping mode. Atomic force microscopy (AFM) imaging of NANP A freshly cleaved mica surface modified with APS (1-(3-aminopropyl) silatrane) was used for AFM imaging, which was performed on the MultiMode AFM NanoScope IV system (Bruker Instruments, Santa Barbara, CA, USA) in tapping mode. A freshly cleaved mica surface modified with APS (1-(3-aminopropyl) silatrane) was used for AFM imaging, which was performed on the MultiMode AFM NanoScope IV system (Bruker Instruments, Santa Barbara, CA, USA) in tapping mode. Loading of exosomes with NANPs Loading of exosomes with NANPs 100 μg of isolated exosomes were loaded with 25 pmoles NANPs using the Exo-Fect siRNA/miRNA transfection kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s manual. The loaded exosomes were cleaned to remove any excess of free NANPs/negative control siRNA, transfection reagent, or complexes. The cleaned NANP loaded exosomes were immediately added onto MDA-MB-231-GFP cells. Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nanoparticle tracking analysis and exosomes labeling for f-NTA Isolated exosomes were labeled with the ExoGlow-NTA fluorescent kit (System Biosciences, Palo Alto, CA, USA). Labeled exosomes were stored at −20°C until the analysis. Unlabeled and labeled samples were analyzed by NTA and f-NTA using ZetaView. Author Manuscript Nuclease protection assay of DNA duplex The loaded exosomes were cleaned to remove any excess of free NANPs/negative control siRNA, transfection reagent, or complexes. The cleaned NANP loaded exosomes were immediately added onto MDA-MB-231-GFP cells. RNA purification and quantitative real time PCR (RT-qPCR) analysis Total RNA was purified using RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. Purified total RNA was reverse transcribed using anchor Oligo (dT)20 primer (Thermo Fisher Scientific, Wilmington, DE, USA) and Superscript III reverse transcriptase (Thermo Fisher Scientific, Wilmington, DE, USA). Real time qPCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Wilmington, DE, USA) with TaqMan gene expression assay eGFP (Thermo Fisher Scientific, Wilmington, DE, Mr04097229_mr) and with TaqMan gene expression assay Beta-2-microglobulin as a reference gene (Thermo Fisher Scientific, Hs00984230_m1, Wilmington, DE, USA) and the QuantStudio 6 FLEX (Applied Biosystems, Thermo Fisher Scientific, Wilmington, DE, USA). Isolation of exosomes Exosomes were isolated from MDA-MB-231 or HUVEC cells using ExoQuick-TC ULTRA (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s manual. Protein concentration was measured by Qubit protein assay and all exosomes were stored at −20°C. Immunoblotting 5 μg of isolated exosomes were run on 4–20% Mini-PROTEAN TGX gel, transferred to PVDF membrane and probed with primary antibodies over-night. Next day, the membranes were washed three times, probed with appropriate secondary antibody and imaged using Molecular Imager ChemiDOC XRS+ Imaging System (Bio-Rad, Hercules, CA, USA). Nanoparticle tracking analysis and exosomes labeling for f-NTA Isolated exosomes were labeled with the ExoGlow-NTA fluorescent kit (System Biosciences, Palo Alto, CA, USA). Labeled exosomes were stored at −20°C until the analysis. Unlabeled and labeled samples were analyzed by NTA and f-NTA using ZetaView. TEM analysis Isolated exosomes were fixed by addition of 4% paraformaldehyde, 5 μl of the sample was dropped on a carbon coated 400 mesh Cu/Rh grid (Ted Pella, Redding, CA, USA) and stained with 5 μl of 1% uranyl acetate (Polysciences, Warrington, PA, USA) prepared in filtered distilled water. The grids were imaged with a FEI Talos L120C TEM with Gatan 4k × 4 k OneView camera. Loading of exosomes with NANPs Author Manuscript Exosomes were isolated from MDA-MB-231 or HUVEC cells using ExoQuick-TC ULTRA (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s manual. Protein concentration was measured by Qubit protein assay and all exosomes were stored at −20°C. Nuclease protection assay of DNA duplex Nuclease protection assay of DNA duplex 125 nM dsDNA labeled with Alexa Fluor 488 at the 5′ sense strand and an Iowa Black Quencher at the 3′ antisense strand was loaded into exosomes, then treated with RQ1 DNase I. The fluorescence resulting from the digestion of the DNA duplex by RQ1 DNase was monitored at 37°C for a total of 60 minutes with measurements at every 30 seconds. Free fluorescently quenched DNA duplex was used as the control. 125 nM dsDNA labeled with Alexa Fluor 488 at the 5′ sense strand and an Iowa Black Quencher at the 3′ antisense strand was loaded into exosomes, then treated with RQ1 DNase I. The fluorescence resulting from the digestion of the DNA duplex by RQ1 DNase was monitored at 37°C for a total of 60 minutes with measurements at every 30 seconds. Free fluorescently quenched DNA duplex was used as the control. Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Page 5 Nordmeier et al. Isolation of exosomes Exosomes were isolated from MDA-MB-231 or HUVEC cells using ExoQuick-TC ULTRA (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s manual. Protein concentration was measured by Qubit protein assay and all exosomes were stored at −20°C. Immunoblotting 5 μg of isolated exosomes were run on 4–20% Mini-PROTEAN TGX gel, transferred to PVDF membrane and probed with primary antibodies over-night. Next day, the membranes were washed three times, probed with appropriate secondary antibody and imaged using Molecular Imager ChemiDOC XRS+ Imaging System (Bio-Rad, Hercules, CA, USA). Nanoparticle tracking analysis and exosomes labeling for f-NTA Isolated exosomes were labeled with the ExoGlow-NTA fluorescent kit (System Biosciences, Palo Alto, CA, USA). Labeled exosomes were stored at −20°C until the analysis. Unlabeled and labeled samples were analyzed by NTA and f-NTA using ZetaView. TEM analysis Isolated exosomes were fixed by addition of 4% paraformaldehyde, 5 μl of the sample was dropped on a carbon coated 400 mesh Cu/Rh grid (Ted Pella, Redding, CA, USA) and stained with 5 μl of 1% uranyl acetate (Polysciences, Warrington, PA, USA) prepared in filtered distilled water. The grids were imaged with a FEI Talos L120C TEM with Gatan 4k × 4 k OneView camera. Loading of exosomes with NANPs 100 μg of isolated exosomes were loaded with 25 pmoles NANPs using the Exo-Fect siRNA/miRNA transfection kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s manual. Cell uptake imaging Cells transfected with NANP loaded exosomes or negative control loaded exosomes were imaged 72 hours post-transfection. For the cancer and primary cells transfected with exosomes loaded with DNA cube-Alexa 488, cells were imaged 24 hours post-transfection. The images were captured using a Leica DMI3000 inverted microscope with a DFC360 FX digital camera (Leica Microsystems, Wetzlar, Germany). Author Manuscript Immunostimulation in vitro HEK-Blue™ hTLR3 and 7 cells, and THP1-Dual™ cells (Invivogen, San Diego, CA, USA) were used for quantifying the activation pf specific toll-like receptor (TLR) and intracellular signaling pathways, respectively. HEK-Blue™ hTLR 3 and 7 cells are HEK cells engineered to stably co-express human TLR and NF-κB-inducible SEAP (secreted embryonic alkaline phosphatase) reporter genes. The TLR activation results in downstream production of SEAP that can be detected and quantified using QUANTI-Blue™. THP1-Dual™ cells are engineered to express SEAP upon NF-kB stimulation, which can also be assessed by QUANTI-Blue™. In order to investigate the conditional activation of NF-kB decoys, Poly (I:C) (polyinosinic-polycytidylic acid, a synthetic analog of dsRNA) and Pam3CSK4 (a synthetic diacylated lipopeptide) were used to stimulate HEK-Blue™ hTLR3 and THP1- Dual™ cells, respectively. For the experiments with the HEK-Blue™ hTLR7 cells, R848 (resiquimod) was used as a positive control. Author Manuscript Flow cytometry Author Manuscript MDA-MB-231-GFP treated cells were trypsinized with 0.25% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA) for 2 minutes at 37 °C, quenched with DMEM supplemented with 10% FBS, and pelleted by centrifugation at 800xg for 2 minutes. The cell pellet was resuspended with 1xPBS supplemented with 1 % FBS into a 12×75mm test tube with a cell strainer cap (Falcon, Durham, NC, USA) and analyzed with the DxP FACScan (Cytek Biosciences, Fremont, CA, USA). The data was analyzed using FCS Express 5 (De Novo Software, Glendale, CA, USA). RNA purification and quantitative real time PCR (RT-qPCR) analysis Total RNA was purified using RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. Purified total RNA was reverse transcribed using anchor Oligo (dT)20 primer (Thermo Fisher Scientific, Wilmington, DE, USA) and Superscript III reverse transcriptase (Thermo Fisher Scientific, Wilmington, DE, USA). Real time qPCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Wilmington, DE, USA) with TaqMan gene expression assay eGFP (Thermo Fisher Scientific, Wilmington, DE, Mr04097229_mr) and with TaqMan gene expression assay Beta-2-microglobulin as a reference gene (Thermo Fisher Scientific, Hs00984230_m1, Wilmington, DE, USA) and the QuantStudio 6 FLEX (Applied Biosystems, Thermo Fisher Scientific, Wilmington, DE, USA). Author Manuscript Page 6 Page 6 Nordmeier et al. Nanomedicine. Author manuscript; available in PMC 2021 November 01. Characterization of isolated exosomes. Extracellular vesicles are cell-derived membrane particles subdivided into: microvesicles, ranging in size from 100–1,000 nm, that are shed from the membrane and exosomes released by fusion of late endosomes with the cell membrane (30–150 nm size range) (Figure 1A) (33–35). Although microvesicles and exosomes are structurally similar and overlap in size, their content and cell origins are different (32). Exosomes secreted by MBA-MD-231(231) were isolated using ExoQuick-TC ULTRA. Prior to the addition of ExoQuick-TC reagent, centrifugation was used to remove cell debris and large particles, such as apoptotic bodies. The isolation method included two steps: (i) precipitation of exosomes with ExoQuick-TC and (ii) their purification with an affinity chromatography column, ULTRA. ExoQuick-TC ULTRA efficiently removes the major non-EV co-isolates, such as bovine albumin and bovine IgG, which usually are overrepresented in the tissue culture media (54). Figure 1B Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nordmeier et al. Page 7 shows high amounts of albumin and IgG after precipitation step and demonstrates significant reduction of the co-isolated after the second step of ULTRA purification. Thus, the use of ExoQuick-TC ULTRA was essential to achieve the higher purity of isolated exosomes. Author Manuscript Auth Author Manuscript The ExoQuick-TC ULTRA isolation method yielded a particle concentration of 4.50E+11 ± 6.80E+10 particles/mL as measured by fluorescent NTA (fNTA) and 4.70E+11 ± 5.10E+10 particles/mL as measured by NTA (Figure 1C). Since NTA measures any particle in solution, including protein aggregates and buffer precipitates, we specifically labeled exosomes with a membrane sensor dye and measured the concentration of intact vesicles using fNTA (55, 56). The mean diameter of the isolated exosomes was 103.5 ± 26.4 nm as fNTA and 123.1 ± 8.1 nm as measured by conventional NTA (Figure 1C). A size distribution curve showed a classical distribution of exosomes ranging in size from 30–150 nm. Common exosomal markers Hsp70, ALIX, and tetraspanin, CD63, were identified (Figure 1B). ALIX is an exosomal protein known for its involvement in the MVB biogenesis (32). CD63 was enriched in exosomal membrane from different origins (57, 58). Hsp70 was released into the extracellular space via exosomes as a membrane- bound protein (59). Calnexin is an integral protein of the endoplasmic reticulum (ER) and was used in as a cellular marker. Absence of calnexin marker in the exosome preparation indicated no cellular contamination was present. Characterization of isolated exosomes. Transmission electron microscopy (TEM) images of isolated exosomes showed normal morphology without any distortion (Fig. 1D) (60). Altogether, we conclude that the isolated sample was enriched in exosomes based on our results. Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Characterization of NANP loaded exosomes and nuclease protection assay. The NANPs used in this study have distinct, strategic designs that confer different compositions, connectivities, shapes, and sizes (61). Both cube and ring RNA scaffolds are assembled from six individual RNAs; cubes are globular (3D), whereas rings are planar (2D). The hydrodynamic radii (based on DLS results) of cubes and rings functionalized with six anti-GFP DS RNAs were estimated to be ~12 and ~15 nm, respectively (16, 62). DNA/RNA fibers are linear (1D) and composed of both DNA and RNA strands. While RNA cubes and RNA/DNA fibers are assembled via intermolecular Watson-Crick base pairing, assembly of RNA rings requires magnesium-dependent, intramolecular Watson-Crick base pairing to facilitate intermolecular kissing loop interactions (63). Although each of these structures has distinct properties, all of them can be functionalized with DS RNAs to silence any gene through sequence specific mRNA targeting. In addition, DNA/RNA fibers are functionalized with NF-kB decoys, inhibiting immune responses through the NF-kB pathway. All NANPs were analyzed by native-PAGE and AFM, as shown in Figure 2A, to confirm the correct assembly. Author Manuscript Author Manuscript To load NANPs into isolated exosomes, Exo-Fect siRNA/miRNA Transfection Kit was used. The Exo-Fect transfection reagent formed a complex with the NANPs and assisted with exosome insertion. Following the transfection, all samples were run through a clean-up column to remove excess Exo-Fect reagent, as well as free NANPs and their complexes. TEM images showed exosomes loaded with functionalized RNA rings, RNA cubes, and RNA/DNA fibers possessed morphology similar to free exosomes (Figure 1D and Figure Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nordmeier et al. Page 8 2B) with no associated structural changes. To verify the integrity of loaded exosomes, the presence of common exosomal markers (Hsp70, ALIX, and tetraspanin, CD63) was confirmed (Figure 2C). Interestingly, NTA analysis indicated that exosomes originally with mean diameter of ~102–103 nm became larger by ~20–30 nm after loading with NANPs (Figure 2D). Author Manuscript Aut Author Manuscript Next, the exosomes’ ability to protect a loaded nucleic acid cargo from nuclease degradation were analyzed using a nuclease protection assay (Figure 3A) developed from previous works (19, 22, 64). As a model system, exosomes loaded with DNA duplexes labeled with Alexa 488 at the 5’-end and Iowa Black quencher at the complementary 3’-end were used. Due to the quencher’s proximity to the fluorophore, the fluorescent signal from the DNA duplex was quenched. Cellular uptake of exosomes loaded with NANP polyplexes. To confirm effective delivery of loaded NANPs, exosomes isolated from human breast cancer MDA-MB-231 (231) cells were loaded with either Alexa 488- or Alexa 546-labeled NANPs and added to 231 cells. Fluorescence microscopy images captured 24 hours post- transfection validated the internalization of loaded exosomes into cells (Figure 3B–C). Flow cytometry and microscopy results demonstrated that exosomes loaded with Alexa 546- labeled DNA duplex internalized into the cells and, with no exosomes present, no significant cellular uptake occurred (Figure 3B). Interestingly, flow cytometry results show different transfection efficiencies of exosome-encapsulated Alexa 488-labeled NANPs, indicating that NANP polyplex shapes may potentially affect cellular uptake efficiency (Figure 3C). Overall, data confirmed that NANP-loaded exosomes can be internalized by human cells with visible effects on transfection efficiency. Interestingly, we noticed that the uptake efficiency for the same number of NANP loaded exosomes is significantly lower for primary cell lines when compared to cancer cells (supporting Figure S1). This may offer additional advantages for exosome mediated delivery of NANP, due to the potential reduction in undesired off-target effects in healthy tissues. Author Manuscript Author Manuscript Characterization of NANP loaded exosomes and nuclease protection assay. However, after RQ1 DNase treatment and subsequent DNA degradation, the fluorophore escaped from the quencher, leading to a progressively increasing fluorescent signal. At the same time, exosome-encapsulated DNA duplexes were protected from DNase digestion and yielded no changes in the fluorescent signal. Indeed, the miniscule increase in fluorescence proves that exosomes can effectively protect nucleic acid cargo from nuclease digestion. Free exosomes were used as controls to show that little to no signal appears in these samples. Results of this experiment suggest that exosomes were able to completely shield their nucleic acid cargos from degradation by enzymatic activity for at least 60 minutes. Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Inhibition of NF-kB pathway. To further verify the intact dual functionality of DNA/RNA fibers that carry NF-kB response specificity and GFP silencing, we conducted a follow-up study using the reporter cell line HEK-Blue™ hTLR3. These cells are designed for the study of human TLR3 stimulation through assessment of NF-kB activation. The latter process induces production of secreted embryonic alkaline phosphatase (SEAP). Poly (I:C), a synthetic mimetic of viral dsRNA, can be used to trigger NF-kB activation and, ultimately, SEAP production (Figure 4C and supporting Figure S3). In this experiment, cognate RNA/DNA fibers (FS and FA) were separately pre-loaded into exosomes which were then mixed together for co-delivery (see FS/Exo+FA/Exo). Individual FS- and FA-loaded exosomes were used as controls (see FS/Exo and FA/Exo). On the same day that the complexes were added to the HEK-Blue™ hTLR3 cells, the cells were challenged with Poly (I:C). Supernatants were then collected and analyzed for presence of secreted alkaline phosphatase (SEAP), which can be easily detected and quantified using QUANTI-Blue™ reagent. Cells treated with poly (I:C) produced significant SEAP signal, and when FS and FA were delivered separately, no inhibition in SEAP production was observed. However, co-delivered of FS and FA significantly reduced SEAP synthesis. To show the generality, another reporter cell line, THP1-Dual™, that also expressed SEAP under the NF-kB promoter was tested (Figure 4D). To induce the activation of the NF-kB pathway, THP1-Dual™ cells were challenged with a TLR agonist (PAM3CSK4). On the same day, exosomes loaded with fibers were added to PAM3CSK4 treated cells and the supernatants were collected and analyzed for the presence of SEAP. The cells treated with Pam3CSK4 induced significant SEAP production, and when FS and FA were delivered separately, no inhibition of SEAP was observed. However, co- delivered FS and FA reduced SEAP production. These results were consistent with Lipofectamine 2000’s actions as a transfection reagent, as described previously (65). When the cells were treated with Poly (I:C) for 24 hours prior to addition of RNA/DNA fibers and then incubated for an additional 24 hours, the extent of SEAP inhibition was decreased (supporting Figure S1). All of these results support intracellular release of NANPs without any loss of their intended function. Author Manuscript Author Manuscript Author Manuscript GFP gene silencing with functionalized NANP loaded exosomes. We achieved successful transfection of labeled NANPs into cells using exosomes as delivery vesicles. To confirm retention of the NANPs’ intended function upon delivery, NANPs decorated with DS RNA against GFP (1, 6, 16, 65) were used. Human breast cancer cells MDA-MB-231-GFP expressing GFP (231-GFP) were treated with a panel of exosomes loaded with anti-GFP-functionalized NANP polyplexes (Figure 4A–B). Based on microscopy, flow cytometry, and RT-qPCR results, we found that 231-GFP cells treated with Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nordmeier et al. Page 9 Page 9 RNA ring- and RNA cube-loaded exosomes demonstrated a marked decrease in GFP expression. Exosomes loaded with sense (FS) or antisense (FA) RNA/DNA fibers did not change GFP expression individually; however, when both exosomes were added onto the same cell, there was a significant decrease in GFP expression. Therefore, fibers can re- associate and release DS RNAs only upon internalization with exosomes. Despite differences in shape, functional RNA cubes, RNA rings, and RNA/DNA fibers performed similarly in GFP silencing experiments (Figure 4A–B), while no cytotoxicity was observed (supporting Figure S2). Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Immunostimulation by exosome loaded NANPs. Another nucleic acid-specific TLR was investigated using HEK-Blue™ hTLR7 cells. Both HEK-Blue™ hTLR 3 and 7 cells were obtained by co-transfection of the hTLR gene into HEK293 cells and engineered to express a single Toll-like receptor. Stimulation of TLR7 with R848, an imidazoquinoline and agonist of TLR7, led to production of SEAP whose levels can be determined using HEK-Blue™ Detection reagent in real time. Here, we used Nordmeier et al. Page 10 these cells to evaluate the potential immunostimulation by the NANP/exosome complexes (Figure 5). Both TLR3 and TLR7 are responsible for RNA detection, while TLR3 activated by dsRNA and TLR7 detects ssRNA (66). Our data confirmed stimulation from both TLR3 and TLR7 cells to be negligible. Author Manuscript DISCUSSION Here, we demonstrated exosome-mediated delivery of different NANPs exhibiting various shapes and structures and investigated their intracellular uptake, post-delivery gene silencing efficiency, and immunostimulation potential. Exosome loading with NANPs of various sizes can be challenging and numerous methods have been developed to efficiently facilitate the transfection. One technique is chemical transfection, shows promising results for direct cargo loading. Exo-Fect™ siRNA/miRNA transfection kit (System Biosciences, Palo Alto, CA, USA) has shown positive results for nucleic acid transfection into exosomes (unpublished results). In order to load the cube, ring, and fiber NANPs into exosomes, Exo- Fect reagent forms a complex with the nucleic acids. We showed that exosomes remain intact post packaging and retain the common exosomal marker such as CD63. Interestingly, we noticed that the exosomes become slightly bigger upon NANPs loading. We demonstrated that NANPs loaded into exosomes with intact shape reassumed their original functionalities upon delivery (Figure 6) and effectively silenced GFP in the cells that constitutively express GFP. In our earlier studies using HEK 293 cells that overexpress human TLR7 and the SEAP genes, we showed that the RNA cubes delivered by L2K and PgP induced SEAP production when placed under the control of an NF-kB and AP-1- inducible promoter (61, 67). In contrast, even for the RNA cubes (known for displaying the greatest immunostimulatory properties) we observed negligible immune response after using exosomes to deliver these structures. Our data suggest that exosome-mediated NANP delivery can potentially “stealth-coat” exosome contents from certain pattern recognition receptors, but further studies are required. In summary, this work may pave the way towards the efficient and safe application of NANPs in personalized medicine by implementing patient-derived exosomes as a novel and promising therapeutic platform. Author Manuscript Author Manuscript Supplementary Material Refer to Web version on PubMed Central for supplementary material. 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A cationic amphiphilic co-polymer as a carrier of nucleic acid nanoparticles (Nanps) for controlled gene silencing, immunostimulation, and biodistribution. Nanomedicine: Nanotechnology, Biology and Medicine. 2020;23:102094. Author Manuscript Author Manuscript Author Manuscript Nordmeier et al. Page 15 HIGHLIGHTS • Exosomes derived from MDA-MB-231 cells delivered NANPs • Exosomes protect NANPs from nuclease degradation • NANPs delivered by exosomes activate RNA interference (RNAi) and NF-kB decoy • Exosome-mediated delivery of NANPs showed high efficiency and low immunostimulation HIGHLIGHTS • Exosomes derived from MDA-MB-231 cells delivered NANPs • Exosomes protect NANPs from nuclease degradation • NANPs delivered by exosomes activate RNA interference (RNAi) and NF-kB decoy • Exosome-mediated delivery of NANPs showed high efficiency and low immunostimulation Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 1: Characterization of exosomes isolated from MDA-MB-231 cells. (A) Schematic representation of exosome formation and release out to the extracellular space. (B) Western blot analysis of exosomal markers for CD63, ALIX and Hsp70, and cellular ER marker Calnexin in the whole cell lysate (WCL) and ExoQuick-TC ULTRA isolated exosomes (Exo). Western Blot analysis of the co-isolating proteins, bovine albumin (bAlbumin) and bovine immunoglobulin (bIgG) after one isolation step of precipitation step with ExoQuick- TC and after two steps of precipitation with ExoQuick-TC ULTRA. (C) The size distribution of isolated exosomes showing concentration in particles/mL. (D) TEM analysis of isolated exosomes. Nordmeier et al. Page 16 Page 16 Nordmeier et al. Nordmeier et al. Fi 1 Author Manuscript Figure 1: Figure 1: Characterization of exosomes isolated from MDA-MB-231 cells. (A) Schematic Characterization of exosomes isolated from MDA MB 231 cells. (A) Schematic representation of exosome formation and release out to the extracellular space. (B) Western blot analysis of exosomal markers for CD63, ALIX and Hsp70, and cellular ER marker Calnexin in the whole cell lysate (WCL) and ExoQuick-TC ULTRA isolated exosomes (Exo). Western Blot analysis of the co-isolating proteins, bovine albumin (bAlbumin) and bovine immunoglobulin (bIgG) after one isolation step of precipitation step with ExoQuick- TC and after two steps of precipitation with ExoQuick-TC ULTRA. (C) The size distribution of isolated exosomes showing concentration in particles/mL. (D) TEM analysis of isolated exosomes. Nanomedicine. Author manuscript; available in PMC 2021 November 01. REFERENCES: Author Manuscript Author Manuscript Author Manuscript Figure 2: Characterization of various functionalized NANPs and NANP loaded exosomes. (A) AFM images and native-PAGE results of RNA rings with six anti-GFP Dicer Substrate (DS) RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, RNA/DNA fibers with anti-GFP DS antisense. (B) TEM images of exosomes loaded with RNA rings with six anti-GFP DS RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, and RNA/DNA fibers with anti-GFP DS antisense. (C) Western blot analysis of exosomal markers for CD63, ALIX, and Hsp70 in ExoQuick-TC ULTRA isolated exosomes (Exo) taken through the loading steps as negative control and exosomes loaded with anti-GFP DS RNAs (Exo/DS RNAs), RNA cubes with six anti-GFP DS RNAs (Exo/RNA cubes), RNA rings with six anti-GFP DS RNAs (Exo/RNA rings), and RNA/DNA fibers with anti-GFP DS sense (Exo/Fibers). (D) NTA analysis of ExoQuick-TC ULTRA isolated exosomes (Exo) taken through the loading steps as negative control and exosomes loaded with anti-GFP DS RNAs (Exo/DS RNAs), RNA cubes with six meier et al. Page 17 Page 17 Page 17 Nordmeier et al. Figure 2: Characterization of various functionalized NANPs and NANP loaded exosomes. (A) AFM images and native-PAGE results of RNA rings with six anti-GFP Dicer Substrate (DS) RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, RNA/DNA fibers with anti-GFP DS antisense. (B) TEM images of exosomes loaded with RNA rings with six anti-GFP DS RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, and RNA/DNA fibers with anti-GFP DS antisense. (C) Western blot analysis of exosomal markers for CD63, ALIX, and Hsp70 in ExoQuick-TC ULTRA isolated exosomes (Exo) taken through the loading steps as negative Author Manuscript Author Manuscript Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. anti-GFP DS RNAs (Exo/RNA cubes), RNA rings with six anti-GFP DS RNAs (Exo/RNA rings), and RNA/DNA fibers with anti-GFP DS sense (Exo/Fibers). Figure 2: Figure 2: Characterization of various functionalized NANPs and NANP loaded exosomes. (A) AFM images and native-PAGE results of RNA rings with six anti-GFP Dicer Substrate (DS) RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, RNA/DNA fibers with anti-GFP DS antisense. (B) TEM images of exosomes loaded with RNA rings with six anti-GFP DS RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, and RNA/DNA fibers with anti-GFP DS antisense. (C) Western blot analysis of exosomal markers for CD63, ALIX, and Hsp70 in ExoQuick-TC ULTRA isolated exosomes (Exo) taken through the loading steps as negative control and exosomes loaded with anti-GFP DS RNAs (Exo/DS RNAs), RNA cubes with six anti-GFP DS RNAs (Exo/RNA cubes), RNA rings with six anti-GFP DS RNAs (Exo/RNA rings), and RNA/DNA fibers with anti-GFP DS sense (Exo/Fibers). (D) NTA analysis of ExoQuick-TC ULTRA isolated exosomes (Exo) taken through the loading steps as negative control and exosomes loaded with anti-GFP DS RNAs (Exo/DS RNAs), RNA cubes with six g Characterization of various functionalized NANPs and NANP loaded exosomes. (A) AFM images and native-PAGE results of RNA rings with six anti-GFP Dicer Substrate (DS) RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, RNA/DNA fibers with anti-GFP DS antisense. (B) TEM images of exosomes loaded with RNA rings with six anti-GFP DS RNAs, RNA cubes with six anti-GFP DS RNAs, RNA/DNA fibers with anti-GFP DS sense, and RNA/DNA fibers with anti-GFP DS antisense. (C) Western blot analysis of exosomal markers for CD63, ALIX, and Hsp70 in ExoQuick-TC ULTRA isolated exosomes (Exo) taken through the loading steps as negative control and exosomes loaded with anti-GFP DS RNAs (Exo/DS RNAs), RNA cubes with six anti-GFP DS RNAs (Exo/RNA cubes), RNA rings with six anti-GFP DS RNAs (Exo/RNA rings), and RNA/DNA fibers with anti-GFP DS sense (Exo/Fibers). (D) NTA analysis of ExoQuick-TC ULTRA isolated exosomes (Exo) taken through the loading steps as negative control and exosomes loaded with anti-GFP DS RNAs (Exo/DS RNAs), RNA cubes with six g Characterization of various functionalized NANPs and NANP loaded exosomes. (A) AFM Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nanomedicine. Author manuscript; available in PMC 2021 November 01. anti-GFP DS RNAs (Exo/RNA cubes), RNA rings with six anti-GFP DS RNAs (Exo/RNA rings), and RNA/DNA fibers with anti-GFP DS sense (Exo/Fibers). Page 18 anti-GFP DS RNAs (Exo/RNA cubes), RNA rings with six anti-GFP DS RNAs (Exo/RNA rings), and RNA/DNA fibers with anti-GFP DS sense (Exo/Fibers). Page 18 Nordmeier et al. Page 18 Page 18 anti-GFP DS RNAs (Exo/RNA cubes), RNA rings with six anti-GFP DS RNAs (Exo/RNA rings), and RNA/DNA fibers with anti-GFP DS sense (Exo/Fibers). Author Manuscript Author Manuscript Author Manuscript Page 19 Nordmeier et al. Figure 3: Exosomes protect nucleic acids from enzymatic degradation and promote their cellular uptake. (A) Schematic diagram of nuclease digestion assay and assay results for DNA duplexes loaded exosomes after incubation with RQ1 DNAse. (B–C) Fluorescent microscope images of human breast cancer MDA-MB-231 show cell uptake of fluorescently labeled DNA duplexes (B) and NANPs (C) and the corresponding flow cytometry analysis. ordmeier et al. Page 19 Author Manuscript Author Manuscript Figure 3: Figure 3: Exosomes protect nucleic acids from enzymatic degradation and promote their cellular Figure 3: Exosomes protect nucleic acids from enzymatic degradation and promote their cellular g Exosomes protect nucleic acids from enzymatic degradation and promote their cellular uptake. (A) Schematic diagram of nuclease digestion assay and assay results for DNA duplexes loaded exosomes after incubation with RQ1 DNAse. (B–C) Fluorescent microscope images of human breast cancer MDA-MB-231 show cell uptake of fluorescently labeled DNA duplexes (B) and NANPs (C) and the corresponding flow cytometry analysis. Author Manuscript Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Page 20 Nordmeier et al. Figure 4: GFP silencing in MDA-MB-231/GFP cells treated with functionalized anti-GFP NANPs loaded into exosomes and inhibition of NF-kB function in HEK-Blue™ hTLR3 and THP1- Dual™ cells. (A) Fluorescent microscope images of MDA-MB-231-GFPs taken 72 hours t t t t ith d l d d ith ith NANP (B) Fl t t Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nanomedicine. Author manuscript; available in PMC 2021 November 01. Figure 4: Figure 4: GFP silencing in MDA MB 231/GFP cells treated with functionalized anti GFP NANPs Figure 4: GFP silencing in MDA-MB-231/GFP cells treated with functionalized anti-GFP NANPs g GFP silencing in MDA-MB-231/GFP cells treated with functionalized anti-GFP NANPs loaded into exosomes and inhibition of NF-kB function in HEK-Blue™ hTLR3 and THP1- Dual™ cells. (A) Fluorescent microscope images of MDA-MB-231-GFPs taken 72 hours post treatment with exosomes and exosomes loaded with either NANPs. (B) Flow cytometry data of the mean GFP fluorescence and RT-qPCR analysis of the relative GFP expression of MDA-MB-231-GFP cells after 72 hours of incubation with NANPs loaded exosomes. Statistical significance for the samples compared to the negative control is denoted by * (*** p<0.001). Statistical significance of Exo/FS, Exo/FA compared to Exo/FS+Exo/FA is denoted by # (### p<0.001). (C) Schematic demonstration of the TLR3 signaling that activates the NF-kB pathway and the assembly of the NF-kB decoys (upon re-association of Author Manuscript Nanomedicine. Author manuscript; available in PMC 2021 November 01. Nordmeier et al. Page 21 the RNA/DNA fibers) that inhibits the nuclear translocation of the activated NF-kB. Poly (I:C) is used to activate the TLR3 ligand, the TLR3 signal resulted in the activation of NF- kB and subsequently NF-kB entered the nucleus, where it will bind to specific sequences of DNA to promote the downstream transcription and translation of secreted embryonic alkaline phosphatase (SEAP). The reporter cell line HEK-Blue™ hTLR3 was transfected with fibers and stimulated with Poly (I:C). The cells were incubated for 24 hours, and the levels of NF-kB-dependent SEAP were measured in the supernatants. (D) Schematic demonstration of the TLR1/2 signaling that activates the NF-kB pathway that can be inhibited with the NF-kB decoys assembled upon re-association of RNA/DNA fibers (F(S) and F(A)). Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 5: Relative levels of immunostimulation of exosomes loaded with NANPs. TLR stimulation is measured via SEAP secretion from (A) HEK-Blue™ hTLR3 and (B) HEK-Blue™ hTLR7 cells. In (A), statistical significance relevant to the cells is denoted by #, statistical significance relevant to poly I:C is denoted by * (# p<0.05, * p<0.001). In (B), statistical significance relevant to R848 is denoted by # ((# p<0.05). ordmeier et al. Page 22 dmeier et al. Page 22 Nordmeier et al. Author Manuscript Figure 5: Figure 5: Relative levels of immunostimulation of exosomes loaded with NANPs. Nanomedicine. Author manuscript; available in PMC 2021 November 01. Figure 4: TLR stimulation is Figure 5: Relative levels of immunostimulation of exosomes loaded with NANPs. TLR stimulation is measured via SEAP secretion from (A) HEK-Blue™ hTLR3 and (B) HEK-Blue™ hTLR7 cells. In (A), statistical significance relevant to the cells is denoted by #, statistical significance relevant to poly I:C is denoted by * (# p<0.05, * p<0.001). In (B), statistical significance relevant to R848 is denoted by # ((# p<0.05). measured via SEAP secretion from (A) HEK-Blue™ hTLR3 and (B) HEK-Blue™ hTLR7 cells. In (A), statistical significance relevant to the cells is denoted by #, statistical significance relevant to poly I:C is denoted by * (# p<0.05, * p<0.001). In (B), statistical significance relevant to R848 is denoted by # ((# p<0.05). Author Manuscript Author Manuscript Author Manuscript Page 23 Page 23 Nordmeier et al. Figure 6: Schematic summary of the proposed mechanism of exosome-mediated delivery of different functionalized NANPs. Exosomes are loaded with NANPs, such as RNA cubes and RNA rings, that are functionalized with 6 DSRNAs, and fiber antisense and fiber sense that carry n numbers of DS RNAs and NF-kB decoys. Those NANPs are delivered by exosomes and released inside the target cells. In the cytosol, the DS RNAs will be further diced by Dicer to result in the generation of siRNAs, by which the RNAi pathway is activated. Meanwhile, the NF-kB decoys are released from the fibers, which in turn bind to NF-kB in order to prevent its nuclear translocation, thus inhibit the downstream production of IRF and IFNs. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 6: Figure 6: Figure 6: Schematic summary of the proposed mechanism of exosome-mediated delivery of different functionalized NANPs. Exosomes are loaded with NANPs, such as RNA cubes and RNA rings, that are functionalized with 6 DSRNAs, and fiber antisense and fiber sense that carry n numbers of DS RNAs and NF-kB decoys. Those NANPs are delivered by exosomes and released inside the target cells. In the cytosol, the DS RNAs will be further diced by Dicer to result in the generation of siRNAs, by which the RNAi pathway is activated. Meanwhile, the NF-kB decoys are released from the fibers, which in turn bind to NF-kB in order to prevent its nuclear translocation, thus inhibit the downstream production of IRF and IFNs. Figure 6: Schematic summary of the proposed mechanism of exosome-mediated delivery of different functionalized NANPs. Exosomes are loaded with NANPs, such as RNA cubes and RNA rings, that are functionalized with 6 DSRNAs, and fiber antisense and fiber sense that carry n numbers of DS RNAs and NF-kB decoys. Those NANPs are delivered by exosomes and released inside the target cells. In the cytosol, the DS RNAs will be further diced by Dicer to result in the generation of siRNAs, by which the RNAi pathway is activated. Meanwhile, the NF-kB decoys are released from the fibers, which in turn bind to NF-kB in order to prevent its nuclear translocation, thus inhibit the downstream production of IRF and IFNs. Author Manuscript

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https://researchonline.lshtm.ac.uk/id/eprint/4646960/1/The%20current%20and%20potential%20health%20benefits%20of%20the%20National%20Health%20Service%20Health%20Check%20cardiovascular%20disease%20prevention%20programme%20in%20England_GOLD%20VoR.pdf

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The current and potential health benefits of the National Health Service Health Check cardiovascular disease prevention programme in England: A microsimulation study

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RESEARCH ARTICLE Abstract Citation: Mytton OT, Jackson C, Steinacher A, Goodman A, Langenberg C, Griffin S, et al. (2018) The current and potential health benefits of the National Health Service Health Check cardiovascular disease prevention programme in England: A microsimulation study. PLoS Med 15 (3): e1002517. https://doi.org/10.1371/journal. pmed.1002517 The current and potential health benefits of the National Health Service Health Check cardiovascular disease prevention programme in England: A microsimulation study Oliver T. Mytton1‡*, Christopher Jackson2‡, Arno Steinacher2, Anna Goodman3, Claudia Langenberg1, Simon Griffin1,4, Nick Wareham1, James Woodcock1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 MRC Epidemiology Unit, University of Cambridge, Cambridge, United Kingdom, 2 MRC Biostatistics Unit, University of Cambridge, Cambridge, United Kingdom, 3 London School of Hygiene and Tropical Medicine, London, United Kingdom, 4 Department of Public Health and Primary Care, University of Cambridge, Cambridge, United Kingdom ‡ These authors are joint first authors on this work. Academic Editor: Aziz Sheikh, Edinburgh University, UNITED KINGDOM Academic Editor: Aziz Sheikh, Edinburgh University, UNITED KINGDOM Received: August 3, 2017 Accepted: January 25, 2018 Published: March 6, 2018 Copyright: © 2018 Mytton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Academic Editor: Aziz Sheikh, Edinburgh University, UNITED KINGDOM Received: August 3, 2017 Accepted: January 25, 2018 Published: March 6, 2018 ‡ These authors are joint first authors on this work. * [email protected] Background The National Health Service (NHS) Health Check programme was introduced in 2009 in England to systematically assess all adults in midlife for cardiovascular disease risk factors. However, its current benefit and impact on health inequalities are unknown. It is also unclear whether feasible changes in how it is delivered could result in increased benefits. It is one of the first such programmes in the world. We sought to estimate the health benefits and effect on inequalities of the current NHS Health Check programme and the impact of making feasi- ble changes to its implementation. ‡ These authors are joint first authors on this work. * otm21@medschl cam ac uk Methods and findings This equates to approximately 300 fewer premature deaths and 1,000 more people living free of these diseases each year in England. We esti- mate that the current programme is increasing QALYs by 3.8 days (95% credible interval 3.0–4.7) per head of population and increasing survival by 3.3 days (2.5–4.1) per head of population over the 60 years of follow-up. The current programme has a greater absolute impact on health for those living in the most deprived areas compared to those living in the least deprived areas (4.4 [2.7–6.5] days of additional quality-adjusted life per head of popu- lation versus 2.8 [1.7–4.0] days; 5.1 [3.4–7.1] additional days lived per head of population versus 3.3 [2.1–4.5] days). Making feasible changes to the delivery of the existing pro- gramme could result in a sizable increase in the benefit. For example, a strategy that com- bines extending eligibility to those with preexisting hypertension, extending the upper age of eligibility to 79 years, increasing uptake of health checks by 30%, and increasing treatment rates 2.5-fold amongst eligible patients (i.e., ‘maximum potential’ scenario) results in at least a 3-fold increase in benefits compared to the current programme (1,360 premature deaths versus 390; 5,100 people free of 1 of the 4 diseases versus 1,370; 37,000 additional QALYs versus 10,000; 33,000 additional years of life versus 9,000). Ensuring those who are as- sessed and eligible for statins receive statins is a particularly important strategy to increase benefits. Estimates of overall benefit are based on current incidence and management, and future declines in disease incidence or improvements in treatment could alter the actual ben- efits observed in the long run. We have focused on the cardiovascular element of the NHS Health Check programme. Some important noncardiovascular health outcomes (e.g., chronic obstructive pulmonary disease [COPD] prevention from smoking cessation and can- cer prevention from weight loss) and other parts of the programme (e.g., brief interventions to reduce harmful alcohol consumption) have not been modelled. Heart Foundation (ES/G007462/1), Cancer Research UK (ES/G007462/1), Economic and Social Research Council (ES/G007462/1), Medical Research Council (ES/G007462/1), the National Institute for Health Research (ES/G007462/1), and the Wellcome Trust (087636/Z/08/Z), under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged. Core MRC Epidemiology Unit (MC_UU_12015) and MRC Biostatistics Unit (U105260566) support is also acknowledged. OTM was funded by a Wellcome Trust fellowship (WT103394) and a National Institute for Health Research Academic Clinical Lectureship. Methods and findings Competing interests: CL receives a stipend as a specialty consulting editor for PLOS Medicine and serves on the journal’s editorial board. NW is a member of the Expert Scientific and Clinical Advisor Panel (ESCAP) for the NHS Health Check Programme and was an editor for a (single) special edition of PLOS Medicine focusing on diabetes. AG received grants and nonfinancial support from Public Health England during the conduct of the study. OTM has an honorary contract with Public Health England (East of England) for the purposes of undertaking health protection on-call duties. Development of the model, as well as analysis, presentation and interpretation of the findings, was independent of the funders. The views expressed in this publication are those of the authors and do not necessarily represent those of Public Health England, the Department of Health, or institutions that fund or support the authors. No funding bodies had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abbreviations: COPD, chronic obstructive pulmonary disease; CrI, credible interval; CVD, cardiovascular disease; ELSA, English Longitudinal Study of Aging; IHD, ischaemic heart disease; NHS, National Health Service; NICE, National Institute for Health and Care Excellence; QALY, quality-adjusted life year. Conclusions Our model indicates that the current NHS Health Check programme is contributing to improvements in health and reducing health inequalities. Feasible changes in the organisa- tion of the programme could result in more than a 3-fold increase in health benefits. Methods and findings We developed a microsimulation model to estimate the health benefits (incident ischaemic heart disease, stroke, dementia, and lung cancer) of the NHS Health Check programme in England. We simulated a population of adults in England aged 40–45 years and followed until age 100 years, using data from the Health Survey of England (2009–2012) and the English Longitudinal Study of Aging (1998–2012), to simulate changes in risk factors for simulated individuals over time. We used recent programme data to describe uptake of NHS Health Checks and of 4 associated interventions (statin medication, antihypertensive medication, smoking cessation, and weight management). Estimates of treatment efficacy and adherence were based on trial data. We estimated the benefits of the current NHS Health Check programme compared to a healthcare system without systematic health checks. This counterfactual scenario models the detection and treatment of risk factors that occur within ‘routine’ primary care. We also explored the impact of making feasible changes to implementation of the programme concerning eligibility, uptake of NHS Health Checks, Copyright: © 2018 Mytton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The work was commissioned and funded by Public Health England. The work was undertaken by the Centre for Diet and Activity Research (CEDAR), a UK Clinical Research Collaboration (UKCRC) Public Health Research Centre of Excellence. Funding from the British PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 1 / 32 The current and potential health benefits of the NHS Health Check programme and uptake of treatments offered through the programme. We estimate that the NHS Health Check programme prevents 390 (95% credible interval 290 to 500) premature deaths before 80 years of age and results in an additional 1,370 (95% credible interval 1,100 to 1,690) peo- ple being free of disease (ischaemic heart disease, stroke, dementia, and lung cancer) at age 80 years per million people aged 40–45 years at baseline. Over the life of the cohort (i.e., followed from 40–45 years to 100 years), the changes result in an additional 10,000 (95% credible interval 8,200 to 13,000) quality-adjusted life years (QALYs) and an additional 9,000 (6,900 to 11,300) years of life. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 What did the researchers do and find? • We developed a longitudinal microsimulation model to simulate the NHS Health Check programme and its impact on health, using epidemiological data for England and per- formance data for the programme. • We estimated that the current NHS Health Check programme is preventing approxi- mately 300 premature deaths (before 80 years) and resulting in an additional 1,000 peo- ple at age 80 years being free of cardiovascular diseases, dementia, and lung cancer each year in England. If risk of cardiovascular disease continues to decline, then these bene- fits will be attenuated by as much as a half. • The benefits were greatest for people living in more deprived areas, and thus, the pro- gramme as a whole is reducing health inequalities. • Making feasible changes to the delivery of the existing programme is likely to result in valuable improvements in health. For example, a strategy that combines extending eligi- bility to those with preexisting hypertension, extending the upper age of eligibility to 79 years, increasing uptake of health checks by 30%, and increasing treatment rates amongst eligible patients 2.5-fold (i.e., ‘maximum potential’ scenario) could result in at least a 3-fold increase in benefits compared to the current programme. Why was this study done? • The National Health Service (NHS) Health Check programme is one of the first such programmes in the world, and there is uncertainty about the impact of the programme on health outcomes and inequalities. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 2 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 The current and potential health benefits of the NHS Health Check programme • The programme is evolving, although there have been no estimates of the impact of pos- sible changes in the way that the programme is delivered on health outcomes or inequalities. • The programme is evolving, although there have been no estimates of the impact of pos- sible changes in the way that the programme is delivered on health outcomes or inequalities. What do these finding mean? • The estimates of overall benefit are broadly in line with an estimate of likely benefit made prior to the programme’s introduction, which provides reassurance that the pro- gramme is meeting those expectations. • The current practice of ensuring a higher attendance amongst people living in more deprived areas appears to be reducing health inequalities. • There appears to be considerable scope to improve the health benefit of the programme by making feasible changes to its delivery, notably by ensuring those who are assessed and eligible for treatments receive appropriate treatment. Focusing on inviting previous nonattenders and widening the eligibility criteria to include those with an existing diag- nosis of hypertension could also make a valuable contribution to increasing the health benefits of the programme. The current and potential health benefits of the NHS Health Check programme To address this, structured vascular risk assessment for adults aged 40–74 years without preexisting diabetes or cardiovascular disease (‘health checks’) was introduced in England in 2009 [7]. The programme sought to systematically identify individuals at risk of cardiovascular disease through a structured risk assessment and an offer of appropriate treatment, either pharmacological or behavioural. Now termed the National Health Service (NHS) Health Check programme, it consists of a defined set of interventions. While other components have been added over time, notably concerning alcohol, it retains a major focus on cardiovascular disease prevention [8]. The programme has been criticised for lacking evidence of benefit [9], and the overall health benefit it offers is unclear. While there have been trials of a ‘general health check’ in the past, many of these studies are old, with some pre-dating the introduction of more effective treatments like statins, and few, if any, of the interventions are comparable to the NHS Health Check programme [10–12]. Published evaluations of the current programme estimate benefit in terms of changes in cardiovascular risk factors (e.g., blood pressure), but these studies are prone to selection bias and do not estimate changes in ‘hard’ health outcomes [13,14]. Previous modelling studies have estimated the health benefit of a vascular check programme in England or the UK. One prior to the programme’s introduction sought to explicitly model the NHS Health Check pro- gramme and was based on an estimate of likely programme performance [7]. A second com- pared a universal (vascular) screening programme (based in part on the NHS Health Check programme) with concentrated screening and other population approaches to cardiovascular disease prevention [15]. The third study compared 7 different models for the delivery of a vas- cular ‘health check’ programme across 6 different European countries [16]. Whilst the vascular check programmes modelled in the latter 2 studies shared similarities with the NHS Health Check programme, neither explicitly modelled the NHS Health Check programme. None of these modelling studies make use of the more detailed emerging empirical data that character- ise uptake by sociodemographic characteristics or the full range of data available on pro- gramme performance (e.g., referral to smoking cessation and weight management services) [13]. Despite concerns about overall benefit [9], the programme remains in place, is legally man- dated as a universal programme [17], receives high-level political support [18,19], and is per- ceived favourably by patients [20,21]. Thus, the programme is likely to continue. A key focus is whether and how the existing programme could be more effective or (further) reduce health inequalities. Whilst there have been local evaluations of different approaches to programme delivery, we are not aware of any studies that have quantified the health impact and/or the effect on inequalities of making systemic changes to the programme’s delivery—for example, changing eligibility criteria, increasing attendance, or increasing uptake of treatments offered through the programme. Given that the programme is now established, it is an opportune time to review how the programme might evolve or change in order to improve impact. We sought to address 2 questions. First, what is the health benefit and effect on health equity of the NHS Health Check programme as it is currently delivered in England? Second, we sought to understand the health benefits (or losses) that might accrue from making changes to the existing programme, considering eligibility criteria (widening or reducing eligibility); increasing the uptake of the programme (either generically or amongst high-risk groups); and improving uptake of treatments offered through the programme. Introduction The prevention of cardiovascular disease remains an important priority in the United King- dom and elsewhere [1–4]. Cardiovascular disease accounts for around a quarter of all deaths and costs around £15 billion annually in the UK [5]. While there are a set of well-established actions to prevent cardiovascular disease, the uptake of these preventive interventions is sub- optimal [6]. 3 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Methods We developed a microsimulation model to assess the effect of, and modifications to, the car- diovascular components of the NHS Health Check programme. The model consists of 2 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 4 / 32 The current and potential health benefits of the NHS Health Check programme Fig 1. Outline of the microsimulation model. https://doi.org/10.1371/journal.pmed.1002517.g001 Fig 1. Outline of the microsimulation model. https://doi.org/10.1371/journal.pmed.1002517.g001 https://doi.org/10.1371/journal.pmed.1002517.g001 modules (Fig 1). The first module (‘population and health’) describes the cardiovascular risk factors, disease status, and mortality of the population over time. The second module (‘Health Check’) simulates the different parts of the NHS Health Check: eligibility and attendance, assessment for treatment, and the effect of treatment. Further technical information on the methods are given as supplementary material (S1 Text), and the data inputs are summarised in Table 1. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 The current and potential health benefits of the NHS Health Check programme Table 1. Summary of data inputs. Part of model Parameter Data source/assumption Population Sociodemographic characteristics Health Survey for England 2009–2012 [31]. Health risk factors at baseline Health Survey for England 2009–2012 [31]. Change in risk factors over time English Longitudinal Study of Ageing (ELSA) 1998–2012 [23]. Disease Epidemiology Ischaemic heart disease (IHD) and stroke Individual 10-year risk of cardiovascular disease was calculated using the QRisk2 score [45]. The 10-year risk was converted to annual risk based on routine data processed using DisMod. The likelihood of a new cardiovascular disease (CVD) event being a stroke or IHD was determined by age- and sex-specific estimates of incidence. Estimates of case fatality were derived from routine data processed using DisMod with further recalibration to account for raised mortality in the first year after diagnosis or presentation (i.e., myocardial infarction: 32% for men and 30% for women [34]; stroke: 7% for men and 5% for women aged under 80 years, 24% for men and 17% for women aged over 80 years) [20]. We also assumed for men and women that 58% and 44%, respectively, of IHD new presentations or diagnoses presented as a myocardial infarction [33] and that 60% of stroke presentations (QRisk2 includes both full strokes and transient ischaemic attacks) were a full stroke rather than a transient ischaemic attack. Routine data sources included the following: mortality statistics for England and Wales [27], the Health Survey for England (prevalence) [31], estimates on case fatality for IHD in England from linked mortality and hospital record data [46], and estimates on case fatality rates for stroke based on primary care records in the UK [47]. Dementia Individual 20-year risk of dementia was calculated using the cardiovascular risk factors, aging, and incidence of dementia risk (CAIDE) score [48]. The 20-year risk was converted to annual risk based on routine data processed using DisMod. Estimates of case fatality were derived from routine data processed using DisMod. Routine data sources included the following: the Cognitive Function and Aging Study II in England (incidence) [29] and a published audit of primary care records in the UK (relative risk of mortality) [30]. Lung cancer Annual estimates of incidence were based on routine data sources processed using DisMod. Population and health module We simulated a closed cohort of 200,000 individuals aged 40–45 years representative of the English population, by sampling individuals from the Health Survey for England 2009–2012 to match the population structure (by gender and ethnicity) in the 2011 census [22]. Each indi- vidual had a set of demographic characteristics (age, sex, ethnicity, deprivation, and education) and a set of cardiovascular risk factors (blood pressure, smoking status, serum cholesterol, and body mass index). We modelled annual change in risk factors using the English Longitudinal Study of Aging (ELSA) (1998–2012) [23], which contains individual data on changes in cardio- vascular risk factors over time and which (mostly) preceded the introduction of health checks. Changes in risk factors were estimated by matching each individual in our simulated popula- tion to one in the ELSA cohort with similar characteristics, with individuals being rematched as their risk factors changed. This nonparametric approach allowed us to generate trajectories that produced realistic results at the population level while also capturing between-individual heterogeneity. Diseases. We estimated incidence and case fatality by age (year increments) and sex for 4 diseases: lung cancer, ischaemic heart disease, stroke, and dementia. We used QRisk2 to PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 5 / 32 Lung cancer cases were attributed to smoking or not, based on published estimates of the proportion of lung cancer cases attributable to smoking in the UK [36]. Routine data sources included the following: mortality statistics for England and Wales [27] and cancer registry data for England (incidence) [26]. National Health Service (NHS) Health Check programme Proportion of eligible population offered a health check 19.7% per year, based on published evaluation [49]. Proportion of people offered a health check who attend Estimates of uptake based on published evaluations and likelihood of attendance varied by age, sex, ethnicity, deprivation, smoking status, and QRisk2 score [13,14,50]. Proportion of people getting a health check who are not eligible on the basis of a chronic condition 5% (95% credible interval [CrI] 2% to 8%): estimated by study team as no data were available. NHS Health Check—initiation of treatment Proportion of smokers at health check who are referred to smoking cessation therapy 3.6% (95% CrI 3.3% to 3.9%), assuming 6.8% of smokers (2,571/37,808) who had a health check were referred to smoking cessation, compared to 3.2% (9,944/310,034) of smokers who do not have a health check based on published programme evaluation [13]. Proportion of obese people (BMI  30) at health check who are referred to weight management interventions 27.5% (95% CrI 26.9% to 28.1%), assuming 38.7% (12,430/32,133) of obese people who had a health check were referred to weight management, compared to 11.2% (4,441/39,774) of obese people who did not have a health check [13]. Only considers weight management, not the additional 31.1% of obese people who were referred to exercise, a group which is assumed to overlap substantially. Proportion who receive statins QRisk2 < 20%: 2.05% (95% CrI 1.97 to 2.13) additional statin prescriptions in health check attenders versus nonattenders. QRisk2  20%: 14.23% (95% CrI 13.71 to 14.76) additional statin prescriptions in health check attenders versus nonattenders. Based on published evaluation [13]. Proportion of people with high blood pressure who receive antihypertensives QRisk2 < 20%: 1.54% (95% CrI 1.46 to 1.62) additional antihypertensive prescriptions in health check attenders versus nonattenders. QRisk2  20%: 2.48% (95% CrI 2.05 to 2.90) additional antihypertensive prescriptions in health check attenders versus nonattenders. Only individuals with hypertension at health check (defined as systolic blood pressure greater than 140 mmHg) were assumed to get antihypertensive treatment in either case. Based on published evaluation [13]. The current and potential health benefits of the NHS Health Check programme Table 1. (Continued) Part of model Parameter Data source/assumption Adherence to treatment Smoking cessation We assumed that 100% of patients referred ‘adhere’ to treatment, as the treatment effectiveness estimates include those who are nonadherent. Weight management programme We assumed 50% attend at least one session, i.e., assuming a lower real-world take- up rate than that in published trials of weight-loss interventions (e.g., 68% in Weight Loss Referrals for Adults in Primary Care [WRAP] trial) [51]. We assumed a 95% CrI of 30% to 70%. Statins 50% adherence to initial prescription (with a 95% CrI of 40% to 60%, from our assumption), based on published estimates [52–54]. An additional 5% (95% CrI 3% to 7%) of people taking statins are assumed to stop taking them each year (our assumption). Antihypertension medication 55% adherence (with a 95% CrI of 45% to 65%, from our assumption), based on published estimates [52,54,55]. An additional 5% (95% CrI 3% to 7%) of people on antihypertensives (AHTs) are assumed to stop taking them each year (our assumption). Treatment effectiveness Smoking cessation Based on an evaluation of an English smoking cessation service, we assumed that 14.6% (95% CrI 13.1% to 16.1%) of those who are referred have quit at 1 year [56]. Relapse after quitting is modelled using ELSA data. Weight management effectiveness Based on a published audit of weight management services in the UK, we assumed a mean BMI change of −1.5 kg/m2 by 1 year for everyone attending at least 1 session [57]. Lost weight assumed to be regained over 5 years, with BMI changes of −1.5, −0.9, −0.6, −0.3, and 0 at 1, 2, 3, 4, and 5 years after health check, respectively (our assumption). Statin effectiveness Based on a meta-analysis of trials of efficacy of statins on cholesterol, we assumed a mean change of −1.22 (95% CrI −1.19 to −1.26) for men and −1.16 (95% CrI −1.10 to −1.23) for women in total cholesterol at 1 year [58] and a mean increase in high- density lipoprotein (HDL) at 1 year of 0.04 (95% CrI 0.028 to 0.052) for men and 0.036 (95% CrI 0.012 to 0.060) for women ([58]). Further adjustment was made to CVD risk for those on statins, reflecting published data on the efficacy of statin treatment (which was not adequately captured by changes in QRisk2 score) [58]. (Continued) Individual 10-year risk of cardiovascular disease was calculated using the QRisk2 score [45]. The 10-year risk was converted to annual risk based on routine data processed using DisMod. The likelihood of a new cardiovascular disease (CVD) event being a stroke or IHD was determined by age- and sex-specific estimates of incidence. Estimates of case fatality were derived from routine data processed using DisMod with further recalibration to account for raised mortality in the first year after diagnosis or presentation (i.e., myocardial infarction: 32% for men and 30% for women [34]; stroke: 7% for men and 5% for women aged under 80 years, 24% for men and 17% for women aged over 80 years) [20]. We also assumed for men and women that 58% and 44%, respectively, of IHD new presentations or diagnoses presented as a myocardial infarction [33] and that 60% of stroke presentations (QRisk2 includes both full strokes and transient ischaemic attacks) were a full stroke rather than a transient ischaemic attack. (Continued) (Continued) 6 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Antihypertensive medication effectiveness We assumed that those under 55 years used an angiotensin-converting-enzyme (ACE) inhibitor and those aged 55 years and over used calcium channel blockers. Based on a meta-analysis of efficacy of Ramipril on blood pressure, we assumed a mean change of −6.29/−4.14 mmHg (95% CrI −9.26 to −3.32/−5.81 to −2.48) [59]. Based on a meta-analysis of the efficacy of a calcium channel blocker on blood pressure, we assumed a mean change of −7.6/−3.1 mmHg (95% CrI −7.95 to −7.25/ −2.75 to −3.45) for men and −9.0/−3.5 mmHg (95% CrI −8.68 to −9.32/−3.18 to −3.82) for women [60]. Published 95% confidence intervals have been used as estimates for 95% credible intervals. We assumed that those under 55 years used an angiotensin-converting-enzyme (ACE) inhibitor and those aged 55 years and over used calcium channel blockers. Based on a meta-analysis of efficacy of Ramipril on blood pressure, we assumed a mean change of −6.29/−4.14 mmHg (95% CrI −9.26 to −3.32/−5.81 to −2.48) [59]. Based on a meta-analysis of the efficacy of a calcium channel blocker on blood pressure, we assumed a mean change of −7.6/−3.1 mmHg (95% CrI −7.95 to −7.25/ −2.75 to −3.45) for men and −9.0/−3.5 mmHg (95% CrI −8.68 to −9.32/−3.18 to −3.82) for women [60]. estimate risk of incident cardiovascular disease for each simulated individual based on socio- demographic and cardiovascular risk factors and the CAIDE score to estimate dementia inci- dence. These tools provide estimates of risk over a 10- and 20-year period, respectively, which were then converted to annual estimates of risk based on average estimates of population inci- dence estimated using DisMod II v1.05 [24] and routine data [25–31]. DisMod was used to generate annual estimates of incidence and case fatality from 2 or more estimates of routine data (e.g., prevalence and mortality). QRisk2 estimates the risk of incident (first) diagnosis of cardiovascular disease (ischaemic heart disease, stroke, or TIA). New cardiovascular events were assigned at random to be either ischaemic heart disease or a stroke, reflecting the relative proportions of these events by age. In doing this, we assumed that the ratio of strokes to TIAs was 60:40 [32]. We assumed that 58% of new diagnoses of ischaemic heart disease for men and 44% for women were an acute myocardial infarction [33]. The current and potential health benefits of the NHS Health Check programme and 5% for women under the age of 80 years, and 24% and 17% for men and women aged 80 years and over) were fatal [34,35]. Making allowance for acute fatality, we recalibrated the annual estimates of case fatality, from DisMod based on routine data sources, to account for acute mortality, i.e., we modelled a higher fatality in the year of diagnosis or presentation. Unadjusted estimates of case fatality by age and sex for dementia and lung cancer from Dis- Mod were used. For smoking, we calculated separate incidence rates for smokers and nonsmokers using DisMod based on reported population attributable fractions [36]. Whilst dementia was not a focus in the original programme [7], it has subsequently been included. Dementia shares many of the same risk factors as ischaemic heart disease and stroke, and vascular pathology in the brain is associated with dementia [37–39]. It is thought that dementia risk is modifiable and that recent reductions in incidence might be attributable to better management of cardiovascular risk [29,40,41]. On this basis, dementia was included in the model, although to date there is no evidence from randomised clinical trials showing that dementia risk can be reduced. Once an individual developed a disease, it was assumed that he or she had the disease for life, adopting the appropriate mortality risk. Mortality from all other causes was estimated using standard life tables [42], after adjusting for cause-specific mortality within the model. The model proceeds in annual increments, with individuals followed until death or 100 years of age. At the beginning of the simulation, a proportion of the population (based on estimates from DisMod) were assumed to have ischaemic heart disease or stroke. The probability of hav- ing cardiovascular disease at baseline depended on the QRisk2 score at baseline. We assumed that nobody had dementia or lung cancer at baseline. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 We further assumed that a proportion of acute events (32% for men and 33% for women for acute myocardial infarction; 7% for men 3.82) for women [60]. Published 95% confidence intervals have been used as estimates for 95% credible intervals. https://doi.org/10.1371/journal.pmed.1002517.t001 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 7 / 32 https://doi.org/10.1371/journal.pmed.1002517.t001 estimate risk of incident cardiovascular disease for each simulated individual based on socio- demographic and cardiovascular risk factors and the CAIDE score to estimate dementia inci- dence. These tools provide estimates of risk over a 10- and 20-year period, respectively, which were then converted to annual estimates of risk based on average estimates of population inci- dence estimated using DisMod II v1.05 [24] and routine data [25–31]. DisMod was used to generate annual estimates of incidence and case fatality from 2 or more estimates of routine data (e.g., prevalence and mortality). QRisk2 estimates the risk of incident (first) diagnosis of cardiovascular disease (ischaemic heart disease, stroke, or TIA). New cardiovascular events were assigned at random to be either ischaemic heart disease or a stroke, reflecting the relative proportions of these events by age. In doing this, we assumed that the ratio of strokes to TIAs was 60:40 [32]. We assumed that 58% of new diagnoses of ischaemic heart disease for men and 44% for women were an acute myocardial infarction [33]. We further assumed that a proportion of acute events (32% for men and 33% for women for acute myocardial infarction; 7% for men 7 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Health check module The full source code is avail- able under licence from a GitHub repository (https://github.com/chjackson/healthchecks). Model calibration and validation We sought to identify the most suitable data for our model and focused on developing the aspects of the model that are most important for the scenarios we sought to explore. During the development of the model, we undertook a series of checks to ensure the model was accu- rately simulating the health check process (i.e., attendance and treatment uptake) and outputs (e.g., blood pressure and QRisk2) by comparing model outputs with empirical data. We also compared trial data for blood pressure medication and statins with published trial data on treatment efficacy. Changes in QRisk2 due to changes in serum cholesterol did not accurately capture the reduction in risk for statin treatment reported in trials, as would be expected, as statins reduce cardiovascular risk by other means (e.g., reducing inflammation) [62]. Conse- quently, we made a further adjustment to cardiovascular disease (CVD) risk for people who were started on a statin by calibrating the modelled reduction in CVD event rates over 5 years achieved by statin so that it was equal to the value observed in trials [58]. To validate the popu- lation and health module, we compared estimates of mortality for ischaemic heart disease and stroke by sex—produced by this part of the model—with published estimates of mortality based on death certification [25]. There was reasonably close agreement (S1 Data). Modelled health benefits In all cases, the effect of changes in risk factors on health was modelled through the respective disease risk scores for cardiovascular disease and dementia. For smoking, we estimated sepa- rate incidence rates for smokers and nonsmokers, as described previously. Body mass index was assumed to have a direct effect on QRisk2 score (rather than through changes in blood pressure and cholesterol). Health check module Simulated individuals were eligible for a health check based on their age and their disease sta- tus (absence of diabetes, cardiovascular disease, and hypertension), reflecting current eligibility criteria. We assumed that the annual probability of an eligible individual being offered a health check was 0.197, based on national programme data [43]. Thus, on average, a person would be offered a health check once every 5 years. We assumed that the likelihood of attendance was determined by sociodemographic char- acteristics (age, ethnicity, and area-level deprivation) and cardiovascular risk factors (smoking status and QRisk2), based on programme data[13], and (in the absence of long-term data) we assumed that past attendance did not affect future attendance. We also assumed that 5% of ineligible individuals attended for an NHS Health Check, e.g., via a drop-in clinic in a pharmacy. We assumed that individuals could be offered 1 or more of 4 treatments: statins, antihyper- tensives, smoking cessation, and weight management [44]. Assumptions regarding who got treated, adherence to treatment, and the effect of treatment are summarised in Table 1. We assumed no interaction between treatments. For all individuals receiving statins, antihypertensives, or weight management, a counter- factual trajectory for each risk factor, without treatment after an NHS Health Check, was simu- lated using the ELSA data. A treatment trajectory was then estimated by adjusting the counterfactual trajectory to represent the effect of treatment. For example, if a woman was compliant with statin therapy (initiated because of a health check), her cholesterol level would be 1.16 mmol/L lower than her background (or ‘counterfactual’) cholesterol. For individuals who quit smoking after attending a smoking cessation programme, their risk of relapse and PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 8 / 32 The current and potential health benefits of the NHS Health Check programme future smoking status were estimated by matching to individuals in the ELSA dataset and adjusting for published evidence on quitting and relapsing [61]. In all cases, we have effectively modelled a decline in treatment effectiveness over time due either to reduced adherence or relapse (see Table 1) such that the health benefits over time may appear to be less than expected from trial data but should be more akin to the benefit attributable to the NHS Health Check programme in the real world. The simulation model was written in Python (version 2.7.6). PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Outcomes Primary outcomes are total incident cases prevented (by age 80 years), premature deaths pre- vented (<80 years), change in quality-adjusted life years (QALYs), and change in survival. We also report incident events prevented by age 100 years (as well as providing a breakdown by disease category) and deaths prevented before 75 years of age. We provide estimates of the total additional quality-adjusted years lived and years lived for the studied cohort of 1,000,000 people and estimates per head of population. We use the latter metric as the primary means to describe the effect of health inequalities, as these measures make allowance for different popu- lation sizes. Standard EQ-5D disutility weights for age, deprivation, and disease status were used to esti- mate QALYs [64]. To provide a comparison with other published estimates that describe the number of events avoided in England each year, we multiply our estimates for events avoided over the life of the cohort by 0.73 (there are approximately 730,000 adults aged 40 in any given year in England). This assumes the benefits observed for the current population in any given year are compara- ble to the benefits that the cohort (aged 40–45 years) experience longitudinally. We also provide estimates for those living in the most and least deprived areas (expressed per head of population to standardise for differences in population size). Each simulated indi- vidual adopted the quintile group of the sampled individual from the Health Survey for England (based on the Index of Multiple Deprivation for the area of residence). The current and potential health benefits of the NHS Health Check programme (3) for smokers, (4) for those at high cardiovascular risk (QRisk2 > 20%), and (5) for nonat- tenders (people who did not attend in the past 5-year cycle). We considered 5 scenarios in which the likelihood of receiving treatment amongst those eli- gible at assessment was increased 2.5-fold for statins alone, antihypertensives alone, smoking cessation alone, weight management alone, and all treatments. The value of 2.5 was selected in light of evidence from Tower Hamlets, London, indicating that the proportion of high-risk (QRisk2 > 20%) health check attenders additionally prescribed statins was around 36%, approximately 2.5 times higher than the national figure of 14% [63]. Whilst it is unclear what the maximum feasible uptake of different treatments is, we have modelled the same relative increase for all treatments and note that programme data suggest that such increases appear, on paper, to be feasible. Finally, to demonstrate the combined benefit of increasing uptake and improving delivery of health checks, we simulated the effect of simultaneously widening eligibility to include those with a diagnosis of hypertension, increasing attendance by 30% for everyone and increasing all treatments by 2.5-fold, which we will term a ‘maximum potential’ scenario. Scenarios First, we compared the present NHS Health Check programme (assuming it continues to operate in its present format) to a counterfactual in which no NHS Health Check programme operated. Second, we explored different scenarios for how the NHS Health Check programme could evolve in the future, considering 3 areas: eligibility criteria, uptake of the programme, and treatment. We considered 4 scenarios in which the eligibility criteria would change: (1) extending the programme to invite those who already have a diagnosis of hypertension, (2) increasing the age at which individuals are first invited for a health check from 40 years to 50 years of age, (3) increasing the upper age at which individuals may be invited to attend the programme from 74 years to 79 years, and (4) changing both the upper and lower age criteria, such that persons aged 50–79 years would be invited. We considered 5 scenarios in which the likelihood of attendance was increased by 30%: (1) for everyone invited, (2) for people living in the most deprived areas (bottom quintile group), PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 9 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Sensitivity analyses We also undertook the following sensitivity analyses: 1. Future attendance was independent of past attendance The model assumes that attendance at a health check is independent of past attendance record. Given the programme has been in existence for less than 10 years, there is limited long-term data to assess the extent to which past attendance predicts future attendance. However, we note that for similar programmes (i.e., screening programmes) past atten- dance can be a predictor of future attendance [65–67]. We thus modelled the current pro- gramme under the assumption that likelihood of attendance was greater (average of 70% per 5-year cycle) if somebody attended after his or her most recent offer of a health check and less if he or she did not (average 30%). We further tested the effect of this assumption on the scenario concerned with increasing invitations to nonattenders. 2. Future changes in CVD incidence and fatality The model assumes that present incidence of and case fatality from CVD continue. How- ever, age-standardised mortality for ischaemic heart disease and stroke has fallen over the past 50 years [68], and while there is uncertainty about the nature of future trends, particu- larly in light of the rising prevalence of obesity and diabetes[69], it seems likely that these trends will continue at least in the short to medium term. We assumed that the recently observed trends would continue for the next 20 years before plateauing. We assumed that the incidence of ischaemic heart disease (IHD) would fall by 4.8% for men and 4.5% for women per year [34] and that stroke incidence would fall by 4.0% per year [47]. We assumed that the case fatality for IHD would fall by 3.6% per year [34] and that the case fatality for stroke would fall by 6.0% per year [35]. We did not model declines for a longer period of time as the observed trends were for a short time period and there is considerable uncertainty about long-term trends. 3. Uncertainty in population cardiovascular risk (at baseline) To understand the extent to which our estimates would change if the model’s estimate of cardiovascular risk in the cohort was too high or too low (i.e., reflect uncertainty in our esti- mate of average risk in the population), we reran our model by multiplying the QRisk2 score by a value chosen from a log-normal distribution with 95% quantiles of 0.8 and 1.2. Sensitivity analyses The value was applied to each simulated individual in any given run of the model (with dif- ferent values drawn from the distribution being applied to different runs). Uncertainty analyses We used probabilistic methods to account for uncertainty in parameters entered into the model. We assigned a probability distribution, rather than a fixed value, for each input param- eter. We then ran our model 100 times, each time sampling each parameter from its stated dis- tribution. This yielded 100 output values, from which we calculated a mean and 95% credible intervals. The number of samples was chosen such that the sampling error in mean outcomes over individuals (due to simulating a finite number of individuals) was small (<5% of that due to parametric uncertainty). In each run, we simulated 200,000 individuals, effectively sampling 20 million individuals for each modelled scenario. We also undertook ‘value of information analyses’ to identify which sources of parametric uncertainty were contributing the most to uncertainty in the results. These are calculated by PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 10 / 32 The current and potential health benefits of the NHS Health Check programme estimating the standard deviation for the result if the exact value of the parameter of interest were to be learnt. This can be compared to the width of the original 95% credible interval to describe the potential value of obtaining perfect information on that parameter. All parameters with uncertainty or credible intervals were considered. We repeated the ‘value of information analysis’ for 2 of the scenarios: our first scenario (comparing the NHS Health Check pro- gramme to a counterfactual in which no programme operated) and the ‘maximum potential’ scenario. interquartile range are given for continuous variables. Deprivation quintile groups are based on the Index of Multiple Deprivation for the area of residence. QRisk2 is the 10-year risk of cardiovascular disease [45]. Abbreviations: HbA1c, haemoglobin A1c; HDL, high-density lipoprotein; TC, total cholesterol. Mean and https://doi.org/10.1371/journal.pmed.1002517.t002 https://doi.org/10.1371/journal.pmed.1002517.t002 Results The baseline characteristics of the population aged 40–45 years are shown in Table 2. In the absence of the NHS Health Check programme, we estimate that per million people aged 40–45 years at baseline in the 60 years of follow-up, there will be 355,000 diagnoses of IHD, 184,000 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 11 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Abbreviations: HbA1c, haemoglobin A1c; HDL, high-density lipoprotein; TC, total cholesterol. Mean and interquartile range are given for continuous variables. Deprivation quintile groups are based on the Index of Multiple Deprivation for the area of residence. QRisk2 is the 10-year risk of cardiovascular disease [45]. Changing eligibility criteria The effect of making changes to the eligibility criteria is shown in Table 4. All the estimates for health impacts (cases prevented, deaths prevented, QALYs, and survival) are changes relative to the current NHS Health Check programme. To estimate changes in these outcomes relative to a counterfactual with no NHS Health Check programme, these values should be added to the corresponding values in Table 3. Options associated with improvements in population health were opening the programme to people with a diagnosis of hypertension and extending the upper age of cutoff to 79 years. Increasing the starting age for eligibility from 40 years to 50 years was associated with a reduc- tion in population health. A hybrid approach, raising the starting age and raising the upper age cutoff, was also associated with an improvement in population health (the loss from increasing the starting age being offset by the gain from increasing the upper age cutoff). Current programme At some point during their 35-year period of potential eligibility, we estimate that 85% of the cohort will be eligible for and 80% will attend for at least 1 health check (Table 3). Whilst the majority (81% of the population) will be eligible for treatment at some point, only a minority (27% of the population) are offered treatments through the NHS Health Check programme. The most common treatment offered is weight management. We estimate that the NHS Health Check programme prevents 390 (95% credible interval [CrI] 290 to 500) premature deaths before 80 years of age and results in an additional 1,370 (95% CrI 1,100 to 1,690) people being free of disease (IHD, stroke, dementia, and lung cancer) at age 80 years per million people aged 40–45 years at baseline. Over the life of the cohort (i.e., followed from 40–45 years to 100 years), we estimate the changes result in an additional 10,000 (95% CrI 8,200 to 13,000) QALYs and an additional 9,000 (6,900 to 11,300) years of life. This is equivalent to 3.8 (3.0 to 4.7) days of quality-adjusted life per head of population and an increase in survival of 3.3 (2.5 to 4.1) days per head of popu- lation. The increase in quality-adjusted life (3.8 days) is greater than the increase in survival (3.3 days), i.e., the intervention results in compression of morbidity. Assuming there are 730,000 people aged 40 years in England each year, this would equate to approximately 300 fewer premature deaths before 80 years of age, 1,000 more people living free of CVD, dementia, and lung cancer at age 80 years, 7,500 additional QALYs, and 6,600 extra years of life each year in England. The current and potential health benefits of the NHS Health Check programme Table 2. Baseline characteristics of the whole population aged 40–45 years and those who go on to participate in the health check programme. Everyone aged 40– 45 years People eligible for a health check at least once (85%) People who go on to attend at least 1 health check (77%) Gender Male 50.4% 51.1% 51.6% Female 49.6% 48.9% 48.4% Ethnicity White 86.1% 86.2% 86.2% Indian 2.5% 2.6% 2.7% Pakistani 2.4% 2.4% 2.4% Caribbean 1.6% 1.5% 1.5% African 2.2% 2.2% 2.1% Other 5.1% 5.1% 5.1% Deprivation 1 (least deprived) 22.1% 23.5% 23.0% 2 22.9% 22.3% 22.2% 3 21.3% 22.6% 22.2% 4 19.8% 19.3% 19.4% 5 (most deprived) 13.9% 12.3% 13.1% Education 10 years or equivalent 44.9% 45.9% 45.7% 7–9 years 45.7% 45.1% 45.2% 6 years 9.4% 9.0% 9.1% QRisk2 2.9 (1.1 to 3.8) 2.5 (1.0 to 3.3) 2.6 (1.1 to 3.4) QRisk2 >20 0.4% 0.2% 0.2% QRisk2 10–20 2.2% 0.5% 0.9% Systolic blood pressure 122.6 (112.5 to 131.5) 120.7 (111.5 to 128) 120.9 (112.5 to 128.5) Diastolic blood pressure 75.2 (68.0 to 81.5) 73.6 (67.0 to 80.0) 73.9 (68.0 to 80.0) Blood pressure >140/90 14.2% 10.3% 10.8% Treated hypertension 4.6% 0.0% 1.1% Cholesterol 5.5 (4.8 to 6.1) 5.5 (4.8 to 6.1) 5.5 (4.8 to 6.1) TC/HDL 4.0 (3.0 to 4.9) 4.0 (2.9 to 4.8) 4.0 (3.0 to 4.8) BMI (kg/m2) 27.5 (24.2 to 30.2) 27.1 (23.9 to 29.6) 27.1 (24.2 to 29.8) Obese 27.1% 23.0% 23.6% HbA1c 5.6 (5.3 to 5.7) 5.5 (5.3 to 5.7) 5.5 (5.3 to 5.7) HbA1c >6.5% 3.4% 4.0% 4.4% Diabetes 3.3% 0.0% 1.0% Smoking Never 52.6% 52.8% 53.3% Ex 22.8% 22% 22.4% Current 24.6% 25.2% 24.3% Abbreviations: HbA1c, haemoglobin A1c; HDL, high-density lipoprotein; TC, total cholesterol. Mean and Table 2. Baseline characteristics of the whole population aged 40–45 years and those who go on to participate in the health check programme. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 12 / 32 The current and potential health benefits of the NHS Health Check programme diagnoses of stroke, 147,000 diagnoses of dementia, 148,000 diagnoses of lung cancer, and 405,000 premature (<80 years) deaths. The current and potential health benefits of the NHS Health Check programme Table 3. Summary of process measures and outcomes for the present National Health Service (NHS) health check programme. Eligibility and uptake (for whole population) Eligible for an NHS Health Check at any time (%) 85.1 Have one or more NHS Health Checks (%) 79.7 Mean health checks (per person) 1.9 Treatment (as a proportion of the whole population) Eligible for an NHS Health Check and any treatment (%) 81.0 Attended for an NHS Health Check and eligible for any treatment (%) 73.3 Offered any treatment (%) 26.6 Offered statins through an NHS Health Check (%) 8.5 Offered antihypertensives through an NHS Health Check (%) 3.3 Referrals for a weight loss programme through an NHS Health Check (%) 17.6 Referrals for smoking cessation services through an NHS Health Check (%) 1.2 Treated with statins through an NHS Health Check (%) 4.3 Treated with antihypertensives through an NHS Health Check (%) 1.8 Attended a weight loss programme after referral from an NHS Health Check (%) 10.0 Attended smoking cessation after referral from an NHS Health Check (%) 0.1 Cases prevented by age 80 (per million) IHD 1,089 (817 to 1,367) Stroke 525 (414 to 671) Dementia 135 (72 to 190) Lung cancer 90 (36 to 147) Additional people living free of one of the diseases listed above at age 80 years 1,371 (1,101 to 1,685) Cases prevented by age 100 (per million) IHD 1,296 (898 to 1,730) Stroke 679 (494 to 958) Dementia 125 (32 to 222) Lung cancer 175 (103 to 259) Additional people living free of one of our diseases at age 100 years 1,235 (956 to 1,566) Premature deaths prevented (per million) <75 years 246 (182 to 331) <80 years 386 (291 to 499) Quality-adjusted life gained over 60 years of follow-up Total (QALYs for whole population) 10,300 (8,170 to 12,900) Days per head of population 3.8 (3.0 to 4.7) Days per eligible person 4.3 (3.4 to 5.4) Days per person screened at least once 4.7 (3.8 to 6) Days per health check 2.0 (1.6 to 2.5) Days per head (most deprived quintile group) 5.1 (3.4 to 7.1) Days per head (least deprived quintile group) 3.3 (2.1 to 4.5) Life gained over 60 years of follow-up Total (years for whole population) 9,700 (6,880 to 11,300) Days per head of population 3.3 (2.5 to 4.1) Days per eligible person 3.7 (2.8 to 4.8) Days per person screened at least once 4.1 (3.2 to 5.2) Days per health check 1.7 (1.4 to 2.2) Days per head (most deprived quintile group) 4.4 (2.7 to 6.5) Days per head (least deprived quintile group) 2.8 (1.7 to 4.0) Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease. Increasing attendance Increasing attendance is associated with improvements in indices of population health (Table 5). Increasing attendance for everyone (by 30%) results in the greatest improvements in population health, although selective approaches (e.g., increasing attendance amongst those at high risk of CVD by 30% or increasing the likelihood of invitation to those who did not attend in the past 5-year cycle) may yield relatively large gains in population health for fewer addi- tional health check appointments. Increasing uptake by 30% amongst those living in the most deprived areas (bottom fifth) is an effective means to reduce inequalities, although it is associated with relatively small gains in measures of average population health. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 13 / 32 Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40–45 years until 80 years of age. Quality-adjusted life gained and life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. https://doi.org/10.1371/journal.pmed.1002517.t003 The current and potential health benefits of the NHS Health Check programme Table 4. Effect of new eligibility criteria on process and outcome measures of the National Health Service (NHS) health check programme. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40–45 years until 80 years of age. Quality-adjusted Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40–45 years until 80 years of age. Quality-adjusted life gained and life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 14 / 32 New eligibility criteria Include people with hypertension Starting age 50 years Upper age of eligibility 79 years Starting age (50 years) and upper eligibility age (79 years) Eligibility and uptake Eligible for HC at any time (%) 92.5 79.7 85.1 79.8 Have one or more HCs (%) 85.3 76.8 79.8 77.2 Mean HCs per head of population 2.1 1.7 2.1 1.9 Treatment (proportion of whole population) Eligible for HC and any treatment (%) 88.3 76.9 81.0 76.9 Attended HC and were eligible for any treatment when attending (%) 79.0 71.5 74.5 72.9 Offered any treatment through HC (%) 29.6 25.1 29.5 28.2 Offered statins through HC (%) 9.4 8.0 10.7 10.3 Offered antihypertensives through HC (%) 3.7 3.0 3.8 3.5 Referred to a weight loss programme through HC (%) 19.9 16.6 18.9 18 Referred to smoking cessation services through HC (%) 1.3 1.1 1.3 1.1 Treated with statins through HC (%) 4.8 4.1 5.4 5.3 Treated with antihypertensives through HC (%) 2.0 1.6 2.1 1.9 Attended a weight loss programme after HC referral (%) 11.3 9.4 10.9 10.3 Attended smoking cessation services after HC referral (%) 0.1 0.1 0.1 0.1 Additional cases prevented by age 80 (per million) IHD 162 (91 to 232) −38 (−81 to −4) 136 (93 to 184) 97 (40 to 160) Stroke 76 (36 to 118) −19 (−49 to 5) 68 (30 to 111) 49 (−1 to 99) Dementia 16 (−11 to 45) −13 (−36 to 8) 0 (−5 to 0) −13 (−36 to 8) Lung cancer 12 (−5 to 30) −8 (−25 to 6) 5 (−1 to 16) −3 (−21 to 16) Additional people living free of one of our diseases at age 80 years 194 (114 to 264) −53 (−94 to −7) 185 (111 to 248) 133 (48 to 203) Additional cases prevented by age 100 (per million) IHD 179 (110 to 279) −36 (−76 to 0) 339 (226 to 442) 303 (198 to 411) Stroke 88 (41 to 155) −17 (−52 to 8) 188 (108 to 272) 171 (90 to 253) Dementia 13 (−21 to 42) −16 (−38 to 14) −25 (−45 to −4) −41 (−73 to 0) Lung cancer 23 (3 to 51) −13 (−39 to 6) 16 (−3 to 39) 3 (−28 to 35) Additional people living free of one of our diseases at age 100 years 160 (89 to 219) −44 (−81 to −4) 330 (235 to 436) 286 (183 to 392) Additional premature deaths prevented (per million) <75 years 34 (9 to 65) −15 (−39 to 1) 0 (0 to 0) −15 (−39 to 1) <80 years 57 (19 to 99) −18 (−44 to 5) 33 (9 to 68) 15 (−24 to 49) Additional quality-adjusted life gained over the 60 years of follow-up Total (QALYs for whole population) 1,400 (688 to 2,010) −604 (−1,090 to −127) 1,480 (868 to 2,050) 876 (185 to 1,670) days per head of population 0.5 (0.3 to 0.7) −0.2 (−0.4 to 0.0) 0.5 (0.3 to 0.7) 0.3 (0.1 to 0.6) days per eligible person 0.6 (0.3 to 0.8) −0.2 (−0.4 to 0.0) 0.6 (0.4 to 0.9) 0.4 (0.1 to 0.8) days per person screened at least once 0.6 (0.3 to 0.9) −0.2 (−0.4 to 0.0) 0.7 (0.4 to 0.9) 0.5 (0.2 to 0.9) days per health check 0.0 (−0.1 to 0.2) 0.1 (0.0 to 0.2) 0.1 (0.0 to 0.2) 0.2 (0.0 to 0.3) days per head (most deprived quintile group) 0.9 (0.2 to 1.7) −0.3 (−0.8 to 0.4) 0.5 (0.1 to 1) 0.2 (−0.5 to 1) days per head (least deprived quintile group) 0.4 (0 to 0.8) −0.2 (−0.5 to 0.1) 0.6 (0.2 to 1) 0.4 (−0.2 to 0.9) Additional life gained over the 60 years of follow-up Total (years for whole population) 1,220 (577 to 1,860) −511(−29.5 to −1,060) 1,320 (706 to 1,870) 813 (83.5 to 1,640) (Continued) p p g on process and outcome measures of the National Health Service (NHS) health check programme. Effect on health equity The current programme has a greater absolute impact on health for those living in the most deprived areas compared to those living in the least deprived areas (gain in quality-adjusted life of 5.1 days for those in the most deprived area versus 3.3 days for those in the least deprived area; gain in life expectancy of 4.4 days versus 2.8 days, respectively). We summarise our estimates on health equity and overall effectiveness in Fig 2. Most modi- fications to the programme that are associated with an improvement in health equity are also associated with an improvement in overall population health. Increasing treatment A 2.5-fold increase in the likelihood of starting treatment amongst those eligible was associated with relatively large improvement in indices of population health (Table 6) compared to increases in attendance or changes in eligibility criteria. The largest gains are seen for a 2.5-fold increase in statin treatment. Increasing all treatments 2.5-fold increases the health benefits of the programme 2- to 3-fold (950 deaths versus 390; 3,400 people free of 1 of the 4 diseases versus 1,370; 25,000 additional QALYs versus 10,000; 22,000 additional years of life versus 9,000). Increasing treatment rates is associated with compression of morbidity (the increase in QALYs is greater than the increase in survival). A strategy that combines extending eligibility to those with preexisting hypertension, extending the upper age of eligibility to 79 years, increasing uptake by 30%, and increasing treatment rates 2.5-fold among eligible patients (i.e., ‘maximum potential’ scenario) results in at least a 3-fold increase in benefits compared to no programme (1,360 premature deaths ver- sus 390; 5,100 people free of 1 of the 4 diseases versus 1,370; 37,000 additional QALYs versus 10,000; 33,000 additional years of life versus 9,000). PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 15 / 32 Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. https://doi.org/10.1371/journal.pmed.1002517.t004 The current and potential health benefits of the NHS Health Check programme Table 4. (Continued) New eligibility criteria Include people with hypertension Starting age 50 years Upper age of eligibility 79 years Starting age (50 years) and upper eligibility age (79 years) days per head of population 0.4 (0.2 to 0.7) −0.2 (−0.4 to 0.0) 0.5 (0.3 to 0.7) 0.3 (0.0 to 0.6) days per eligible person 0.5 (0.2 to 0.7) −0.2 (−0.4 to 0.0) 0.6 (0.3 to 0.8) 0.4 (0.1 to 0.8) days per person screened at least once 0.5 (0.2 to 0.8) −0.1 (−0.3 to 0.0) 0.6 (0.3 to 0.9) 0.5 (0.2 to 0.8) days per health check 0.0 (−0.1 to 0.2) 0.1 (0.0 to 0.2) 0.1 (0.0 to 0.2) 0.1 (0.0 to 0.3) days per head (most deprived quintile group) 0.8 (0.1 to 1.7) −0.2 (−0.8 to 0.4) 0.5 (0.0 to 1.0) 0.2 (−0.5 to 1.1) days per head (least deprived quintile group) 0.3 (0.0 to 0.8) −0.2 (−0.5 to 0.1) 0.5 (0.2 to 1.0) 0.4 (−0.2 to 0.8) Table 4. (Continued) Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 The current and potential health benefits of the NHS Health Check programme Table 5. Effect of increasing attendance on process and outcome measures of the National Health Service (NHS) health check programme (n = 1,000,000). Value of information analyses A value of information analysis that considered the different sources of parametric uncertainty captured within the model (Table A in S1 Data and Table B in S1 Data) showed that the 16 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 The current and potential health benefits of the NHS Health Check programme Table 5. Uptake increased by 30% for everyone Uptake increased by 30% for the most deprived quintile group Uptake increased by 30% for smokers Uptake increased by 30% for those at high risk of CVD± Increase likelihood of offer of a health check to previous nonattenders by 30% Eligibility and uptake Eligible for HC at any time (%) 85.1 85.1 85.1 85.1 85.1 Have one or more HCs (%) 83.0 80.0 80.4 80.4 81.4 Mean HCs per head of population 2.2 1.9 2.0 2.0 2.1 Treatment (proportion of whole population) Eligible for HC and any treatment (%) 81.0 81.0 81.0 81.0 81.0 Attended HC and were eligible for any treatment when attending (%) 77.0 73.6 74.1 74.3 75.3 Offered any treatment through HC (%) 29.8 27 27.3 27.7 28.4 Offered statins through HC (%) 9.6 8.6 8.7 9.2 9.0 Offered antihypertensives through HC (%) 3.8 3.4 3.4 3.5 3.6 Referred to a weight loss programme through HC (%) 19.8 17.9 18 18.1 18.9 Referred to smoking cessation services through HC (%) 1.4 1.2 1.4 1.3 1.3 Treated with statins through HC (%) 4.9 4.4 4.4 4.7 4.6 Treated with antihypertensives through HC (%) 2.1 1.8 1.9 1.9 2.0 Attended a weight loss programme after HC referral (%) 11.4 10.2 10.2 10.3 10.8 Attended smoking cessation services after HC referral (%) 0.2 0.1 0.2 0.1 0.1 Additional cases prevented by age 80 IHD 149 (60 to 244) 23 (−10 to 65) 42 (2 to 97) 93 (37 to 154) 90 (28 to 154) Stroke 78 (25 to 146) 12 (−11 to 45) 25 (−10 to 64) 49 (12 to 91) 43 (−5 to 96) Dementia 29 (−3 to 63) 6 (−9 to 21) 7 (−6 to 28) 2 (−10 to 14) 19 (−4 to 43) Lung cancer 15 (−12 to 44) 2 (−5 to 13) 16 (−9 to 45) 3 (−9 to 15) 9 (−15 to 30) Additional people living free of one of our diseases at age 80 years 200 (82 to 282) 28 (−13 to 66) 58 (19 to 107) 104 (42 to 164) 122 (49 to 191) Additional cases prevented (by age 100) IHD 172 (74 to 290) 26 (−16 to 64) 41 (−16 to 94) 125 (50 to 207) 95 (11 to 170) Stroke 99 (19 to 171) 15 (−18 to 57) 26 (−14 to 75) 72 (15 to 140) 53 (−12 to 118) Dementia 34 (−22 to 83) 5 (−19 to 28) 5 (−22 to 41) −7 (−36 to 15) 25 (−21 to 73) Lung cancer 30 (−5 to 76) 3 (−12 to 22) 31 (−3 to 74) 9 (−11 to 35) 19 (−14 to 53) Additional people living free of one of our diseases at age 100 years 181 (63 to 308) 26 (−7 to 63) 41 (−8 to 88) 100 (39 to 180) 107 (24 to 191) Additional deaths prevented (all cause) <75 years 40 (6 to 76) 6 (−12 to 21) 18 (−10 to 45) 18 (−10 to 46) 25 (−1 to 56) <80 years 60 (14 to 115) 10 (−9 to 33) 22 (−11 to 55) 30 (0 to 64) 37 (2 to 79) Additional quality-adjusted life gained over the 60 years of follow-up Total (QALYs for whole population) 1720 (781 to 2,760) 268 (−128 to 658) 594 (−27.1 to 1,140) 805 (284 to 1,280) 990 (302 to 1,740) (Continued) PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 17 / 32 Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, and days of quality-adjusted life and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40–45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. ± High risk of CVD was defined as QRisk2 > 20% Abbreviations: CVD, cardiovascular disease; HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, and days of quality-adjusted life and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40–45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. ± Hi h i k f CVD d fi d QRi k2 > 20% ± High risk of CVD was defined as QRisk2 > 20%. https://doi.org/10.1371/journal.pmed.1002517.t005 parameters contributing most to the uncertainty in the results were the initial adherence to statin prescription (23% of variance) and the annual dropout rate from statins (15% of vari- ance), both for analyses estimating the overall contribution of the current NHS Health Check programme and analyses estimating the benefit of the ‘maximum potential’ scenario. (Continued) Uptake increased by 30% for everyone Uptake increased by 30% for the most deprived quintile group Uptake increased by 30% for smokers Uptake increased by 30% for those at high risk of CVD± Increase likelihood of offer of a health check to previous nonattenders by 30% days per head of population 0.6 (0.3 to 1) 0.1 (0 to 0.2) 0.2 (0 to 0.4) 0.3 (0.1 to 0.5) 0.4 (0.1 to 0.6) days per eligible person 0.7 (0.3 to 1.2) 0.1 (−0.1 to 0.3) 0.2 (0 to 0.5) 0.3 (0.1 to 0.5) 0.4 (0.1 to 0.7) days per person screened at least once 0.8 (0.4, 1.2) 0.1 (−0.1 to 0.3) 0.3 (0 to 0.5) 0.4 (0.1 to 0.6) 0.4 (0.1 to 0.8) days per health check 0.0 (−0.2 to 0.2) 0.0 (−0.1 to 0.1) 0.0 (−0.1 to 0.1) 0.1 (0 to 0.2) 0.0 (−0.1 to 0.1) days per head (most deprived quintile group) 0.7 (−0.3 to 1.7) 0.7 (−0.3 to 1.7) 0.2 (−0.3 to 0.9) 0.4 (−0.2 to 1) 0.3 (−0.4 to 1.2) days per head (least deprived quintile group) 0.6 (0 to 1.2) 0.0 (0.0 to 0.0) 0.1 (−0.1 to 0.5) 0.3 (−0.1 to 0.7) 0.4 (−0.2 to 0.9) Additional life gained over the 60 years of follow-up Total (years for whole population) 1,510 (574 to 2,540) 242 (−208 to 626) 542 (−88.2 to 1,140) 716 (177 to 1,240) 868 (171 to 1,630) days per head of population 0.6 (0.2 to 0.9) 0.1 (−0.1 to 0.2) 0.2 (0 to 0.4) 0.3 (0.1 to 0.5) 0.3 (0.1 to 0.6) days per eligible person 0.6 (0.2 to 1.1) 0.1 (−0.1 to 0.3) 0.2 (0 to 0.5) 0.3 (0.1 to 0.5) 0.4 (0.1 to 0.7) days per person screened at least once 0.7 (0.3 to 1.1) 0.1 (−0.1 to 0.3) 0.2 (0 to 0.5) 0.3 (0.1 to 0.6) 0.4 (0.1 to 0.7) days per health check 0.0 (−0.2 to 0.2) 0.0 (−0.1 to 0.1) 0.0 (−0.1 to 0.1) 0.1 (0.0 to 0.2) 0 (−0.1 to 0.1) days per head (most deprived quintile group) 0.6 (−0.5 to 1.7) 0.6 (−0.5 to 1.7) 0.2 (−0.3 to 0.9) 0.4 (−0.4 to 1.0) 0.3 (−0.5 to 1.2) days per head (least deprived quintile group) 0.5 (−0.1 to 1.1) 0.0 (0.0 to 0.0) 0.1 (−0.1 to 0.5) 0.2 (−0.1 to 0.7) 0.3 (−0.2 to 0.9) Abbreviations: CVD, cardiovascular disease; HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 The current and potential health benefits of the NHS Health Check programme Table 6. Effect of increasing treatment on process and outcomes measures of the National Health Service (NHS) health check programme (n = 1,000,000). Sensitivity analyses Assuming that past attendance predicted future attendance (i.e., those who previously attended were more likely to attend in the future) resulted in a smaller proportion of the popu- lation attending for one or more health checks (75% versus 80%), fewer health checks (1.6 ver- sus 1.9 per head of population), and a marginally smaller overall improvement in population health (340 versus 390 premature deaths avoided; 1,200 versus 1,400 people living free of the 4 diseases at age 80 years). Under this assumption, the added benefit from a strategy of increas- ing likelihood of invitation to ‘non-attenders’ was similar (40 deaths versus 40; 130 people free of 1 of the 4 diseases at age 80 years versus 120; 940 additional QALYs versus 990; and 800 additional years of life versus 870). PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 18 / 32 Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. https://doi.org/10.1371/journal.pmed.1002517.t006 Assuming that recent trends in incidence and case fatality for stroke and IHD continue would reduce the estimate of benefit of the current NHS Health Checks programme by around half (170 deaths versus 390; 890 people free of 1 of the 4 diseases versus 1,370; 5,700 additional QALYs versus 10,000; 4,600 additional years of life versus 9,000; Table 7). The current and potential health benefits of the NHS Health Check programme Table 6. (Continued) 2.5-fold increase in the likelihood of starting treatment at assessment amongst eligible patients Statins Antihypertensives Smoking cessation services Weight loss programme All treatments Additional life gained over the 60 years of follow-up Total (years for whole population) 9,970 (7,080 to 13,400) 913 (512 to 1,420) 1,750 (855 to 2,780) 632 (229 to 1,120) 13,300 (10,300 to 16,800) days per head of population 3.6 (2.6 to 4.9) 0.3 (0.2 to 0.5) 0.6 (0.3 to 1.0) 0.2 (0.1 to 0.4) 4.8 (3.8 to 6.1) days per eligible person 4.1 (2.9 to 5.4) 0.4 (0.2 to 0.6) 0.7 (0.4 to 1.1) 0.3 (0.1 to 0.4) 5.5 (4.3 to 6.9) days per person screened at least once 4.6 (3.3 to 6.0) 0.4 (0.2 to 0.7) 0.8 (0.4 to 1.3) 0.3 (0.1 to 0.5) 6.1 (4.8 to 7.6) days per health check 1.9 (1.4 to 2.5) 0.2 (0.1 to 0.3) 0.3 (0.2 to 0.5) 0.1 (0.0 to 0.2) 2.6 (2.0 to 3.2) days per head (most deprived quintile group) 5.2 (3.3 to 7.9) 0.4 (0.1 to 0.9) 0.6 (−0.2 to 1.5) 0.3 (0.0 to 0.8) 6.5 (4.2 to 9.6) days per head (least deprived quintile group) 3.3 (2.1 to 5.0) 0.3 (0.1 to 0.7) 0.5 (−0.1 to 1.1) 0.2 (0 to 0.5) 4.3 (2.8 to 6.1) Table 6. (Continued) Table 6. 2.5-fold increase in the likelihood of starting treatment at assessment amongst eligible patients Statins Antihypertensives Smoking cessation services Weight loss programme All treatments Eligibility and uptake Eligible for HC at any time (%) 85.1 85.1 85.1 85.1 85.1 Have one or more HCs (%) 79.6 79.6 79.6 79.6 79.6 Mean HCs per head of population 1.9 1.9 1.9 1.9 1.9 Treatment (proportion of whole population) Eligible for HC and any treatment (%) 81.0 81.0 81.0 81.0 81.0 Attended HC and were eligible for any treatment when attending (%) 73.1 73.2 73.2 73.2 73.1 Offered any treatment through HC (%) 34.2 29.4 27.5 39.7 47.4 Offered statins through HC (%) 19.5 8.4 8.4 8.4 19.5 Offered antihypertensives through HC (%) 3.3 8.1 3.3 3.3 8.0 Referred to a weight loss programme through HC (%) 17.6 17.6 17.6 33.3 33.3 Referred to smoking cessation services through HC (%) 1.2 1.2 2.9 1.2 2.9 Treated with statins through HC through HC (%) 10.3 4.3 4.3 4.3 10.3 Treated with antihypertensives through HC (%) 1.8 4.5 1.8 1.8 4.5 Attended a weight loss programme after HC referral (%) 9.9 9.9 9.9 21.2 21.2 Attended smoking cessation services after HC referral (%) 0.1 0.1 0.3 0.1 0.3 Additional cases prevented by age 80 IHD 1,493 (1,084 to 1,910) 67 (28 to 110) 16 (-17 to 48) 14 (0 to 31) 1,589 (1,184 to 2,020) Stroke 698 (484 to 911) 37 (9 to 67) 10 (−10 to 39) 8 (−4 to 25) 753 (528 to 979) Dementia 53 (9 to 98) 77 (43 to 123) −3 (−17 to 10) 76 (38 to 123) 202 (132 to 281) Lung cancer 0 (−5 to 0) 0 (0 to 0) 127 (79 to 182) 0 (0 to 0) 127 (77 to 182) Additional people living free of one of our diseases at age 80 years 1,697 (1,255 to 2,147) 124 (72 to 179) 108 (58 to 162) 67 (26 to 107) 1,995 (1,550 to 2,440) Additional cases prevented (by age 100) IHD 1,825 (1,254 to 2,486) 52 (10 to 94) 1 (−37 to 42) 1 (−20 to 17) 1,878 (1,292 to 2,538) Stroke 921 (613 to 1,264) 31 (1 to 63) −2 (−31 to 29) 3 (−18 to 23) 952 (647 to 1,309) Dementia −20 (−84 to 49) 109 (52 to 174) −22 (−44 to 1) 129 (57 to 208) 195 (65 to 316) Lung cancer 0 (−5 to 0) 0 (0 to 0) 255 (161 to 366) 0 (0 to 0) 255 (159 to 366) Additional people living free of one of our diseases at age 100 years 1,503 (1,049 to 2,073) 96 (54 to 147) 120 (49 to 176) 66 (24 to 119) 1,787 (1,334 to 2,368) Additional deaths prevented (all cause) over the 60 years of follow-up <75 years 265 (191 to 363) 23 (3 to 50) 64 (26 to 106) 12 (0 to 35) 364 (276 to 474) <80 years 423 (300 to 553) 37 (15 to 75) 79 (30 to 128) 23 (4 to 50) 563 (423 to 731) Additional quality-adjusted life gained over the 60 years of follow-up Total (QALYs for whole population) 11,600 (8,260 to 15,000) 1,120 (704 to 1,690) 1,730 (840 to 2,660) 782 (324 to 1,330) 15,200 (11,700 to 19,030) days per head of population 4.2 (3 to 5.5) 0.4 (0.3 to 0.6) 0.6 (0.3 to 1.0) 0.3 (0.1 to 0.5) 5.6 (4.3 to 7) days per eligible person 4.7 (3.4 to 6.1) 0.5 (0.3 to 0.7) 0.7 (0.4 to 1.1) 0.3 (0.1 to 0.5) 6.3 (4.9 to 7.9) days per person screened at least once 5.3 (3.8 to 6.8) 0.5 (0.3 to 0.8) 0.8 (0.4 to 1.2) 0.4 (0.1 to 0.6) 7 (5.5 to 8.7) days per health check 2.2 (1.6 to 2.9) 0.2 (0.1 to 0.3) 0.3 (0.2 to 0.5) 0.2 (0.1 to 0.3) 2.9 (2.3 to 3.6) days per head (most deprived quintile group) 6.0 (3.8 to 8.6) 0.5 (0.1 to 1) 0.6 (−0.1 to 1.5) 0.4 (0.0 to 0.9) 7.5 (5.1 to 10.4) days per head (least deprived quintile group) 3.8 (2.5 to 5.6) 0.4 (0.2 to 0.8) 0.5 (−0.1 to 1.1) 0.3 (0.0 to 0.6) 4.9 (3.5 to 6.9) (Continued) PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 19 / 32 Principal findings We estimate the current NHS Health Check programme is preventing approximately 300 pre- mature deaths per year and resulting in 1,000 additional people aged 80 years being free of CVD, dementia, and lung cancer each year in England. It is increasing quality-adjusted life by around 4 days and life expectancy by around 3 days per person aged 40–45 years. There is potential to considerably improve the benefits of NHS Health Checks, notably by increasing uptake of treat- ments, increasing attendance, and extending the programme to include those with diagnosed hypertension. In keeping with the benefits of the current programme, most of these modifica- tions are associated with an improvement in health equity and compression of morbidity. The estimate of ‘maximum potential’ (relative to present performance) also halved under the same assumption (400 deaths versus 970; 2,300 people free of 1 of the 4 diseases versus 3,700; 14,000 additional QALYs versus 27,000; 11,000 additional years of life versus 24,000; Table 7). Assuming considerable uncertainty in baseline CVD risk (multiplying the QRisk2 score by a value chosen from a log-normal distribution with 95% quantiles of 0.8 and 1.2) does not change the conclusion, although the 95% CrIs widen (approximately doubling; Table C in S1 Data). (Continued) 2.5-fold increase in the likelihood of starting treatment at assessment amongst eligible patients Statins Antihypertensives Smoking cessation services Weight loss programme All treatments Additional life gained over the 60 years of follow-up Total (years for whole population) 9,970 (7,080 to 13,400) 913 (512 to 1,420) 1,750 (855 to 2,780) 632 (229 to 1,120) 13,300 (10,300 to 16,800) days per head of population 3.6 (2.6 to 4.9) 0.3 (0.2 to 0.5) 0.6 (0.3 to 1.0) 0.2 (0.1 to 0.4) 4.8 (3.8 to 6.1) days per eligible person 4.1 (2.9 to 5.4) 0.4 (0.2 to 0.6) 0.7 (0.4 to 1.1) 0.3 (0.1 to 0.4) 5.5 (4.3 to 6.9) days per person screened at least once 4.6 (3.3 to 6.0) 0.4 (0.2 to 0.7) 0.8 (0.4 to 1.3) 0.3 (0.1 to 0.5) 6.1 (4.8 to 7.6) days per health check 1.9 (1.4 to 2.5) 0.2 (0.1 to 0.3) 0.3 (0.2 to 0.5) 0.1 (0.0 to 0.2) 2.6 (2.0 to 3.2) days per head (most deprived quintile group) 5.2 (3.3 to 7.9) 0.4 (0.1 to 0.9) 0.6 (−0.2 to 1.5) 0.3 (0.0 to 0.8) 6.5 (4.2 to 9.6) days per head (least deprived quintile group) 3.3 (2.1 to 5.0) 0.3 (0.1 to 0.7) 0.5 (−0.1 to 1.1) 0.2 (0 to 0.5) 4.3 (2.8 to 6.1) Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. The current and potential health benefits of the NHS Health Check programme Table 7. Sensitivity analysis showing the impact of a reduction in cardiovascular disease (CVD) risk (incidence and case fatality) on the estimate of benefits attribut- able to the National Health Service (NHS) health check programme. Strengths and limitations Our work has several strengths. We have estimated the additional benefit of the NHS Health Check programme, over and above routine care, by using data that reflect changes in risk 20 / 32 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 The current and potential health benefits of the NHS Health Check programme Fig 2. Health equity plot showing the effect of modifications to the existing programme on over (health gain) and on health equity. https://doi.org/10.1371/journal.pmed.1002517.g002 Fig 2. Health equity plot showing the effect of modifications to the existing programme on overall effectiveness (health gain) and on health equity. https://doi org/10 1371/journal pmed 1002517 g002 Fig 2. Health equity plot showing the effect of modifications to the existing programme on overall effectiveness (health gain) and on health equity. https://doi.org/10.1371/journal.pmed.1002517.g002 Fig 2. Health equity plot showing the effect of modifications to the existing programme on overall effectiveness (health gain) and on health equity. https://doi.org/10.1371/journal.pmed.1002517.g002 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 21 / 32 Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. The ‘maximum potential’ scenario models the effect of simultaneously widening eligibility to include those with a diagnosis of hypertension, increasing attendance by 30% for everyone and increasing all treatments by 2.5-fold. https://doi.org/10.1371/journal.pmed.1002517.t007 Current NHS Health Check programme (compared to no programme) ‘Maximum potential’ scenario (compared to current programme) No decline in CVD risk Decline in CVD risk No decline in CVD risk Decline in CVD risk Additional cases prevented by age 80 (per million) IHD 1,089 (817 to 1,367) 592 (465 to 794) 2,802 (2,236 to 3,459) 1,533 (1,263 to 1,915) Stroke 525 (414 to 671) 269 (181 to 371) 1,335 (1,063 to 1,656) 704 (531 to 933) Dementia 135 (72 to 190) 126 (53 to 208) 320 (220 to 462) 299 (155 to 447) Lung cancer 90 (36 to 147) 94 (44 to 151) 207 (133 to 293) 221 (145 to 310) Additional people living free of one of our diseases at age 80 years 1,371 (1,101 to 1,685) 886 (697 to 1,122) 3,562 (2,903 to 4,253) 2,286 (1,891 to 2,716) Additional cases prevented by age 100 (per million) IHD 1,296 (898 to 1,730) 811 (580 to 1,129) 3,837 (2,833 to 4,962) 2,407 (1,840 to 3,009) Stroke 679 (494 to 958) 400 (269 to 564) 1,993 (1,518 to 2,565) 1,215 (904 to 1,619) Dementia 125 (32 to 222) 267 (141 to 393) 235 (75 to 384) 625 (421 to 853) Lung cancer 175 (103 to 259) 186 (113 to 281) 435 (311 to 567) 469 (331 to 596) Additional people living free of one of our diseases at age 100 years 1,235 (956 to 1,566) 1,044 (784 to 1,379) 3,654 (2,842 to 4,591) 3,007 (2,400 to 3,721) Additional premature deaths prevented (per million) <75 years 246 (182 to 331) 101 (47 to 164) 544 (424 to 713) 211 (137 to 294) <80 years 386 (291 to 499) 167 (82 to 253) 967 (777 to 1,215) 402 (287 to 516) Additional quality-adjusted life gained over the 60 years of follow-up Total (QALYs for whole population) 10,300 (8,170 to 12,900) 5,700 (4,180 to 7,730) 27,400 (22,400 to 33,500) 14,400 (11,700 to 17,600) days per head of population 3.8 (3.0 to 4.7) 2.1 (1.5 to 2.8) 10.0 (8.2 to 12.2) 5.2 (4.3 to 6.4) days per eligible person 4.3 (3.4 to 5.4) 2.5 (1.8 to 3.3) 10.6 (8.7 to 12.9) 5.7 (4.7 to 6.9) days per person screened at least once 4.7 (3.8 to 6.0) 2.7 (2.1 to 3.5) 11.3 (9.3 to 13.7) 6.0 (5.0 to 7.3) days per health check 2.0 (1.6 to 2.5) 1.1 (0.8 to 1.4) 3.0 (2.5 to 3.8) 1.5 (1.2 to 1.9) days per head (most deprived quintile group) 5.1 (3.4 to 7.1) 2.7 (1.2 to 4.2) 12.9 (9.0 to 16.7) 6.9 (4.7 to 9.7) days per head (least deprived quintile group) 3.3 (2.1 to 4.5) 1.7 (0.7 to 2.8) 9.0 (6.6 to 11.3) 4.5 (2.7 to 6.4) Additional life gained over the 60 years of follow-up Total (years for whole population) 9,700 (6,880 to 11,300) 4,620 (3,120 to 6,450) 24,000 (19,400 to 29,200) 11,500 (9,360 to 14,200) days per head of population 3.3 (2.5 to 4.1) 1.7 (1.1 to 2.4) 8.8 (7.1 to 10.7) 4.2 (3.4 to 5.2) days per eligible person 3.7 (2.8 to 4.8) 2.0 (1.4 to 2.7) 9.3 (7.6 to 11.3) 4.6 (3.7 to 5.6) days per person screened at least once 4.1 (3.2 to 5.2) 2.2 (1.6 to 2.9) 9.9 (8.1 to 12) 4.8 (3.9 to 5.9) days per health check 1.7 (1.4 to 2.2) 0.9 (0.6 to 1.2) 2.7 (2.1 to 3.3) 1.2 (0.9 to 1.5) days per head (most deprived quintile group) 4.4 (2.7 to 6.5) 2.1 (0.7 to 3.6) 11.3 (7.6 to 15.3) 5.5 (3.3 to 7.8) days per head (least deprived quintile group) 2.8 (1.7 to 4.0) 1.4 (0.4 to 2.5) 7.8 (5.5 to 9.9) 3.6 (1.9 to 5.5) Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. The current and potential health benefits of the NHS Health Check programme factors over time considering changes due to routine healthcare and by using estimates of additional treatments attributable to an NHS Health Check. By comparing NHS Health Checks with a counterfactual situation of no NHS Health Checks on the same population, we have eliminated the selection bias (i.e., healthy people who are concerned about their health choosing to attend a health check) that may affect observational studies of the programme. We have also made allowance for nonadherence and relapse of treatment. As a result, the model effectively simulates the impact of treatment decisions dissipating over time, resulting in a smaller estimate of benefit compared to approaches assuming fixed adherence over time. The microsimulation approach gives substantial flexibility, allowing exploration of different sce- narios, testing of assumptions, and description of outcomes by subgroup. By modelling differences in uptake by deprivation and disease incidence by deprivation (due to differences in risk factor prevalence and using QRisk2 score, which includes depriva- tion and ethnicity as predictive factors), we have been able to model health impacts by depriva- tion. However, we have not made allowance for likely differences in survival (case fatality) by deprivation, which may have led to an underestimation of the programme’s impact on reduc- ing health inequalities [70,71]. We have also assumed the same level of adherence to treatment by deprivation. Different levels of adherence by deprivation might also affect the programme’s impact on inequalities. We have represented uncertainty around multiple input parameters. Nonetheless, as with all models, there are parts for which we could find limited data (e.g., likelihood of repeat atten- dance for an NHS Health Check, attendance at a weight management programme after refer- ral, and long-term compliance with medication), and the measured ‘parametric’ uncertainty may not capture the ‘true’ uncertainty in some of the parameters (e.g., disutility weights). We have not made any allowance for migration, although we note that inward migration is less common in the age group (over 40 years) that we studied [72]. We have also assumed that the present background incidence of disease continues and that no major new treatments will be developed. Different assumptions about future disease inci- dence or risk factors would alter the estimates of overall health benefit and might affect the overall estimate of relative benefit of the different scenarios. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. The ‘maximum potential’ scenario models the effect of simultaneously widening eligibility to include those with a diagnosis of hypertension, increasing attendance by 30% for everyone and increasing all treatments by 2.5-fold. https://doi.org/10.1371/journal.pmed.1002517.t007 Abbreviations: HC, an NHS Health Check; IHD, ischaemic heart disease; QALY, quality-adjusted life year. Deprivation quintile groups are based on the Index of Multiple Deprivation score for the area of residence. Health outcomes (cases prevented, premature deaths prevented, days of quality-adjusted life, and days of life gained) are expressed relative to the existing programme. Cases prevented are all cases prevented when following a cohort of 1 million adults aged 40–45 years until either 80 or 100 years of age. Premature deaths prevented are all-cause deaths prevented before age 75 or 80 years, when following a cohort of 1 million adults aged 40– 45 years until 80 years of age. Additional quality-adjusted life gained and additional life gained are over the 60 years of follow-up, i.e., the remaining lifetime of the cohort. Estimates of 95% credible intervals (due to parameter uncertainty) are shown in parentheses. The 95% credible intervals are the 2.5th and 97.5th percentile of all estimates from 100 simulations. The ‘maximum potential’ scenario models the effect of simultaneously widening eligibility to include those with a diagnosis of hypertension, increasing attendance by 30% for everyone and increasing all treatments by 2.5-fold. https://doi.org/10.1371/journal.pmed.1002517.t007 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 22 / 32 The current and potential health benefits of the NHS Health Check programme COPD [75]. Given this and the exclusion of other health outcomes, our estimate of health gain is likely to underestimate the overall health gain from the programme. We focused on the car- diovascular elements of the NHS Health Check because this was the original focus of the pro- gramme, because of the large burden of disease attributable to CVD, and because of the well- developed evidence base describing treatment uptake and effectiveness. We note that the cardiovascular benefits attributable to weight loss interventions (Table 6) are less than for other treatments, despite a large number of referrals to weight management. This is in part because it was assumed that any weight loss was fully regained over a 5-year period. It may in part reflect the cardiovascular benefits of weight being modelled through QRisk2 score, which includes BMI. We did not additionally explicitly model the impact of BMI on other risk factors (e.g., cholesterol and blood pressure), as changes in these risk factors through changes in BMI have been implicitly modelled through the matching process that models change in risk factors over time (see Section 2.1 in S1 Text). Nonetheless, this approach may have underestimated the benefits of weight loss on CVD. We have primarily considered benefits at the population level, which are different to bene- fits at the individual level. Stopping smoking is highly beneficial for health at an individual level, but that component of the programme is currently delivering limited population benefit because few individuals are being referred to smoking cessation services (through the NHS Health Check programme). This may reflect national declines in demand for smoking cessa- tion services, in part due to declines in smoking prevalence and in part due to the availability of e-cigarettes. The individual benefit for an individual who is referred to smoking cessation services and who quits will be substantial. Whilst we have tried to choose scenarios based on what may be realistically or ideally attainable, we do not know what is possible. We note others have commented on the suboptimal uptake of treatments amongst people attending a health check [14]. Low uptake of treatments may reflect variation in quality of care but may be due to clinical factors (e.g., patient choice, side effects, or contraindications).[76] This work concerns one aspect of secondary prevention of CVD. In terms of reducing mor- bidity and mortality from CVD other aspects of prevention, such as population-level approaches (e.g., cigarette taxation and reformulation to reduce salt) [75,77,78], and aspects of clinical care (e.g., management of acute coronary syndromes) are valuable. This work does not attempt to consider the relative contributions of these different elements, which others have done [15]. In practice, countries are likely to adopt a spectrum of approaches from primary prevention, at the population level, to the delivery of high-quality clinical care to individuals to reduce the burden of CVD [79]. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 It is noticeable that the estimate of overall benefit falls by as much as half if the present downward trend in CVD continues for another 20 years. This underscores the challenges in making forecasts about the benefits in health attributable to the programme in the long term. There are also differences in disease incidence and variation in programme delivery between local authorities, which may result in different estimates of absolute benefit, at a local level, to those presented here. Whilst dementia risk is modifiable [29,41], there is no trial evi- dence to demonstrate the effects of lipid-lowering therapy, weight loss, or smoking on demen- tia incidence, and consequently, the effects on dementia should be treated with caution. There are aspects of the programme we have not considered. We have not estimated costs, nor have we considered harms of treatments. Notably, we have not considered the additional risk of type 2 diabetes attributable to statins, which might influence the modelled benefits of statins used in primary prevention [73,74]. Other side effects are likely to be short-lived, and thus, their overall contribution to (lifelong) disutility would be small, and allowance has been made for patients not starting and discontinuing medication (which may be a response to side effects and thus limit the duration of side effects). Elements of the programme we have not modelled include brief interventions around alcohol, benefits from early diagnosis (e.g., chronic kidney disease and type 2 diabetes). With the exception of lung cancer, other (noncar- diovascular) health benefits (e.g., weight loss on cancer and smoking on other cancers and chronic obstructive pulmonary disease [COPD]) have not been modelled. In some settings, the largest contributor to QALY benefit from smoking cessation is through reduction in PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 23 / 32 Comparison to other work We are not aware of any other work that has sought to estimate the impact of making changes to the NHS Health Check programme. We note that the original modelling work, undertaken by the Department of Health, suggested that 40 was the optimal age to start screening [80], and consistent with this, we found a small health loss associated with increasing the starting age to 50 years. The original modelling undertaken by the Department of Health estimated that the pro- gramme could prevent 1,600 heart attacks and strokes and at least 650 premature deaths each year [81]. Kypridemos et al. estimated that a ‘vascular check programme’ in the UK might pre- vent approximately 1,000 nonfatal and 200 fatal cases of CVD annually [15]. While there are important differences between the models (e.g., effectiveness of statins and trends in CVD incidence), we note that despite this, these estimates and our own (1,700 events, of which 1,400 are attributable to CVD, and 300 premature deaths prevented per annum) are relatively simi- lar. Our estimates for the increase in QALYs (3.8 days per head of population) are of a similar order of magnitude but are less than half of the estimates of a ‘health check’ programme (8.6 days per head of population) when following a population for 30 years using the Archimedes model, which has been validated for diabetes treatments [16,82]. While we cannot be sure how ‘valid’ these other model estimates are, if they are ‘valid’, these comparisons provide some reas- surance concerning the validity of our model’s output. Our work can also be compared to observational reports of the current programme. One study estimated the health benefits for those who attend for a health check compared to a matched control group who do not attend [83]. The paper reported similar levels of treatment to those that we modelled and reported changes in cardiovascular risk factors (e.g., 2.5 mmHg reduction in blood pressure) for attenders relative to nonattenders. We note that these differ- ences are relatively large (our respective estimates being 0.07 mmHg for those who attend for a health check), at a population-level, and would result in much greater health improvements than those that we estimated. Model validation There are different views on the appropriateness and meaning of the concept of validity in model development. We take the position that validity needs to be considered in the context of use. It is not possible for a model to be universally valid. Models can be used to answer multiple questions, and validity should be considered specifically for each question. Validation through comparing a model’s findings with empirical research is usually only partially possible, as models are typically used to answer questions that no single empirical study can answer. Other factors need to be considered when deciding whether a model is ‘valid’ or robust. We think our model is robust because of (1) the use of good data sources, e.g., national data on disease epidemiology, published data on programme performance, and estimates of treatment efficacy from trials; (2) the steps taken during model development, e.g., calibration and use of expert advice; (3) the comparisons of some model outputs with mortality data, i.e., effectively validat- ing the population health module; (4) extensive stochastic uncertainty analysis and sensitivity PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 24 / 32 The current and potential health benefits of the NHS Health Check programme analysis; and (5) comparisons with other modelling studies (described below). The uncertainty and sensitivity analyses suggest that the pattern of findings and the estimates of order of mag- nitude are broadly similar under a range of different assumptions for the comparisons of most interest to us. Substantial changes in CVD risk, now or in the future, and statin adherence have been identified as factors most likely to have a marked impact on estimate of benefit. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 Interpretation and implications Broadly, our work suggests the programme is contributing to benefits in population health and is also serving to reduce health inequalities. As the gain in time lived in full health is greater than the increase in survival, the programme is adding more good quality life years than it is adding years to life (i.e., it is compressing morbidity). We have presented 2 contrasting metrics of benefit, one based around events avoided and a second based on increase in life expectancy (including QALYs). QALYs are important for making comparisons with other health interventions and making judgements about cost-effec- tiveness. We have presented numbers for the population, as a whole, and per head of popula- tion. The latter is standardised for the population size and is a common metric used to describe the benefit of other similar programmes, i.e., screening programmes. However, the mean benefit per person can be misleading, as the benefits are not evenly distributed. The events avoided may be a better way to communicate how the programme may offer a substan- tial benefit (prevention of major disease or premature death) for a small proportion of the population. If the programme were optimised, the health benefits of the programme could be even greater. Our work suggests a number of approaches that might enable this. Increasing the uptake of treatments, principally statins, through the programme may be a more important strategy to increase the health benefits of the programme. This appears to be more important than increasing programme attendance, which has been a focus of efforts to improve the pro- gramme to date. It might be a more efficient use of resources to ensure the programme is opti- mally managing patients before seeking to increase attendance. The benefits of statins offered through the programme were particularly pronounced. Given the recent recommendations from the UK’s National Institute for Health and Care Excellence (NICE) to lower the threshold for initiating statin therapy for primary prevention [87], it seems likely that there will be scope to increase statin therapy. Other interventions are estimated to have a smaller impact. Nonetheless, there is scope for their impact to be greater. For example, smoking cessation is highly beneficial but had a very low uptake [88]; the benefit of this component of the programme could be greater if more smokers were referred. Comparison to other work These discrepancies in health outcomes may be explained by residual confounding of the observational data (i.e., those who attend for a health check are healthier than those who do not) or may suggest other pathways through which the health check may be influencing cardiovascular risk, such as stimulating participants to adopt health- ier habits (e.g., being physically active or eating a healthier diet). Comparisons with other population-level interventions should be interpreted cautiously, as methods may vary and the nature and scale of the interventions can be very different. Struc- tural interventions (e.g., taxing unhealthy foods, 2,300 CVD deaths averted; salt reformulation, 6,000 CVD deaths averted per annum) may have a greater impact on CVD [78,84]. Such inter- ventions can be politically difficult to implement, and individual interventions may be better seen as a complement to structural approaches [79]. Comparisons with screening programmes may be instructive, as the programme shares some characteristics with screening programmes. Breast cancer screening in the UK is estimated to increase life expectancy by 6.9 days and QALYs by 2.0 days per eligible women [85], whilst bowel cancer screening is estimated to increase life expectancy by 4.6 to 9.6 days (depending on the screening model adopted) and QALYs by 3.8 to 10.2 days per eligible person [86]. Currently, the NHS Health Check PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 25 / 32 The current and potential health benefits of the NHS Health Check programme programme is performing at the lower end of these estimates, although our work suggests it has the potential to exceed the benefits of these screening programmes. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 S1 Text. Methods supplement (technical appendix). (DOCX) S1 Text. Methods supplement (technical appendix). (DOCX) Summary Our work suggests that the current NHS Health Check programme is contributing to improve- ments in population health, a narrowing of health inequalities, and compressing morbidity. There appears to be considerable scope to improve the health benefit of the programme, par- ticularly by ensuring those who are assessed and eligible for treatment receive appropriate treatment. Focusing on inviting previous nonattenders and widening the eligibility criteria to include those with an existing diagnosis of hypertension could also make a valuable contribu- tion to increasing the health benefits of the programme. Future research We have primarily considered health benefits. Whilst a detailed assessment of health impact is important in making decisions about the NHS Health Check programme, future work should integrate cost data and undertake a cost-effectiveness or cost-utility analysis, which will pro- vide a stronger basis for decision making. Future modelling work may want to consider other scenarios around changes in delivery, e.g., targeting known smokers or opportunistic health checks in primary care. Our scenarios on the potential increase in uptake of different treat- ment for statins are based on what is being achieved in one well-performing area. However, there is considerable uncertainty about what could be achieved (at scale) and how much it would cost. What our study does provide is an indication of how much additional health bene- fit could be realised if these scenarios were achieved. Further work might want to explore why referral and treatment rates are apparently low and how these rates might be increased. As data on the operation of the programme improve, researchers will be better able to estimate the current and potential future health impacts. Programme data should also allow direct observation of benefit (e.g., change in blood pressure), although in the absence of randomised controlled trials, issues concerning selection bias will limit the use of such comparisons. How- ever, observational studies may lack power to detect the differences in ‘hard’ health outcomes at a population level that we report in this paper. Modelling will continue to be necessary to investigate longer-term outcomes of the current programme and to address ‘what if’ questions about the possible benefits of making changes to the programme. Other similar models do exist [15,16,73]. Formal comparisons between models may serve as a form of validation and means to identify the respective strengths and weaknesses of the different models. Interpretation and implications It seems likely that weight loss interventions would be more beneficial if the weight loss was maintained. Overall, there may be a need for quality assurance around elements of the pro- gramme, as there are in screening programmes, to enable the programme to maximise its potential. National data suggest some targeting of high-risk groups (e.g., based on deprivation or eth- nicity) already occurs [15,44], and this approach has appeared to be successful in reducing health inequalities. Now that the programme is established, targeting those who did not attend in the previous 5-year cycle would also appear to be a sensible strategy, as a complement to existing targeting strategies. Given the relatively large incremental gain for extending the pro- gramme to include those with an existing diagnosis of hypertension, this change in eligibility criteria merits further exploration. Given that we have not considered costs, it would be inappropriate to draw conclusions about the overall cost-effectiveness of the overall programme or the marginal cost-effectiveness of the change scenarios we explore. However, we note our estimates of benefits in terms of new cases of CVD prevented are similar to the estimates from the original modelling prior to programme’s introduction, which suggested that the programme was cost-effective [80]. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 26 / 32 The current and potential health benefits of the NHS Health Check programme Supporting information S1 Data. Comparison of observed and modelled mortality for ischaemic heart disease and stroke. S2 Data. Results supplement. (DOCX) S2 Data. Results supplement. (DOCX) Acknowledgments We are grateful to John Robson for sharing his preliminary research findings on the NHS Health Check programme and his expertise on the programme; Amy Ahern, who provided advice on the effectiveness of weight loss interventions; and the staff at Public Health England, who gave advice on the scenarios modelled. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002517 March 6, 2018 27 / 32 The current and potential health benefits of the NHS Health Check programme Author Contributions Conceptualization: Oliver T. Mytton, Anna Goodman, Claudia Langenberg, Simon Griffin, Nick Wareham, James Woodcock. Data curation: Oliver T. Mytton, Christopher Jackson, Anna Goodman, James Woodcock. Formal analysis: Christopher Jackson, Arno Steinacher, James Woodcock. Funding acquisition: Nick Wareham, James Woodcock. Investigation: Oliver T. Mytton, Christopher Jackson, James Woodcock. Methodology: Oliver T. Mytton, Christopher Jackson, Arno Steinacher, Anna Goodman, Nick Wareham, James Woodcock. Project administration: Oliver T. Mytton, Christopher Jackson, James Woodcock. Resources: Nick Wareham. Resources: Nick Wareham. Software: Christopher Jackson, Arno Steinacher. Supervision: Christopher Jackson, Nick Wareham, James Woodcock. Validation: Oliver T. Mytton, Christopher Jackson, Anna Goodman, Claudia Langenberg, Simon Griffin, Nick Wareham, James Woodcock. Visualization: Oliver T. Mytton, Christopher Jackson, James Woodcock. Writing – original draft: Oliver T. Mytton, Christopher Jackson. Writing – review & editing: Oliver T. Mytton, Christopher Jackson, Arno Steinacher, Anna Goodman, Claudia Langenberg, Simon Griffin, Nick Wareham, James Woodcock. References 1. Frieden TR, Berwick DM. The “Million Hearts” initiative—preventing heart attacks and strokes. N Engl J Med. 2011; 365: e27. https://doi.org/10.1056/NEJMp1110421 PMID: 21913835 2. Murray CJL, Richards MA, Newton JN, Fenton KA, Anderson HR, Atkinson C, et al. UK health perfor- mance: findings of the Global Burden of Disease Study 2010. Lancet. 2013; 381: 997–1020. https://doi. org/10.1016/S0140-6736(13)60355-4 PMID: 23668584 3. Public Health England. From evidence into action: opportunities to protect and improve the nation’s health [Internet]. London; 2014. 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Facebook's Potential for Collaborative e-Learning

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http://rusc.uoc.edu ARTICLE Facebook’s Potential for Collaborative e-Learning Francesc Llorens Cerdà [email protected] Lecturer-Consultant, Fundamentals of e-Learning and Educational Design, Open University of Catalonia (UOC) Neus Capdeferro Planas [email protected] Tutor, Graduate Programmes, IDEC-Pompeu Fabra Submitted in: June 2010 Accepted in: November 2010 Published in: July 2011 Recommended citation LLORENS, Francesc; CAPDEFERRO, Neus (2011). “Facebook’s Potential for Collaborative e-Learning” [online article]. Revista de Universidad y Sociedad del Conocimiento (RUSC). Vol. 8, No 2, pp. 197-210. UOC. [Accessed: dd/mm/yy]. <http://rusc.uoc.edu/ojs/index.php/rusc/article/view/v8n2-llorens-capdeferro/v8n2-llorenscapdeferro-eng> ISSN 1698-580X Abstract Today, Facebook (www.facebook.com) is probably the most palpable example of environments known as ‘social networks’ or ‘Web 2.0’. Social networking sites are platforms that facilitate information sharing, interaction and collaboration among their users. However, Facebook’s success is not solely dependent on its capacity to connect people, although this was its initial orientation. The platform’s power for sharing resources and linking content on the Internet to user profiles, as well as its evolution towards lifestreaming and microblogging, enable it provide support for complex, continuous interaction experiences and, consequently, to structure collaborative-learning processes. The platform’s communication tools, combined with the option to enhance its potential by installing third-party modules and applications, allow members of a community or work team to carry out very diverse activities. On the basis of theoretical underpinnings represented by the socio-constructivist perspective on communities of practice, the Web2Learn work group analysed and assessed the features that enable RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 197 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning Facebook to be used as a platform for carrying out collaborative online activities from two angles: technological and educational. Keywords Facebook, social networks, collaborative learning, communities of practice, Web 2.0 Posibilidades de la plataforma Facebook para el aprendizaje colaborativo en línea Resumen Facebook (www.facebook.com) quizás sea hoy el ejemplo más «palpable» de los entornos denominados redes sociales o 2.0. Las redes sociales son plataformas que facilitan el intercambio de información, la interacción y la colaboración entre sus usuarios. El éxito de Facebook como red social, sin embargo, no depende sólo de su capacidad para conectar personas, aunque sea esta su orientación inicial. La potencia de la plataforma para compartir recursos, para vincular contenidos presentes en internet a los perfiles de los usuarios y su evolución hacia el lifestreaming y el microblogging la facultan para dar soporte a experiencias de interacción complejas y continuas, y, con ello, para estructurar procesos de aprendizaje colaborativo. Las herramientas comunicativas de la plataforma, así como la opción de enriquecer sus potencialidades mediante la instalación de aplicaciones y módulos de terceros permiten a los miembros de una comunidad o equipo de trabajo desarrollar actividades heterogéneas. Sobre el marco teórico representado por la perspectiva socioconstructivista de las comunidades de práctica, el grupo de trabajo Web2Learn ha analizado y valorado, desde una doble óptica, tecnológica y pedagógica, las características que hacen posible la utilización de Facebook como plataforma para el desarrollo de actividades colaborativas en línea. Palabras clave Facebook, redes sociales, aprendizaje colaborativo, comunidades de práctica, web 2.0 1. Introduction: theoretical underpinnings Scientific and technological progress forces people to make a constant effort to adapt to changes and to be permanently prepared to learn. Individuals are confronted with new needs: they have to rise to unique social and professional challenges, and participate more actively in their own learning processes. The structures and demands springing up all around us, in both educational and professional arenas, call for greater interaction among people. The success of modern organisations does not stem so much from individual creative genius as it does from the organisational and participatory capacities of professional groups and the collective generation of shared knowledge; in short, from talents that are practically impossible to find in one individual alone. One of the key features of Web 2.0 application is collaboration, not only between machine and user, but also among users. These social applications have the capacity to function as ‘intellectual RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 198 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning partners’ to promote critical thinking and facilitate cognitive processing (Voithofer et al., 2007). Text, voice, music, graphics, photos, animation and video are combined to promote users’ thinking and creativity when undertaking high-level tasks. They offer a wide range of resources that can be used for problem solving, critical thinking collaboration and so on (Dillenbourg, 1999), in both physical classrooms and virtual learning environments. In addition, Web 2.0 technologies, with their interactivity potential, foster active participation and student-centred learning. Collaboration among students is a defining feature of constructivist classrooms (Jonassen et al., 1993), and Web 2.0 has wide-ranging potential for social interactivity and the promotion of collaboration and collective learning. Virtual communities of students can be organised on the Internet, allowing them to work in small groups to attain shared objectives and to strengthen their commitment to the values inherent to collaborative working. The more or less diverse grouping of students for the purpose of undertaking tasks may favour the creation of ‘zones of proximal development’ (Vygotsky, 1978) and provide students with opportunities to construct shared meaning for their practices (Dillon, 2004). Facebook is an example of a Web 2.0 social networking site, which has enormous potential in the field of education despite the fact that it was not designed as an environment for constructing and managing learning experiences. It operates as an open platform, unlike other systems organised around courses or formally structured content. In fact, while Facebook is not a learning environment, either in its underlying concept or the design of its tools, it can serve as a very valuable support for the new social orientations now prevailing in approaches to educational processes. According to Garrison et al. (2005), learning communities represent a fusion between the individual realm (subjective) and the shared realm (objective). In this context, Facebook represents a great opportunity to generate knowledge and inter-group cohesion. The theoretical and practical underpinnings for considering collaboration processes in virtual environments are now in a phase of expansion. Together with the classic theory of communities of practice, as described by Lave et al. (1990) and Wenger (1998) in the context of ‘situated learning’, it is necessary to consider the ‘learning by doing’ concept, the cornerstone of these communities, which may be better suited to the model of interaction and collaboration expressed in social networks. Jyri Engeström (1999) has tried to reconceptualise learning by doing in a paradigm called ‘learning by expanding’. For Engeström, the ‘community’ concept is the central nucleus on which various dynamics converge, be they human (subjects), instrumental (objects and tools) or communicational (rules, division of labour). The result of these dynamics is necessarily a shared, agreed outcome. The structure underlying learning communities therefore implies the production of knowledge and the consideration of ‘social knowledge’ as an added value rather than the simple sum of many pieces of individual knowledge. Facebook constructs ‘sociality’ by means of a strategy that connects users not only with each other, but also with numerous circles of sub-networks, events and groups. It assumes that the production of creative experiences is a social event, which is based on pooling resources and content contributed by people and processed using shared-use tools. In this article, our intention is to focus attention on a particular type of experience – educational – and to present the results of an analysis of the potential and, of course, the limitations of Facebook. RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 199 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning 2. Work plan: objectives and activities on the platform This article describes the work carried out by the Web2Learn1 group in the second term of 2010, in the context of the master’s degree in Education and Information and Communication Technologies (e-Learning) offered by the Open University of Catalonia (UOC). The group undertook a project, the main aim of which was to generate knowledge on Facebook’s potential for carrying out collaborative e-learning activities. Web2Learn developed a theoretical design specifying the objectives and activities that had to be carried out, and then implemented them; to do that, it created a private group on Facebook and transferred the process of collaborative working to it. The online activities carried out, described in relation to the initial objectives set by the group (in italics), were as a follows: •• To stimulate the development of basic technical and social skills in order to participate in the social network in particular, and in contemporary society in general. o Creating individual accounts on Facebook. o Creating a group space (Web2Learn work group) to carry out collaborative activities, and configuring it properly. •• To promote peer-to-peer learning and working. o Individually exploring the features of the environment and pooling the results, making a note of the research results in the space itself. o Proposing strategies, resources and information sources to other members of the group in connection with the educational use of Facebook and its potential as a support for collaborative working. •• To produce knowledge in the very process of collaboration among group members. o Documenting the working process using external applications, for both written and multimedia formats (text, video, presentations, screencasts and mind maps). o Expanding the environment’s connectivity through external data storage services (GDocs, Scribd, Box.net), showing the generative and expansive capacity of the network. •• To assess the potential of the chosen environment as a means for collaborative working and to selfassess the group’s work. o Elaborating a mind map summarising the research results, the activities carried out and the conclusions drawn by the group. o Performing an assessment of the platform and a self-assessment of the group’s work, specifically developing a tool for assessing the collaborative environment and its technological and educational potential. 1. The Web2Learn group was formed within the framework of the subject ‘Learning based on virtual collaborative activities’. Members were grouped together on the basis of the professional e-learning experience, and the topic of the study emerged from the final activity of the subject, which anticipated carrying out a project for learning and collaborative work in a virtual environment. Besides the authors, the work group included Jaume Solans Cases and José Luis Soler Domínguez, who gave us their permission to use materials and conclusions drawn from the group work. RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 200 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning 3. Facebook for online collaborative working: strengths and weaknesses The process of interaction among individuals is key in the analysis of virtual collaborative environments from a socioculturally-oriented constructivist perspective (Barberà et al., 2008). It is vital to focus on the assessment of the quality of joint activities carried out by participants on certain types of content and tasks, and on the way technological resources mediate, transform and optimise those activities. We have analysed whether a work group created on Facebook can incorporate the necessary learning resources to carry out collaborative activities in a safe, functional environment that is easy to manage and configure. Our assessment has been split into technological aspects (the platform’s native and expansive capacities) and educational aspects (the development of communication and interaction methods suited to learning objectives). The results are described below. 3.1. Technological aspects When Mark Zuckerberg created Facebook in 2004, his objective was to offer the general public a communication model that had initially been developed as an environment for use by students at Harvard University. This environment provided simple tools for students to share news. Today, Facebook has over 500 million registered users; it has become a true paradigm for the development of virtual social relationships and has definitively outstripped Myspace (www.myspace.com), its main competitor, especially in Europe. In the United States, the number of people aged over 65 registered on social networking sites has increased by 53% in the last two years, and Facebook is the out-andout winner, with a total of 7.2 million users (Nielsen, 2009). There is no question whatsoever that social networking is a global phenomenon that is far more than a craze or a fashion. It represents a new way of relating to others, which does not discriminate on grounds of age, gender or culture. Facebook is a paradigm of inclusive interaction. One of the keys to success of the site is its orientation towards what we could describe as ‘extended technological development’. A feature of Facebook is its open architecture. More than one million independent people in 180 countries worldwide collaborate on the development of applications that can be integrated into Facebook to enhance its native functions. Extended technological development not only broadens the technological potential of the site, but also consolidates its expansion and uptake worldwide. Thanks to easy-to-develop application protocols (APIs), programmers and entrepreneurs everywhere can contribute to the creation of modules to meet very different needs. Facebook has become a model of global sociality. However, one of the best aspects, in our opinion, is the initial simplicity of the platform for new users. From a functional viewpoint, and despite having evolved tremendously since its launch, the environment has not lost sight of the fact that the main objective of virtual communication is to share texts, links, photos and videos. Thus, Facebook’s native tools are the ones required to immediately start creating a community of friends that is based on sharing these basic objects. RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 201 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning Regarding Facebook groups, the functional unit analysed in our study, the platform initially provides technological support for the following items: –– The group’s profile, administered by the group creator/page owner. –– The group’s wall for members. –– The group’s discussion boards. –– The group’s photos. –– The group’s videos. –– The group’s events. The various modules all work in more or less the same way. When administrator rights are given to members of a group, any of them can take unrestricted action on any of the sections apart from events, which is reserved for the group creator/page owner. This anomaly does not allow members to interact on a shared calendar, which shows that the collaborative-working philosophy of Facebook is not the same as that of other collaborative-working environments – or at least those that have traditionally been considered as such. Facebook also permits three levels of privacy, classified as open, closed and secret. Everyone on Facebook can view and join open groups. Only members can view closed groups, and joining them is by invitation only. Secret groups cannot be found in searches. For most kinds of task-based collaborative projects, the ideal format is a closed group. Open groups allow interactions to be focused on long-term objectives, and the potential for generating viral knowledge to be increased. From a technological viewpoint, Facebook’s strengths for collaborative working are as follows: •• Simplicity and speed of creating and administering a work group. From his or her private profile, an individual user creates a new work group and invites other participants to join. •• Simplicity of use of native tools. Facebook’s basic functions (wall, discussion boards, photos, etc.) are easy to use, accessible, intuitive and visually well-structured. A group can start interacting immediately after it has been created. •• Chat, messaging and image tagging. These functions, which are particular to Web 2.0 environments, are available on the social networking site. However, tagging is only available for images, and the main objective is to tag people in photos. However, there is nothing stopping us from using this function to document graphics, for example. •• A high degree of external connectivity. Due to Facebook’s extraordinary expansion, the social presence of brands, advertising and events has compelled many external content supply services to implement Facebook connectivity. Indeed, it is possible to assert that this social networking site is a Web 2.0 product and that its enormous uptake has forced many external services to adapt to the new social philosophy or, in other words, to fit into the Web 2.0 environment. •• Internal expansion capacity. Thanks to the development of the site’s own applications and to independent programmers, both users and groups can expand its native capacities by using RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 202 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning add-on modules. For example, we can install modules for Google Calendar, To-do lists, blog sites, Flickr photos, Slideshare slides, etc. •• Microblogging and lifestreaming features. In each successive redesign of the platform, its adaptability to the ‘real-time Web’ concept has become apparent. This concept is particular to microblogging services. The rise of other social networking sites aimed at real-time communication, such as Twitter (www.twitter.com), caused Facebook to improve its news feeds (the home page that opens by default for every user), offering faster, more effective refresh and tracking technologies. •• A powerful support for mobile learning. Support for mobile devices and operating systems (iPhone, Android, Maemo, etc.) is one of the platform’s most spectacular advances. Since September 2007, when Facebook launched the service, more than 100 million users now access Facebook via mobile devices, and it is a fact that these users are twice as active on the platform as users accessing it via their PCs. This flexibility is an indisputable advantage for developing mobile learning experiences that would not be very – or at all – functional in other environments. The support features that we have just described demonstrate that what we are witnessing is a platform that is technologically rich, adaptable and expandable, capable of supporting collaborative-working experiences in learning communities. However, in order to issue a more accurate diagnosis, we must also take account of the site’s shortcomings in relation to the objectives of our study. When taking an in-depth, critical look at Facebook, we find that there are several elements that tend to hinder the implementation of learning experiences, either because some of the native tools are not well-developed, or because certain tools are quite simply missing. Let’s take a look at some of them: •• Presence of ‘noise’ and distracting elements. When Facebook is used to create a network for group working, certain elements that are justifiable for individual recreational use can become distracting. The environment shows advertising, warnings, suggestions and requests, which are the basis of its interactive richness, but they are also superfluous in teamworking environments. •• The drop-down comments system on walls tends to make it hard to see information. If a user is not a habitual user, the presence of new comments may be overlooked. Likewise, the difference between ‘read’ and ‘unread’ items is not easy to see, and is occasionally too spurious. •• Facebook lacks a true system for tagging, filtering, searching and organising information. The speed at which news is produced, and the same speed at which it disappears into older posts, are not compatible a priori with the task of undertaking ‘storage work’, whereby knowledge generated can be classified, saved, and easily and quickly retrieved. Collaborative working on complex projects over long periods of time is demanding with regard to the meta-tagging of documents and requires effective storage, organisation and look-up systems. Despite the fact that Facebook allows photos to be tagged, as already mentioned, this function is not very RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 203 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning useful for anything other than indicating the one-off presence of elements in them (such as friends’ faces), and it does not extend to other types of object. •• Facebook’s group discussion boards are too basic. On the one hand, they lack a text editor offering the functions required by a minimally complex presentation of a topic. On the other, they do not allow any type of reply nesting, so, despite their name, they simply become lists of replies to the main topic. Once again, the site lacks a system for organising messages to facilitate searches on them. Trying to locate a message that we know is somewhere can often be very chaotic. •• Facebook lacks functions that are native to environments specifically oriented towards work groups. It does not support file uploads (apart from photos and videos), multi-user file editing, task list creation or task assignment (to members), or monitoring. A truly shared events calendar does not exist, nor does the option (which is desirable under certain circumstances) to control the amount of time users spend working in the group space. It also lacks a good log of actions for individual members of a work team, which would otherwise allow their contributions to the group to be reviewed in an orderly manner, rather than being mixed up with their general contributions to the network. •• There is no way of installing, in one go, an application for a work group. To make an application available to group members, each member needs to install it individually and configure it accordingly. Consequently, the application is available for each user under his or her private profile, and is therefore inappropriate when it comes to separating personal elements from elements shared by the group. The proliferation of applications does not mean that they always work properly, or that they behave in a stable manner. Managing applications requires greater technological competency, which cannot always be taken for granted. The configuration of Facebook applications, especially the really powerful ones, often requires a high level of skill and a prior understanding of what is expected to happen when the application is integrated into the social network. •• Individual members of a group cannot create events. This is the case, even when they are group administrators. In its current state of development, this function is minimally operational because, if a decision is taken for several users to be group administrators, it is only to be expected that they should be able to manage events and the group’s calendar. •• Facebook does not natively provide synchronous bidirectional audio and video. While users can leave messages and video recordings, it is not possible to use this channel for bidirectional real-time conversations. It is necessary to resort to installing third-party, browser-dependent plug-ins to implement this functionality. Facebook’s native chat is a simple tool and, therefore, is generally too slow. As can be seen, a fair diagnosis of Facebook’s potential for supporting collaborative-learning experiences should take account of the fact that the world’s most widespread social networking site works differently depending on the viewpoint: it is not the same from a user-centric perspective as it is from the professional-requirement perspective of a learning or research community. However, RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 204 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning any considerations bearing negatively on the technological features of the site as applied to work groups should not mar a final judgment on it potential, since Facebook was not designed as a groupworking environment, and cannot, therefore, be strictly assessed in the same way as a dedicated platform could. Some of the shortcomings we have highlighted may be irrelevant to the needs of many work teams. Despite the lack of certain features, Facebook is, for these work groups, a great support for their interactions, especially if they are based on discussing and sharing resources, and integrating external content available on the Internet. 3.2. Educational aspects From the viewpoint of group working, Facebook provides a virtual space in which collectives involved in a shared objective can discuss topics, give their opinions, organise events, send information, share ideas and proposals, elaborate content, etc. Thus, a virtual community emerges. However, such virtual communities are not solely limited to sharing texts, photos, links or videos. Rather, a social sense of belonging to a group arises in them (McMillan et al., 1986), thus shaping a social aggregation that emerges from the network itself, since the group holds public discussions that are sufficiently widespread, with enough human feeling, to form networks of personal relationships in cyberspace. When the main motive for the existence of a community switches from simple information sharing to learning and professional development, what we then find is a virtual learning community. Unlike the classic conception of a virtual community centred a priori on objectives or interests, Facebook is initially user-centric. In other words, it is a social networking site that starts with the user and is configured around him or her, without any precise objective or norms other than the dissemination of his or her own virtual existence. A user is the owner of his or her own space: the profile. He or she has a list of friends/contacts, and everything that he or she does, if he or she so decides, is visible to that list, which creates a thread of dynamic communication with others, giving them the chance to reply. Thus, complex and enriching lines of social interaction are generated. This dissemination mechanism is called ‘virality’, that is to say, the impact that our actions have on the accounts of others, through news and notifications. Learning and collective intelligence networks can be built in this way, based on communication and on open, shared knowledge. Facebook groups, whose transmission methods are identical to those on individual accounts, become spaces that are independent from the main network of interactions. This allows communication and cooperation processes to be focused on and directed towards the goals and objectives set by group members. Work groups can therefore benefit from the interaction tools available on the social networking site and from its dynamic capacities. Moreover, open groups are subject to virality. Detailed below are the main aspects of the educational potential that Facebook offers for learning and collaborative working: •• It fosters a virtual community culture and social learning. From a pyschosociological viewpoint, this culture is rooted in values that emerge from the users, who interact in the network on a shared objective or topic, and generate interpersonal links of trust and support, as well as RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 205 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning feelings of belonging and social identity. Furthermore, the existence of exchange networks and information flows is a significant aspect when it comes to shaping and maintaining a social network. The importance of collaboration should be underscored (Prendes, 2003): the objective is to create a ‘shared experience’ rather than an ‘experience that is shared’. According to Cabero (2003), such online collaborative working is characterised by: o A social situation of interaction between groups of not very diverse subjects. o The attainment of objectives by (individually and jointly) carrying out tasks. o A positive interdependence among the subjects. o Cooperative working, which requires participants to have communication skills, symmetrical and reciprocal relationships, and a desire to share the solution to a problem. •• It supports innovative learning approaches. Facebook a suitable platform for promoting informal learning, and it allows individuals to move towards the lifelong-learning ideal, user-managed open learning and collaborative learning. It is a permanent environment in which users can stay in touch after completing an educational action. Informal learning expands the potential to construct knowledge and develop skills. Rounded off with the presence of other teaching figures, such as learning mentors and facilitators, Facebook offers the potential to support lifelong learning. Likewise, it can be of great help for professional refresher training through collaboration among peers. When using a network, users negotiate ideas. They are the centre of learning activities and become active knowledge builders, since the network would otherwise be devoid of meaning. Furthermore, the core nature of interactions requires students to develop the necessary competencies for group working. Facebook allows users to work in groups and create shared experiences, thus promoting collaborative learning. •• It motivates students. Although Facebook is a relatively new tool, it has an extraordinary level of penetration in society. Younger generations are very interested in using these new technologies for sharing information and communicating. Students tend to be more motivated by taking part in a learning environment in which they are the active users, the protagonists. The incorporation of new users may be a factor that encourages students to take part in this environment. In addition, social interaction may provide those with low self-esteem with greater benefit (Ellison et al., 2007). Furthermore, the platform stimulates creativity and makes learning more spontaneous and fun. •• It allows significant content to be presented by means of genuine materials. Groups created on Facebook work on real projects and problems connected, for example, with professional experiences. From these, they are able to access information and work on suitable concepts. According to Jonassen (1999), the objective of learning is to think like any other member of the professional community on the topic in question. The task or problem that needs to be solved collectively is the thread of the to-ing and fro-ing of information transmitted within the social network. This consists of materials from users themselves, such as videos, podcasts, multimedia products, links to documents, Flash files and blog articles. These materials can be integrated into the learning environment by inserting hyperlinks or embedding them as objects. Thus, knowledge is articulated in the ‘cogs’ of connections. RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 206 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning •• Facebook supports synchronous and asynchronous communication. The individual or group wall and discussion boards are examples of asynchronous communication. This provides students with important benefits and promotes critical thinking, allowing them to reflect on concepts for longer, and even encourages shy learners to express their ideas freely (Hara et al., 2000). Facebook also supports synchronous communication, although it does have its limitations, as described in the previous section. It is able to detect which members or friends are online. Thus, a user can initiate a real-time chat with other members. In addition, Facebook sends notifications of everything that is happening in a group by e-mail and feeds. Interaction and communication via this social networking site is really effective and continuous. 4. Conclusions New frameworks for developing interactions between the actors of learning processes require new ways of gaining an understanding of such processes. Today, these frameworks are of a technological kind, and their prime expression is found in social networking sites, which have already been called ‘social operating systems’ (Piscitelli et al., 2010). In turn, these interaction models require a revision of classic paradigms of analysis, as well as the development of methodologies leading to the proposal of new knowledge-containing objects – Personal Learning Environments (PLEs), case methods, e-Transfolio (Barberà et al., 2009) and so on. The most recent educational methods (e.g. connectivism) tend to blur the rigid boundaries between formal and informal learning as a consequence of the manifest potential of these new collective knowledge repositories – social networks. As we have shown, their educational potential is extraordinary, and particularly so if a fresh look is taken at the concepts of education and training, and emphasis is placed on the social nature of knowledge construction. From the viewpoint of its design and the degree of development of its native tools, it could be said that Facebook is not the best option for implementing collaborative-working projects, especially if they have high demands in terms of time control, information organisation and task-management flexibility. However, there is no question whatsoever that ‘people are Facebook’. In other words, the spread of the site also brings about a democratisation of access to its resources (and to the thousands of ‘essential’ applications made compatible with Facebook to survive). This is something worth considering, in many learning experiences, before deciding on other platforms or dedicated systems. Creating an initiative and disseminating it is simple and immediate. Even though Facebook lacks a collaborative-project orientation (which is not its original vocation), the incredibly high uptake of this social networking site, combined with its external connectivity, the exponential enhancement of open projects, the innovative learning approaches it supports and its capacity to foster inclusive learning, makes Facebook an option that is worthy of serious consideration when proposing online collaborative-working experiences. RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 207 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning References BARBERÀ, E.; Mauri, T.; Onrubia, J. [et al.] (2008). Cómo valorar la calidad de la enseñanza basada en las TIC. Pautas e instrumentos de análisis. Barcelona: Graó. BARBERÀ, E.; GUÀRDIA, L.; VALL-LLOVERA, M. 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Learning as a social system”. In: Systems Thinker. <http://www.co-i-l.com/coil/knowledge-garden/cop/lss.shtml> About the Authors Francesc Llorens Cerdà [email protected] [email protected] Lecturer-Consultant, Fundamentals of e-Learning and Educational Design, Open University of Catalonia (UOC) Lecturer-Consultant in Fundamentals of e-Learning and Educational Design on the master’s degree in Education and Information and Communication Technologies (e-Learning) offered by the UOC. Lecturer in Web Design and Educational Multimedia Application Programming on the master’s degree in Information and Communication Technologies offered by the University of Alicante (UA). Teacher of Philosophy at IES Dr. Lluís Simarro, Xàtiva, Valencia. Master’s degree in Information and Communication Technologies (e-Learning) awarded by the UOC. Degree in Philosophy and Education Sciences (Philosophy section) awarded by the University of Valencia (UV). Expert in e-learning and programmer of virtual environments and multimedia applications. Author of the book Postecnología. ¿El final del sueño? (Novadors-Edicions, Valencia, 2008). He has published numerous articles on the relationships between information technologies and the knowledge society. He has received several national awards for educational innovation (Santillana, Ministry of Education of the Government of Valencia, Caixa Popular…). Estudis de Psicologia i Ciències de l’Educació Universitat Oberta de Catalunya Rambla del Poblenou, 156 08018 Barcelona Spain RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 209 CC Francesc Llorens Cerdà and Neus Capdeferro Planas http://rusc.uoc.edu Facebook’s Potential for Collaborative e-Learning Neus Capdeferro Planas [email protected] Tutor, Graduate Programmes, IDEC-Pompeu Fabra. Corporate Solutions Division. Registered with the Professional Association of Pedagogues of Catalonia, number 1105. Degree in Psychopedagogy awarded by the UOC. Diploma in Information Technology awarded by the University School of Technology Mataró. Diploma in Education (primary and secondary) awarded by the University of Barcelona. University expert in the design of multimedia applications for the Spanish National University of Distance Education (UNED). She is currently studying for a master’s degree in Education and e-Learning at the UOC. As her ‘hybrid’ education and training demonstrate, her interests have always focused on the application of her technological know-how to solve educational problems. She has undertaken several research projects on psychopedagogical practice in the field of informal education and, specifically, on the design and elaboration of educational resources for technology-mediated environments. IDEC Universitat Pompeu Fabra C. Balmes, 132 08008 Barcelona Spain The texts published in this journal are – unless indicated otherwise – covered by the Creative Commons Spain Attribution 3.0 licence. You may copy, distribute, transmit and adapt the work, provided you attribute it (authorship, journal name, publisher) in the manner specified by the author(s) or licensor(s). The full text of the licence can be consulted here: http://creativecommons.org/licenses/by/3.0/es/deed.en. RUSC Vol. 8 No 2 | Universitat Oberta de Catalunya | Barcelona, July 2011 | ISSN 1698-580X 210 CC Francesc Llorens Cerdà and Neus Capdeferro Planas 

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THE FEATURES OF PARALLEL TRAINING SYSTEM AT HIGHER EDUCATION INSTITUTIONS IN THE MODE OF STUDENTS` INDEPENDENT WORK

Modern engineering and innovative technologies

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Issue 25 / Part 3 Issue 25 / Part 3 Modern engineering and innovative technologies http://www.moderntechno.de/index.php/meit/article/view/meit25-03-046 DOI: 10.30890/2567-5273.2023-25-03-046 http://www.moderntechno.de/index.php/meit/article/view/meit25-03-046 DOI: 10.30890/2567-5273.2023-25-03-046 UDC 378 THE FEATURES OF PARALLEL TRAINING SYSTEM AT HIGHER EDUCATION INSTITUTIONS IN THE MODE OF STUDENTS` INDEPENDENT WORK Kulaeva Z. A. master. Hryshchuk Iryna Anatoliivna master. The National University of Physical Education and Sport of Ukraine, Kiev, Physcultura Street,1, 01027 The National University of Physical Education and Sport of Ukraine, Kiev, Physcultura Street,1, 01027 Abstract. The internal system-forming factors that allow us to consider the model of activity from the standpoint of an integrated approach are the forms of organization of independent work of students and ways to control it. They act as a separate subsystem and have their own internal structure. In our case, the alternative learning model is a step-by-step process of introducing an activity model through various types of tasks that turn into a system of business games. The mastery of communication and activity skills by students with a focus on promising creative activity is the main goal of training. There are two levels of methods of activity - technological and professional. The technological level determines the skills that underlie the mastery of work techniques. The professional level reflects the area of professional content, obtaining generalized systematized knowledge. In the process of educational and professional activities of students, the technological and professional levels are in constant unity, which reflects the didactic unity of the operational- procedural and logical-content aspects of education. Key words: educational business games, extracurricular work, didactic task, integrated independent learning activity, mastering professional skills, cognitive self-regulation, tools and methods, behavioral foundations, development. ISSN 2567-5273 Introduction. To a large extent, the quality of student training at a university is determined by the organization, planning and management of their independent work. Only in the process of independent activity cognitive interests are formed, high-quality assimilation of knowledge takes place, and creative abilities develop. Therefore, increasing the efficiency of organizing students' independent work is one of the main tasks of modern pedagogy. But independent work in a foreign language in a non- linguistic university should be based on a clear interdisciplinary integration and, first of all, should be aimed at the formation of professional communication skills. The formation of a modern specialist, combining the role of a leader and an executor in one person, aimed at positive highly professional behavior, depends entirely on the formation of his creative abilities, self-education and self-education. The study of the structure and content of the communicative skills and abilities of the students of the department "Finance and Credit" served as the basis for the development and construction of an integrated model of parallel teaching of a foreign language in the mode of independent work, capable of ensuring the effective formation of relevant skills and abilities in students. Building training on the principles of unity, integrity and phased sequence, we used a systematic approach to building a training model, considering it as a systematized set of interdependent elements, united by common functional and managerial goals, identified on the basis of certain features. With a www.moderntechno.de ISSN 2567-5273 102 Issue 25 / Part 3 Issue 25 / Part 3 Modern engineering and innovative technologies Issue 25 / Part 3 systematic approach, it is important to search for system-forming factors, which are divided into external and internal. External factors contribute to the emergence and development of the system under given conditions; internal ones are generated by individual elements combined into a system, groups of elements, or the entire set. In this case, the external system-forming factor is the professional and communicative activity of the manager-economist, presented in the form of a developed structure of professional skills. These skills act as a set of elements that are in relationships and connections with each other, representing a model of professional activity. From a pedagogical point of view, it acts as the goal of training and as an evaluation criterion for the quality of training. Introduction. Its structure includes an integral system of qualities of the human individual, allowing him to perform the required professional functions and act in accordance with the requirements of society. The activity model combines professional and moral and ethical tasks that a graduate of an educational institution must solve. It defines the social and moral, professional, intellectual knowledge, skills and abilities necessary for its successful activity and acquired through training within the framework of the pedagogical model. The formation of a modern specialist, combining the role of a leader and an executor in one person, aimed at positive highly professional behavior, depends entirely on the formation of his creative abilities, self-education and self-education. The study of the structure and content of the communicative skills and abilities of the students of the department "Finance and Credit" served as the basis for the development and construction of an integrated model of parallel teaching of a foreign language in the mode of independent work, capable of ensuring the effective formation of relevant skills and abilities in students. Building training on the principles of unity, integrity and phased sequence, we used a systematic approach to building a training model, considering it as a systematized set of interdependent elements, united by common functional and managerial goals, identified on the basis of certain features. With a systematic approach, it is important to search for system-forming factors, which are divided into external and internal. External factors contribute to the emergence and development of the system under given conditions; internal ones are generated by individual elements combined into a system, groups of elements, or the entire set. In this case, the external system-forming factor is the professional and communicative activity of the manager-economist, presented in the form of a developed structure of professional skills. These skills act as a set of elements that are in relationships and connections with each other, representing a model of professional activity. From a pedagogical point of view, it acts as the goal of training and as an evaluation criterion for the quality of training. Its structure includes an integral system of qualities of the human individual, allowing him to perform the required professional functions and act in accordance with the requirements of society. The activity model combines professional and moral and ethical tasks that a graduate of an educational institution must solve. ISSN 2567-5273 Introduction. It defines the social and moral, professional, intellectual knowledge, skills and abilities necessary for its successful activity and acquired through training Main text 1. The internal system-forming factors www.moderntechno.de ISSN 2567-5273 103 Issue 25 / Part 3 Modern engineering and innovative technologies Issue 25 / Part 3 The internal system-forming factors that allow us to consider the model of activity from the standpoint of an integrated approach are the forms of organization of independent work of students and ways to control it. They act as a separate subsystem and have their own internal structure. In our case, the alternative learning model is a step-by-step process of introducing an activity model through various types of tasks that turn into a system of business games. The mastery of communication and activity skills by students with a focus on promising creative activity is the main goal of training. There are two levels of methods of activity - technological and professional. The technological level determines the skills that underlie the mastery of work techniques. The professional level reflects the area of professional content, obtaining generalized systematized knowledge. In the process of educational and professional activities of students, the technological and professional levels are in constant unity, which reflects the didactic unity of the operational- procedural and logical-content aspects of education. The allocation of the structural elements of the system occurs according to the following criteria: on the basis of the need for a particular part of the whole and on the principle of their functional correspondence to each other. If we have in mind the system of educational business games, it is necessary: to single out a phased transition from teaching general, superficially professional skills and abilities to professional communicative foreign language competence. In our case, this is a phased, multi-stage transition from classroom independent work to extracurricular work, from reproductive to creative work. In practice, this happens in the form of the following chain of species. Work with foreign texts and activation of professional and communicative skills of independent work of students, special and linguistic knowledge through the performance of problematic tasks; the inclusion of elements of a role-playing game; participation in business games in a foreign language. www.moderntechno.de Introduction. of a role-playing game; participation in business games in a foreign langua p y g g p p g g g The model developed by us uses the following types of business game The model developed by us uses the following types of business games: • games "Connoisseurs" - the main didactic task is to master the knowledge that determines the formation of professional skills; • games "Connoisseurs" - the main didactic task is to master the knowledge that determines the formation of professional skills; • games "Performers" - in the didactic aspect are aimed at mastering professional skills in standard situations; • games "Performers" - in the didactic aspect are aimed at mastering professional skills in standard situations; • games "Creators" - didactically solve the problem of creative transfer, professional knowledge and skills in situations as close as possible to production; • games "Profi" - solve the didactic problem of the complex application and use of professional skills in situations as close as possible to real production. The experimental model of parallel learning in the mode of integrated independent learning activity can be defined as developed by professional and foreign language communication skills acquired within the framework of a parallel learning system based on problem-based gaming technologies. In our experiment, business games are both the content and form of learning and a type of control. The basis for determining and selecting the content of training is the model of the specialist's activity, presented in a business game. The content of education is the content and volume of educational information presented to students for study and assimilation, a set of tasks, tasks and exercises necessary for mastering professional skills and abilities. The content of academic disciplines is one of the elements of the didactic www.moderntechno.de ISSN 2567-5273 ISSN 2567-5273 104 Issue 25 / Part 3 Issue 25 / Part 3 Modern engineering and innovative technologies Issue 25 / Part 3 system, the main function of which is to teach how to solve problems with a focus on professional and general scientific knowledge. Tasks are arranged in accordance with the logic of the formation of professional and linguistic knowledge on the basis of a gradual increase not only in the complexity of linguistic and professional content, but also an increase in the level of independence and creativity in solving them. Introduction. The problem of training a modern specialist - a generalist in his field requires the selection and determination of the content of training, but also the choice of methods, ways, means, forms of training, distribution of study time. The decisive influence on the choice and expediency of combining the components of the didactic system is exerted by the goals, objectives and content of education. 2. Teaching methods A special place among the listed components of the educational process is occupied by teaching methods. We consider the method as a set of forming principles and organization of educational material, based on pedagogically competent interaction between the teacher and students to solve fixed didactic tasks. In the course of the study, a system of problem-developing teaching methods was applied. The specificity of our time implies a wide variability in the scope of application of professional skills and abilities; a modern specialist has to face the problem of finding the most appropriate solutions and taking appropriate measures to implement them. Students should develop personal experience in the formation of logically sound decisions based on the acquired knowledge. The choice of teaching methods in our case is determined by the following factors: • the level of general education, mental and physical indicators of the student's development; • the level of general education, mental and physical indicators of the student's development; • learning objectives, consisting in the preparation of a bilingual specialist of a sufficient level of qualification, learning objectives arising from the learning objectives; • the content of the educational material, selected according to the objectives; content of science and practice. • the content of the educational material, selected according to the objectives; content of science and practice. With the increasing role of independent work of students in the educational process, the responsibility of the teacher for the application of the appropriate range of methods and teaching aids, applied depending on the age and level of independence of the students, increases. Since not only the activity of acquiring educational information is carried out in the process, but also the process of control and self-control, appropriate methods of control, stimulation and motivation of educational and cognitive activity were used. When choosing and combining teaching methods, we were guided by certain criteria. The correspondence of the methods to the principles of teaching, the goals and objectives of independent work of students, the nature of the subject being studied, the educational opportunities of the student, the level of training of the study group as a whole, the characteristics of the student team, the compliance with the existing learning conditions and the time allotted for independent educational activities were taken into account; professional and personal capabilities of the teacher. ISSN 2567-5273 2. Teaching methods The choice of the optimal combination of teaching methods took place in several www.moderntechno.de www.moderntechno.de ISSN 2567-5273 105 Issue 25 / Part 3 Issue 25 / Part 3 Modern engineering and innovative technologies Issue 25 / Part 3 stages. The combination of the main groups of methods was determined depending on the nature of teaching the subject and the didactic and methodological tasks of the educational process. The learning technology was tested, in the process of which the necessary groups of methods and means were determined for organizing training in the mode independent work of students. The process was carried out on the basis of cooperation between teachers of special and language disciplines at the secondary levels of education of non-linguistic universities. All teachers used uniform forms, methods and means of organizing independent work, which was a significant reserve for increasing its effectiveness. The following methods of organizing students' independent work were used: o methods of organization and implementation of educational and cognitive activities; o methods of organization and implementation of educational and cognitive activities; o methods of stimulation and motivation of educational and cognitive acti hods of stimulation and motivation of educational and cognitive activity; o methods of control and self-control over the effectiveness of educational and cognitive activities. o methods of control and self-control over the effectiveness of educational and cognitive activities. Building an effective learning technology is impossible without the use of a variety of learning tools. The main purpose of using a set of teaching aids is to form in the minds of students a "polymodal" image of the process, object, phenomenon that regulates human activity. This image is formed through the system of human analyzers. In the course of the study, an important place was given to organizational forms of training, which streamlined the sequence of acquiring knowledge, skills and abilities. The organizational form of education is the types of training sessions that differ from each other in didactic goals, composition of students, venue, duration, content of the teacher and student. Within their framework, a system of interaction between a teacher and a student was implemented, carried out according to a certain, pre-established order and mode. In our observation, this is the provision of information for study with a set of tasks - monitoring consultations according to a predetermined schedule (individual or in mini groups) - final control (in the form of a business game). 2. Teaching methods Control is represented by its only acceptable form - reflexive. Consultations of each substantive stage performed the function of intermediate control and, in parallel, preparation for the business game. Due to the fact that in our system the games are arranged in accordance with the increase in the complexity of educational and gaming tasks and the level of independence of their implementation, there is a stimulation of a creative approach to the implementation of the proposed tasks. In fact, three types of independent work of students were activated: reproductive, combined and productive. The last two types involve the variation of already acquired knowledge, acquired skills and abilities, while the first one is a process of reproducing the information received accumulated knowledge. Orientation to it leads to insufficient independence of thinking, inability to independently acquire knowledge and apply it in creative activity. ISSN 2567-5273 3. The system of business games. The solution of a specific cognitive task in the independent work of students of the combined type involves a productive process of searching for new information with elements of creative activity. The highest level of cognitive independence of a student occurs at the stage of a productive type of independent extracurricular www.moderntechno.de www.moderntechno.de 106 Issue 25 / Part 3 Issue 25 / Part 3 Modern engineering and innovative technologies Issue 25 / Part 3 educational activity. The student is busy searching for new principles for solving the problems he faces. He carries out the processes of designing, combining new knowledge, skills and abilities from previously learned ones. Independent work of this type is carried out in the process of preparing and implementing a business game. The basis of independent work of a productive type is the research method of teaching, the essence of which is to organize the creative, search activities of students, to solve new problems for them. The system of business games ensures the development of mental skills and behavioral skills in the course of productive independent learning activities. For example, the games "Connoisseurs" were used by us at the stage of consolidating, deepening and expanding basic professional knowledge in a foreign language. The game "Consultant" determines the correctness of the governing model for the optimal solution of business problems. The game "Performers" was held at the stage of transition to a productive type of independent work. Students acted in standard or slightly changed situations, activating the acquired knowledge, skills and abilities. The game "Creators" was used at the stage of a productive form of independent work of students, when transferring the acquired knowledge, skills and abilities to a situation that simulates real and practical conditions that require a creative approach to solving problems. Games can be held using the elements of "brainstorming" or the performance of certain roles with the condition of a pre- prepared database. In the first case, the game is organized in accordance with the stages of collective problem solving: the generation of ideas, their criticism and constructive study. There may be a rigid division of participants into "generators", "managers", "analysts". In another option, flexible transition of functions is allowed during discussions. In the first case, maintaining role correspondence is fundamentally important. In the second, only the optimal solution of the problem is important. ISSN 2567-5273 www.moderntechno.de Summary and conclusions. Have been considered the integrated model of parallel learning is based on the system-multilevel organization of students' independent activities, in which the connection between classroom and extracurricular forms of students' independent work is carried out. The system of business games based on problem-based learning combines the functions of learning and monitoring independent learning activities. It is aimed at mastering by students the professional skills they need in their future activities, foreign language, professional communication skills and abilities. This system of such an organization of the parallel line fully satisfies the basic pedagogical conditions, since it interconnects with systems of a higher and lower order, takes into account the psychophysiological characteristics of students is implemented in the mode of cognitive self-regulation, involves limiting the external algorithmization of students' activities and is carried out through the use of a set of tools and methods for students' independent work. The learning control system through the system of business games of business games has an open dynamic character. Its content and organizational elements depend on the qualifications and competence of teachers, on the student's ability to work independently, due to the relationship between classroom and extracurricular independent work of students. Outside of activity there is no development. Were received the means, the methods and the tools of the self-education, the organization of an interactive communication mode during the implementation and control of the educational process, the differentiation of learning, ensuring the student's independence in regulating the nature and volume of the studied material, the pace and timing of educational activities, ensuring the continuity of classroom and extracurricular forms of the students` independent work. Therefore, the student needs to be involved in a professionally oriented organized educational activity. It is especially important to realize the content side of professional activity and the formation of a conscious positive attitude towards it. If this process is limited by the scheme of information accumulation within the framework of the traditional educational process without didactic modeling of independent cognitive and behavioral activity, then the formation of the student's professional readiness will be more successful, since the necessary transition of external influences into internal ones, quantities - into quality is carried out. In modern society, it is necessary that a person's personal mode, his needs, interests, beliefs, evaluation criteria contribute to the formation and regulate normal social and professional relationships. 3. The system of business games. Depending on the organization of the game, criteria for evaluating the activities of teams are proposed - the quality of the performance of the role, compliance with the rules of the game, originality of decisions, communication style. As part of our project, games are held only in a foreign language. In the games with an emphasis on game distribution, the composition and functions of the participants are determined in advance depending on the proposed game situation. Players offer and justify solutions from the standpoint of the services, divisions, and positions they represent. The correctness of the approach to solving the problem, the depth of the solution found is very important. In games built on the principle of "brainstorming", the teacher plays the role of an active leader. In the games of the second type, he only an observer who can correct the course of the game without interfering with the course of its development. At the first stage, the teacher should clearly present the plot of the game to the students, emphasizing its meaning and educational value. It creates a positive emotional background for students, contacts and discussions are carried out in an atmosphere of ease, collective search. Students must understand the purpose of the game, know or independently propose the stages of its implementation, terms, conditions and rules. Game tasks, personal interests, expectations, assessment of the level of professional readiness, implementation of the proposed role should be www.moderntechno.de 107 Issue 25 / Part 3 Issue 25 / Part 3 Modern engineering and innovative technologies Issue 25 / Part 3 discussed with the participants. Participants are warned about the possibility of an unexpected change in the course of the game, without devoting them to the essence of the intended problem situation. It is permissible to conduct a trial drawing of game fragments at supervisory consultations through the solution of communicative and professional tasks. Groups of participants are formed in advance, the tasks of game groups and individual functionalities are specified, the course of preparation is corrected, and the results of the game are negotiated. Participants can independently adjust all these components, but in practice this rarely happens, which shows the lack of initiative and desire for self-realization. ISSN 2567-5273 www.moderntechno.de Summary and conclusions. Education by the method of conflicts through "breaks in activity" creates such model situations in www.moderntechno.de 108 Issue 25 / Part 3 Issue 25 / Part 3 Modern engineering and innovative technologies Issue 25 / Part 3 which behavioral, informational is necessary and psychological activation. The process of personality` development is carried out on the basis of socialization and self-development and the mechanism of reflection. The formation of behavioral foundations that ensure these processes is the goal of a closed didactic system for building a professionally oriented organized educational activity, since it provides a programmed, informationally personal, behavioral approach to training and education. Education within the framework of the proposed model of independent work of students, based on directly experienced experience, differentiates students according to an individual type of activity, teaches problematic, conceptual thinking. The forms of intermediate and final control of independent learning activity that we offer through solving problematic tasks in the course of individual and group discussions, discussions, business games mobilize the entire amount of knowledge gained, transferring them to the phase of abstract perception. References References 1. Andreev VI (1988). Dialectics of education and self-education of creative personality: Fundamentals of pedagogy of creativity. Kazan: Kazan University. 288 p 1. Andreev VI (1988). Dialectics of education and self-education of creative personality: Fundamentals of pedagogy of creativity. Kazan: Kazan University. 288 p 2. State standard of basic and complete general secondary education. № 1392 [Effective from 2011-11-23]. URL: http://zakon4.rada.gov.ua/laws / show / 1392- 2011-n (accessed 14 October 2020). 3. The concept of specialized education in high school. Order of the Ministry of Education and Science №1456 dated October 21, 2013. Labor training in a modern school. 2013. № 10. S. 2–10. 4. Piekhota О.М., Kiktenko A.Z., Lyubarska O.M. and others (2004). Educational technologies. Teaching method way. /; For order. OHM. Infantry. Kyiv: ASK, 256 p. 4. Piekhota О.М., Kiktenko A.Z., Lyubarska O.M. and others (2004). Educational technologies. Teaching method way. /; For order. OHM. Infantry. Kyiv: ASK, 256 p. 5. Sysoeva S.O (1994). Fundamentals of pedagogical creativity of the teacher. Teaching. manual. Kyiv: ISDOU. 112 p. 5. Sysoeva S.O (1994). Fundamentals of pedagogical creativity of the teacher. Teaching. manual. Kyiv: ISDOU. 112 p. 6. Pometun О.І., Pyrozhenko L.V. (2004). Modern lesson. Interactive learning technologies: Scientific method. manual. For order. OI Sweeper. Kyiv: ASK, 192 p. 6. Pometun О.І., Pyrozhenko L.V. (2004). Modern lesson. Interactive learning technologies: Scientific method. Summary and conclusions. manual. For order. OI Sweeper. Kyiv: ASK, 192 p. 8. Trofimchuk L.O. (2013). Psychological and pedagogical features of the development of creative abilities of students. Labor training in a modern school. № 1. S. 14–18. 8. Trofimchuk L.O. (2013). Psychological and pedagogical features of the development of creative abilities of students. Labor training in a modern school. № 1. S. 14–18. 9. Chytak О.М. (2010). Modern interactive technologies in the lessons of labor training. Labor training at school. № 9 (21). Pp. 10–16. 9. Chytak О.М. (2010). Modern interactive technologies in the lessons of labor training. Labor training at school. № 9 (21). Pp. 10–16. g g ( ) p 10. Shadrikov V.D. (1994) Activity and abilities. Moscow: Ed. LOGOS Corporation, 320 p. 10. Shadrikov V.D. (1994) Activity and abilities. Moscow: Ed. LOGOS Corporation, 320 p. Article sent: 17.02.2023 © Kulaeva Z. A. Article sent: 17.02.2023 © Kulaeva Z. A. ISSN 2567-5273 www.moderntechno.de 109

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Portuguese

Avaliação da amônia emitida de camas sobrepostas e piso concretado utilizados na criação de suínos

Revista Brasileira de Engenharia Agrícola e Ambiental

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Revista Brasileira de Engenharia Agrícola e Ambiental v.13, n.2, p.210–213, 2009 Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 133.06 – 20/10/2006 • Aprovado em 04/08/2008 Revista Brasileira de Engenharia Agrícola e Ambiental v.13, n.2, p.210–213, 2009 Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 133.06 – 20/10/2006 • Aprovado em 04/08/2008 Revista Brasileira de Engenharia Agrícola e Ambiental v.13, n.2, p.210–213, 2009 Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br Protocolo 133.06 – 20/10/2006 • Aprovado em 04/08/2008 RESUMO A criação de suínos em camas sobrepostas surgiu como alternativa ao uso excessivo de água utilizada na atividade, mas veio, também, o problema da emissão de gases poluentes, principalmente a amônia. Objetivou-se, com este trabalho, avaliar a quantidade de amônia emitida pelas camas de maravalha, casca de arroz e pelo piso de concreto, comparando- a com os níveis aceitáveis para a produção de suínos criados em camas, entre dezembro de 2002 e abril de 2003, no município de Concórdia, SC; três edificações (12,0 x 10,0 m) foram utilizadas, cada uma com 216 animais (Landrace X Large White) e se avaliou a qualidade do ar quantificando-se a emissão diária de amônia, às dez e quatorze horas; a cama de maravalha, por sua vez, apresentou melhor eficiência na absorção das dejeções em relação à de casca de ar- roz; entretanto, nela foram encontrados os menores valores de emissão da amônia, enquanto no piso de concreto foram observados os maiores valores para o gás; desta forma, indica-se a cama de maravalha como a mais apropriada para a criação de suínos em condições de verão, para o Sul do Brasil; enfim, a análise estatística lançou mão de um delinea- mento experimental inteiramente casualizado e 4 repetições por tratamento. Palavras-chave: maravalha, casca de arroz, gases Palavras-chave: maravalha, casca de arroz, gases Evaluation of the emitted ammonia from deep beddings and concrete floor used in the swine production Evaluation of the emitted ammonia from deep beddings and concrete floor used in the swine production Avaliação da amônia emitida de camas sobrepostas e piso concretado utilizados na criação de suínos1 Robson M. de Paulo2, Ilda de F. F. Tinôco3, Paulo A. V. de Oliveira4, Cecília de F. Souza4, Fernando da C. Baêta4 1 Parte da Dissertação de Mestrado do primeiro autor 2 UENF, Laboratório de Engenharia Agrícola, Av. Alberto Lamego 2000, Parque Califórnia, CEP 28013-602, Campos dos Goytacazes, RJ. Fone: (22) 2726-1543. Fax: (22) 2726-1549. E-mail: [email protected] 3 DEA/UFV, Av. P.H. Rolfs s/n, CEP 36571-000, Viçosa, MG. Fone: (31) 3899-1884. Fax: (31) 3899-2735. E-mail: [email protected] 4 Embrapa Suínos e Aves, CP 21, CEP 89700-000, Concórdia, SC. Fone: (49) 442-8555. Fax: (49) 442-8559, E-mail: [email protected] MATERIAL E MÉTODOS O experimento foi realizado no Centro Nacional de Pes- quisa de Suínos e Aves da EMBRAPA (EMBRAPA/CNPSA), localizado no Distrito de Tamanduá, na cidade de Concór- dia, Estado de Santa Catarina, situada a 584,0 m de altitu- de, com Latitude de 27° 18’ S e Longitude de 51° 59’ O, no período de dezembro de 2002 a abril de 2003. As instala- ções se compunham de três galpões, medindo 12,0 x 10,0 m e pé direito de 3,25 m; cada galpão foi dividido em quatro baias com dimensões de 5,0 x 6,0 m; cobertura de telhas de fibrocimento com lanternim. Os fechamentos laterais, com 1,0 m de altura, eram constituídos de placas vazadas de con- creto e/ou grades de ferro A distância entre as instalações era de 10,0 m. Na primeira instalação utilizou cama de marava- lha; na segunda, casca de arroz e, na terceira, piso de con- creto. Foram utilizados 216 animais das raças Landrace e Large White (machos castrados e fêmeas), com peso médio inicial de 25 kg e idade média de 63 dias, compreendendo as fases de crescimento e terminação. Os animais receberam, durante o período experimental (120 dias) a mesma dieta. Segundo Paulo (2003) deve-se considerar que o uso de camas sobrepostas na suinocultura proporciona economia significativa de água utilizada nos processos produtivos e evita o lançamento das águas residuárias nos cursos d’água. Dartora et al. (1998) concluíram que boa parte dos siste- mas de criação de suínos existentes no Brasil resulta em ele- vada produção de dejetos líquidos, gerando problemas de manejo, armazenamento, distribuição e poluição ambiental. Ainda de acordo com eles, somente no Estado de Santa Ca- tarina 80% das fontes de água e a maioria dos rios e riachos do oeste e meio oeste estão contaminados. Levantamentos realizados em zonas rurais das regiões produtoras de suínos de Santa Catarina, revelam que 85% das fontes de água es- tão contaminados por coliformes fecais, oriundos do lança- mento direto de esterco de suínos em cursos ou mananciais d’água (Lohmann et al., 1999). Para efeito de estudo da emissão de amônia dividiu-se o período experimental em três fases, que compreendiam os intervalos de pesagem: 1) 25-50; 2) 50-75 e 3) 75-135 kg. INTRODUÇÃO Com base no ganho de peso, consumo de ração e consu- mo de água, Paulo (2003) concluiu que o desempenho zoo- técnico não foi influenciado pelas camas de maravalha, cas- ca de arroz nem pelo piso de concreto; entretanto, os animais mantidos no piso de concreto apresentaram resultados ligei- ramente melhores que os alojados nas camas sobrepostas. A amônia é o mais importante gás encontrado em insta- lações para a criação de suínos, podendo ocorrer em níveis bastante altos, além de ser um irritante ao sistema respira- tório; pode, também, afetar a saúde dos animais e dos traba- lhadores que atuam na atividade. O desempenho zootécnico dos animais criados no siste- ma de camas sobrepostas, é similar ao daqueles criados no piso convencional, além de melhor bem-estar, sustentabili- dade ambiental e menor custo inicial (Hill, 2000). Segundo Heber et al. (2002) a amônia pode causar pneu- monias e diminuição da taxa de crescimento nos animais, visto que o gás tem odor distinto e é detectado pelo homem em concentração de 5 ppm. A concentração máxima recomen- dada pelo NIOSH (1996) é da ordem de 25 ppm; por outro lado, pesquisadores europeus sugerem um limite da ordem de 10 ppm. Tendo como base os resultados do índice de temperatura de globo negro e umidade (ITGU), carga térmica de radia- ção (CTR) e umidade relativa do ar (UR), Paulo (2003) ob- servou discreta vantagem para o piso de concreto. Dentre as camas, a casca de arroz se mostrou mais eficiente; a umida- de relativa do ar esteve sempre acima do valor considerado limite para suínos em crescimento e terminação, que é de 75%. Oliveira (2000) em experimento realizado no Laborató- rio de Bioclimatologia do INRA-França e comparando o sis- tema de cama sobreposta com o sistema de piso ripado, cons- tatou que as concentrações médias de amônia observadas foram significativamente diferentes, sendo que o sistema de cama apresentou as menores concentrações. Objetivou-se, com o presente trabalho, avaliar se a quan- tidade de amônia (NH3) emitida pelas camas sobrepostas compostas de maravalha e de casca de arroz e pelo piso de concreto, utilizadas na criação de suínos em crescimento e terminação, para as condições de verão do Sul do Brasil, é prejudicial aos animais e ao homem, de acordo com níveis tolerados internacionalmente. INTRODUÇÃO A cama absorve o componente líquido do resíduo, sepa- rando-o dos sólidos, reduzindo a velocidade do processo de decomposição, que produz combinações como amônia e sul- fito de hidrogênio, sendo assim mínimo o odor nessas insta- lações (Chapin et al., 1998). Paulo (2003) avaliando a temperatura de camas sobrepos- tas e piso de concreto, concluiu que no período da manhã (9 h), a temperatura da cama de maravalha foi maior que a cama com casca de arroz. Entre a cama de casca de arroz e o piso de concreto, não houve diferença de temperatura. A alta produção de calor gerada nas camas tende a influenciar no comportamento e desempenho dos animais. As tempera- turas elevadas indicam que a atividade bacteriana produz calor suficiente para evaporar a água da cama, favorecendo a emissão de gases produzidos nas camas, pelos animais. A cama gera grandes quantidades de calor por meio da degra- dação da matéria orgânica. ABSTRACT Swine production in deep bedding appeared as an alternative to the excessive use of water in the activity, however the problem of emission of pollutant gases appeared, mainly ammonia. This research aimed at evaluating the amount of ammonia emitted by wood shavings and rice husks deep beds and concrete floor, compared to acceptable levels for swine production in beds, during December 2002 to April 2003, in Concórdia, Santa Catarina State. Three constructions with dimensions of 12.0 x 10.0 m, divided in 4 boxes with 5.0 x 6.0 m were used, each with a total of 216 animals (Landrace X Large White). The evaluation of air quality was based on the quantification of the ammonia emission, daily measurements, at 10:00 a.m. and 2:00 p.m. The wood shaving bed presented better efficiency in the absorption of the wastes compared to the rice husk, which presented the lowest values of ammonia emission. In the concrete floor the highest values were observed for the gas. As such, the wood shaving bed is indicated as the most appropriate for the swine production in summer conditions for the South of Brazil. For the statistical analysis an entirely randomized experimental design, with 4 replicates for each treatment was used. Key words: wood shavings, rice husks, gases Key words: wood shavings, rice husks, gases 211 R. Bras. Eng. Agríc. Ambiental, v.13, n.2, p.210–213, 2009. RESULTADOS E DISCUSSÃO Por meio das médias diárias realizadas nos dois tratamen- tos estudados (cama de maravalha e cama de casca de ar- roz), apresentadas na Tabela 1, observa-se que não houve diferença significativa (P < 0,05) quando comparados ao tra- tamento convencional com piso de concreto. Confirmando os valores mais altos para as condições de inverno, Sampaio et al. (2006) concluíram que as mais altas concentrações de amônia foram observadas no inverno e mais especificamente nos horários da tarde, correspondendo aos horários de maior temperatura do ar. Tabela 1. Médias* dos valores de emissão de amônia (NH3), em partes por milhão (ppm), medidas nas camas de maravalha e casca de arroz comparadas com o piso de concreto Tabela 1. Médias* dos valores de emissão de amônia (NH3), em partes por milhão (ppm), medidas nas camas de maravalha e casca de arroz comparadas com o piso de concreto Tabela 1. Médias* dos valores de emissão de amônia (NH3), em partes por milhão (ppm), medidas nas camas de maravalha e casca de arroz comparadas com o piso de concreto otn e m ata rT s aid é M a hla v ara M A 0 3,3 z orra e d a c s a C A 8 9,2 oterc n o c e d o siP A 0 2,5 * As médias seguidas de pelo menos uma mesma letra não diferem entre si pelo Teste de Tukey (P < 0,05) Andersson (1996) observou maiores valores para emissão de amônia em cama com palha quando comparado com ou- tros tratamentos semelhantes e em tempos diferentes. É importante observar que, em média, os valores de con- centração de amônia (NH3) no tratamento piso de concreto foram 37% maiores quando comparados com o tratamento cama de maravalha e 44% quando a comparação se refere ao tratamento cama de casca de arroz. Pode-se observar, na Figura 1, os valores encontrados nas camas e no piso con- vencional comparados àquele considerado padrão. Os picos foram observados de 16, 15 e 22 ppm para as camas de maravalha, arroz e piso de concreto, respectiva- mente; os resultados encontrados nas camas permaneceram abaixo daqueles constatados por Oliveira (2000) que traba- lhou com suínos mantidos em piso ripado, ou seja, de 15,20 ± 6,40 ppm. MATERIAL E MÉTODOS Os animais foram alojados em grupo de 72 por instalação (18 animais por baia); as baias possuíam área útil de 21,0 m2 (4,2 x 5,0 m) e profundidade de 0,50 m (instalações com camas); as baias foram equipadas com um bebedouro e um comedouro, recebendo água e ração à vontade. Para quanti- ficar a emissão de amônia foram feitas medições de concen- tração instantânea (ppm) a 0,25 m do piso, correspondente O uso de camas sobrepostas sobre o piso como alternati- va ao tradicional de piso de concreto, vem-se tornando uma prática constante na criação de suínos nas fases de cresci- mento e terminação, pois evita a utilização de lagoas para tratamento de dejetos. R. Bras. Eng. Agríc. Ambiental, v.13, n.2, p.210–213, 2009. Robson M. de Paulo et al. 212 ao nível médio do dorso dos animais (focinho do suíno). Tabela 2. Médias* dos valores de emissão de amônia (NH3), em partes por milhão (ppm), medidas em cada fase do experimento, para os tratamentos As medições foram realizadas em dois horários (10 e 14 h), em períodos diários, sendo uma leitura por baia na área considerada crítica (local das dejeções), durante o pe- ríodo experimental. Para a coleta de dados utilizou-se um detector eletroquímico portátil de leitura direta da marca Crowcon com faixa de leitura de 0-50 ppm, temperatura de operação de -20 a 50 °C e umidade de 0 a 95%. Os dados foram analisados por meio de regressão, através do progra- ma SAEG 5.0. MATERIAL E MÉTODOS e s a F s aid é M ) 1 d 8 9 - 3 6 A 0 2,2 ) 2 d 6 2 1 - 8 9 A 6 0,2 ) 3 d 3 8 1 - 6 2 1 B 5 1,5 * As médias seguidas de pelo menos uma mesma letra não diferem entre si pelo Teste de Tukey (P < 0,05) e s a F s aid é M ) 1 d 8 9 - 3 6 A 0 2,2 ) 2 d 6 2 1 - 8 9 A 6 0,2 ) 3 d 3 8 1 - 6 2 1 B 5 1,5 As médias seguidas de pelo menos uma mesma letra não diferem entre si pelo Teste de ukey (P < 0,05) Maiores valores para emissão de amônia em cama de ma- ravalha foram observados por Cordeiro (2003), que trabalhan- do com suínos em crescimento e terminação em condições de inverno, observou diferença significativa na emissão de amô- nia, sendo os maiores valores para as camas de maravalha e casca de arroz, quando comparados com o piso de concreto; o autor concluiu que os valores ficaram abaixo de 20 ppm. R. Bras. Eng. Agríc. Ambiental, v.13, n.2, p.210–213, 2009. RESULTADOS E DISCUSSÃO Os valores médios para o piso de concre- to estão de acordo com os resultados obtidos por Oliveira (2000), ou seja, 9,20 ± 4,20 ppm para os valores de emissão de amônia (NH3). 0 1 2 3 4 5 6 Maravalha Casca de arroz Piso de concreto Tratamento Concentração (ppm) Figura 1. Comparação dos valores de amônia emitidos pelas camas de maravalha e casca de arroz com o piso de concreto e o mínimo tolerado pelos suínos (padrão) LITERATURA CITADA De maneira geral, os níveis médios de amônia encontra- dos nas instalações em condições de verão não tendem a causar conseqüências mais graves aos animais, por estarem abaixo dos níveis críticos. Andersson, M. Performance of bedding materials in affecting ammonia emissions from pig manure. Journal of Agricultural Engineering Research, v.65, n.3, p.213-222, 1996. Chapin, A.; Boulind, C.; Moore, A. Controlling odor and gaseous emission problems from industrial swine facilities – Recent laws and new ideas. Yale: Environmental Protection Clinic, 1998. 81p. Os valores observados estão de acordo com os valores es- tipulados por organismos internacionais, a exemplo da NI- OSH (1996), que recomenda que a concentração máxima do gás não seja superior a 25 ppm, bem mais elevada que aquela sugerida por pesquisadores da Europa, que é da ordem de 10 ppm (Heber et al., 2002); geralmente, os níveis da amô- nia são mais elevados no verão; esta elevação nos níveis de amônia pode estar associada ao fato dos animais ingerirem uma quantidade maior de água e a necessidade de constante alimentação, quadro que gera, também, maior produção de dejetos sólidos e líquidos e, por conseqüência, maior produ- ção de gases nas instalações. Segundo Harmon & Xin (1995) apud Santos (2001), o nível máximo tolerado para a amônia (NH3) nas instalações para suínos, é de 10 ppm; o gás pode ser facilmente detectado por meio do odor, na concentração de 5 ppm ou mais; a partir de 50 ppm, passa a afetar o cres- cimento e a saúde dos animais e, em sendo mais leve que o ar, tende a concentrar-se junto à cobertura das instalações; sua concentração depende muito da higiene local e da efici- ência da ventilação. Cordeiro, M. B. Avaliação de sistema de camas sobrepostas quanto ao conforto térmico e ambiental e ao desempenho zootécnico para suínos nas fases de crescimento e terminação. Viçosa: UFV, 2003. 63p. Dissertação Mestrado Dartora, V.; Perdomo, C. C.; Tumelero, I. L. Manejo de dejetos de suínos. Boletim Informativo Pesquisa BIPERS, Embrapa Suínos e Aves/Extensão-EMATER-RS, n.11, ano 7, 1998. Heber, A.; Jones, D.; Sutton, A. Indoor air quality: Controlling ammonia gas in swine buildings. Purdue University Coopera- tive: Extension Service. http://cdc.gov/niosh/nasd/docs4/ in98003.html. 20 Ago. 2002. Higarashi, M. M.; Coldebella, A.; Oliveira, P. A. V.; Kunz, A.; Mattei, R. M.; Silva, V. S.; Amaral, A. L. Concentração de ma- cronutrientes e metais pesados em maravalha de unidade de suínos em cama sobreposta. AGRADECIMENTOS Por ser rugoso, o piso de concreto usado no compara- tivo pode ter influenciado nos maiores valores médios de concentração de amônia; a parte ripada com o canal con- dutor, situado sob a instalação, pode ser outra explicação para esses valores. Outra explicação reside no fato de os animais de maior porte possuírem maior capacidade de revolver as camas e, portanto, aeração mais eficiente, com isso as perdas atmosféricas seriam mais pronunciadas (Higarashi et al., 2008). Os autores expressam seus agradecimentos ao apoio con- cedido pelo CNPq, CAPES, FAPEMIG e EMBRAPA Suinos e Aves. Agradecimento especial à professora Dra. Ilda Tinôco (DEA/UFV) pela dedicação e incentivo. Concentração (ppm) No comparativo entre as três fases de observação, ocor- reu diferenciação na emissão de amônia, por parte das ca- mas. Nas fases 1 (63 – 98) e 2 (98 – 126d) não se constatou diferença significativa entre os dois tratamentos, porém os valores para a cama de maravalha foram ligeiramente supe- riores, talvez pelo fato do material absorver mais as dejeções que a cama de casca de arroz, além da constante movimen- tação pelos animais; entretanto, esses valores para a emis- são do gás não comprometem a saúde dos animais; na ter- ceira fase se observaram os maiores valores médios quando comparados com as duas primeiras (Tabela 2). Possivelmente em razão desta fase compreender um período maior de ob- servação (57 dias, contra 35 e 28 para as fases 1 e 2, respec- tivamente) outra explicação pode ser dada com base no peso maior dos animais e, por conseqüência, maior volume de dejeções produzido por animal nas camas, uma vez que houve aumento no consumo de água e alimento, diferente do que ocorre no inverno, quando temperaturas menores não favo- recem tanto o consumo de água quanto no verão. Figura 1. Comparação dos valores de amônia emitidos pelas camas de maravalha e casca de arroz com o piso de concreto e o mínimo tolerado pelos suínos (padrão) Os valores da concentração de amônia obtidos nas camas de maravalha e de casca de arroz estão abaixo daquele reco- mendado por pesquisadores da área, que é da ordem de 10 ppm; o que também pode ter influenciado diretamente os resultados, é a incidência da ventilação nas instalações (aber- tas) durante todo o período de criação, o que não ocorre em países da Europa nem nos Estados Unidos, onde as instala- ções são completamente fechadas. A insolação direta sobre as camas também pode ter efeito benéfico pois não permi- tia, em algumas partes, a umidade das dejeções. R. Bras. Eng. Agríc. Ambiental, v.13, n.2, p.210–213, 2009. 213 LITERATURA CITADA Engenharia Agrícola e Ambiental, v.12, n.3, p.311-317, 2008. Hill, J. D. Deep bed swine finishing. In: Seminário Internacional de Suinocultura, 5, São Paulo. Anais... Concórdia: Embrapa Suínos e Aves, 2000. p.83-88. Em algumas ocasiões os valores instantâneos superaram os limites considerados críticos por pesquisadores e organis- mos internacionais devido, sem dúvida, à agitação das ca- mas pelos animais, comportamento considerado normal. Lohmann, O.; Silva, R. F.; Fontoura, T. B.; Silva, D. M.; Jaques, R. J. S.; Fries, M. R.; Aita, C. Determinação de microrganis- mos fecais em solo, sob campo nativo, submetidos a aplicações periódicas de esterco suíno. In: Congresso Brasileiro de Ciên- cia do Solo, 27, 1999, Brasília. Anais... Brasília: Embrapa Cer- rado, 1999. CD Rom. CONCLUSÕES NIOSH – National Institute of Occupational Safety and Health. Safety in swine production systems. North Carolina: Cooperative Extension Service Publications. n. PIH – 104, 1996. 1. A maravalha apresentou melhor eficiência na absorção das dejeções, sendo mais indicada, portanto, para compor as camas para a suinocultura. Oliveira, P. A. V. Produção de suínos em sistema “deep bedding”: experiência brasileira. In: Seminário Internacional de Suinocul- tura, 5, 2000, São Paulo. Anais... Concórdia: EMBRAPA/CNP- SA, 2000. p.101-107. 2. Tanto a cama de maravalha quanto a de casca de arroz apresentaram valores para a emissão de amônia abaixo dos limites estabelecidos por pesquisadores e organismos inter- nacionais. Paulo, R. M. Uso de camas sobrepostas durante as fases de cres- cimento e terminação de suínos em condições de verão. Viço- sa: UFV, 2003. 65p. Dissertação Mestrado 3. A utilização de camas sobrepostas na criação de suí- nos possibilita economia de água, uma vez que não há ne- cessidade de se lavar constantemente as baias para limpeza das dejeções produzidas pelos animais. Sampaio, C. A. P.; Nääs, I. A.; Salgado, D. D. Amônia, gás sulfí- drico, metano e monóxido de carbono na produção de suínos. Ciências Agroveterinárias, v.5, n.2, p.156 -164, 2006. 4. Ao final da criação, a cama fornece material orgâ- nico pronto para utilização nas plantações, em decorrên- cia do manejo do esterco seco e do acúmulo dos nutrien- tes N, P e K. Santos, M. A. A. Efeitos da ventilação natural em instalações de suínos em crescimento e terminação. Campinas: UNICAMP, 2001. 90p. Tese Doutorado R. Bras. Eng. Agríc. Ambiental, v.13, n.2, p.210–213, 2009.

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Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions

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RESEARCH ARTICLE Hiromitsu Ota1,2, Yumi Ito-Matsuoka1, Yasuhisa MatsuiID1,2,3* 1 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan, 2 The Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Chuo-ku, Tokyo, Japan, 3 Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan * [email protected] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miR- NAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/ Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogen- esis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 30-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in sper- matogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development. OPEN ACCESS OPEN ACCESS Citation: Ota H, Ito-Matsuoka Y, Matsui Y (2019) Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions. PLoS ONE 14(2): e0211739. https://doi.org/10.1371/journal. pone.0211739 Editor: Jean-Pierre Rouault, Centre de Recherche en Cancerologie de Lyon, FRANCE Received: October 4, 2018 Accepted: January 18, 2019 Published: February 1, 2019 Copyright: © 2019 Ota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Ota H, Ito-Matsuoka Y, Matsui Y (2019) Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions. PLoS ONE 14(2): e0211739. https://doi.org/10.1371/journal. pone.0211739 Editor: Jean-Pierre Rouault, Centre de Recherche en Cancerologie de Lyon, FRANCE Received: October 4, 2018 Accepted: January 18, 2019 Published: February 1, 2019 Copyright: © 2019 Ota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Ota H, Ito-Matsuoka Y, Matsui Y (2019) Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions. PLoS ONE 14(2): e0211739. https://doi.org/10.1371/journal. pone.0211739 Editor: Jean-Pierre Rouault, Centre de Recherche en Cancerologie de Lyon, FRANCE Copyright: © 2019 Ota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions Hiromitsu Ota1,2, Yumi Ito-Matsuoka1, Yasuhisa MatsuiID1,2,3* * [email protected] Introduction the Japan Agency for Medical Research and Development8https://www.amed.go.jp/en/index. html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Germ cells first arise as primordial germ cells (PGCs) in early embryos [1], and they proliferate and migrate into the genital ridges during embryonic development [2]. After arriving at the genital ridges, male germ cells enter G0 mitotic arrest and differentiate into prospermatogonia, which resume proliferation after birth [3, 4]. Subsequently, they further differentiate into sper- matogonial stem cells (SSCs), and a subpopulation of SSCs starts the first wave of spermato- genesis [5, 6]. Spermatogenesis is a highly complex differentiation process, during which gene expression is highly orchestrated and strictly regulated [7]. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. In addition to transcriptional regulation, post-transcriptional regulation also plays an important role during spermatogenesis. MicroRNAs (miRNAs) are small non-coding RNAs 18–23 nucleotides in length that play a critical role in the regulation of development and differ- entiation in many organisms and different tissues [8, 9]. For example, miR-125a modulates self-renewal of hematopoietic stem cells and protects them from apoptosis [10]. miR-1 and miR-206 inhibit cell proliferation and promote myoblast differentiation [11]. miR-29 has mul- tiple activities at different stages of osteoblast differentiation [12]. miRNAs repress gene expression by binding the 30-untranslated region (UTR) of their target mRNAs, thereby decreasing mRNA stability and translational efficiency [13]. Dicer1 encodes an RNase III endonuclease, which is a key enzyme for the biogenesis of miRNAs. Germ cell-specific Dicer1 knockout affects PGC proliferation, spermatogenesis, and fertility. For example, specific removal of Dicer1 in male germ cells with Ddx4-Cre results in deficient transition from the leptotene to zygotene stage in meiosis, increased apoptosis in the pachytene stage, and morphological defects in spermatozoa [14, 15]. Germ cell-specific knock- out mice of Drosha, which encodes an RNase III endonuclease that processes primary miRNAs to pre-miRNAs, with germ cell-specific Stra8-Cre also leads to progressive loss of pachytene spermatocytes and spermatids [16]. In addition, previous studies reported that various miR- NAs were expressed in germ cells and are involved in their development [17]. For instance, survival and/or proliferation [18–22], and differentiation [23–25] of SSCs are controlled by several miRNAs via inhibiting their target gene expression. miRNAs function in the spermatogenesis Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: H.O. was supported by a Grant-in-Aid (KAKENHI) for Young Scientists (B) (grant #JP16K20908) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp/en/index.htm). Y.M. was supported by KAKENHI in the Innovative Areas, “Mechanisms regulating gamete formation in animals” (grant #16H06530) from MEXT, and by AMED-CREST (grant #JP17gm0510017h) from 1 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Introduction However, miRNAs-mediated regu- lation of complex spermatogenic processes have not been fully understood. WNT/β-catenin signaling is a highly conserved pathway that is essential for embryonic development and cellular differentiation. For example, WNT5A stimulates the proliferation and self-renewal of hematopoietic stem cells in vitro [26], and WNT10B plays an important role in bone development [27]. In the WNT/β-catenin signaling pathway, WNTs bind to members of the Frizzled (FZD) family of receptors and form a stable ligand-receptor complex [28, 29]. This complex phosphorylates the intra-cellular Dishevelled protein and inhibits glyco- gen synthase kinase-3β, which induces ubiquitination and subsequent degradation of β-cate- nin via phosphorylation; thereby, WNTs reduce degradation of β-catenin [30]. Hence, cytoplasmic levels of β-catenin rise, and β-catenin translocates to the nucleus where it associ- ates with T-cell factor/lymphoid enhancer binding factor transcription factors to activate the expression of downstream genes [31, 32]. WNT/β-catenin signaling stimulates proliferation of SSCs but represses their differentiation potential [33–35], and is also involved in later stages of spermatogenesis [36–38]. WNT/β-catenin signaling is a highly conserved pathway that is essential for embryonic development and cellular differentiation. For example, WNT5A stimulates the proliferation and self-renewal of hematopoietic stem cells in vitro [26], and WNT10B plays an important role in bone development [27]. In the WNT/β-catenin signaling pathway, WNTs bind to members of the Frizzled (FZD) family of receptors and form a stable ligand-receptor complex [28, 29]. This complex phosphorylates the intra-cellular Dishevelled protein and inhibits glyco- gen synthase kinase-3β, which induces ubiquitination and subsequent degradation of β-cate- nin via phosphorylation; thereby, WNTs reduce degradation of β-catenin [30]. Hence, cytoplasmic levels of β-catenin rise, and β-catenin translocates to the nucleus where it associ- ates with T-cell factor/lymphoid enhancer binding factor transcription factors to activate the expression of downstream genes [31, 32]. WNT/β-catenin signaling stimulates proliferation of SSCs but represses their differentiation potential [33–35], and is also involved in later stages of spermatogenesis [36–38]. In this study, we identified germ cell-specific, X chromosome-linked miRNAs (XmiRs; miR- 741-3p, miR-871-3p, miR-880-3p). We generated knockout mice of these miRNA genes. The phenotype of the mice suggested that miR-871-3p and miR-880-3p were functionally redundant and that their deficiency caused spermatogenic failure by abnormally activating the WNT/β-cate- nin signaling pathway. In vitro studies by using germ stem cells (GSCs) revealed that WNT/β- catenin signaling was under the control of miR-871-3p and miR-880-3p to regulate GSC number. PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Construction of gRNA expression vectors and preparation of gRNAs and Cas9 mRNA Target sequences of gRNAs were selected by using a web program CRISPRdirect (https:// crispr.dbcls.jp/) [43]. DNA oligonucleotides, which have targeting sequences with BsaI cutting sites at 50end were synthesized by Fasmac Co., Ltd. (Atsugi, Japan) and were annealed. The annealed oligonucleotides were cloned into BsaI site of a gRNA expression vector pDR274 [44]. The oligonucleotide sequences for gRNA constructs were shown in S9 Table. gRNAs were transcribed from the DraI-digested gRNA expression vectors as templates by using MEGAshortscript kit (Invitrogen AM1354). The Cas9 mRNA was transcribed from SalI- digested Cas9 expression vector, pSP64-hCas9, by using mMESSAGE mMACHINE SP6 Tran- scription Kit (Invitrogen AM1340). Following completion of transcription, the samples were treated by DNase I according to the manufacturer’s instructions. Both the gRNA and the Cas9-encoding mRNA were purified by MEGA clear Transcription Clean-Up Kit (Invitrogen AM1908). Purified RNAs were concentrated by ethanol precipitation and re-dissolved in Opti-MEM I Reduced Serum Medium (Gibco 31985062). miRNA sequence analysis Small RNA sequence data using in this study are as follows [40–42]: GSE40499; Brain (SRR553582), Cerebellum (SRR553583), Heart (SRR553584), Kidney (SRR553585), Testes (SRR553586), GSE52950; ES cells (SRR1042095, SRR1042096, SRR1042097), MEF cells (SRR1042098, SRR1042099), GSE59254; PGC (SRR15097510), Spermatogonia (SRR1509750), Spermatozoa (SRR1509748). Cutadapt (Ver 1.8.3) was used to clip adapter sequences from raw small RNAs sequencing data. Prinseq (Ver. 0.20.4) was then used to filter low quality reads out from clipped reads. The processed reads were aligned to mouse-genomic reference (mm10) or miRBase 21 reference by using aligner program Bowtie (Ver 1.1.2). Heatmaps were built using the ‘regHeatmap’ function of the ‘Heatplus’ package of R. Animals MCH and B6D2F1 (C57BL/6 x DBA2 F1) were purchased from CLEA Japan Inc. and Japan SLC., respectively. Oct4-deltaPE-GFP [39] transgenic mice were maintained in a C57BL/6J genetic background. All animal care and experiments were carried out in according to the guidelines for experimental animals defined by the facility, the Animal Unit of the Institute of Development, Aging and Cancer (Tohoku University). Animal protocols were reviewed and approved by the Tohoku University Animal Studies Committee. Introduction 2 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis Generating XmiR-deficient mice by genome editing Fertilized eggs were collected from B6D2F1 females mated with Oct4-deltaPE-GFP transgenic male mice in mWM medium (ARK Resource), and were washed with Opti-MEM I Reduced Serum Medium (Gibco 31985062) three times. Eggs were then subjected to electroporation according to a described method [45]. Eggs were aligned in a line in an electrode chamber filled with Opti-MEM I Reduced Serum Medium containing 400 ng of Cas9 mRNA and 200 ng of gRNA (total 5 μl). A condition of electroporation was at 30 V (3 msec ON + 97 msec OFF) × 7 times. After electroporation, the eggs were immediately collected from the electrode and washed with mWM medium three times, and then cultured overnight in mWM medium at 37˚C and 5% CO2 incubator. Two-cell embryo were collected and transplanted to pseudo- pregnant MCH mice. 3 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis Histological examination Testes were fixed in Bouin at 4˚C overnight. The fixed testes were dehydrated in a graded series of ethanol (70% to 100%), cleared in xylene, and embedded in paraffin wax. Embedded tissue samples were sectioned at a thickness of 5 μm and then mounted on slides. These sec- tions were deparaffinized with xylene two times, rehydrated with a graded series of ethanol (100% to 70%) and distilled water, and then stained with hematoxylin for 10 min. Sections were rinsed with water for 20 min, stained with 1% eosin for 5min, and then rinsed with water briefly. These sections were dehydrated with a graded series of ethanol (70% to100%) followed by xylene two times and mounted with Permount (Falma). Diameter of seminiferous tubules that were round or nearly round were measured by using Image-J software. Prediction of target genes of XmiRs Candidate target mRNAs of each XmiR and of neighboring miRNA were predicted by the three web programs (miRDB (http://www.mirdb.org), TargetScan (http://www.targetscan. org), and microT-CDS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r= microT_CDS/)) based on complementarity of sequences between miRNAs and 3’-UTR of mRNAs[46–48]. Candidate target mRNAs selected by at least one program were applied for further analysis of hierarchical clustering based on similarity of their target sequences by hclust in R program. To predict common target mRNAs of miR-871-3p and miR-880-3p, their target mRNAs predicted by at least the two programs were selected, and common mRNAs between target mRNAs of miR-871-3p and miR-880-3p were identified. PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Immunostaining Testes of WT and ΔXmiRs mice were fixed with 2% paraformaldehyde for overnight and embedded with Optimum Cutting Temperature (O.C.T.) compound (Sakura Finetek 4583). The embedded samples were sectioned using Cryostat CM1900 (Leica) with a section thick- ness of 10 μm. The sectioned samples were permeabilized and blocked in 5% bovine serum albumin (BSA) and 1% Triton X-100 in PBS for 1 h at room temperature. The sections were then incubated with the primary antibodies diluted by 1% BSA and 0.1% Triton X-100 in PBS overnight at 4˚C, and were incubated with the secondary antibodies in the same buffer with 1 μg/ml DAPI for 2 h at 4˚C. Samples were washed for 5 min × 3 by 0.1% Triton X-100 in PBS after the primary and the secondary antibody treatments. Samples were mounted with VEC- TASHIELD (VECTOR H-1000) and observed with confocal laser scan microscope TCS SP8 (Leica). The primary antibodies were: SCP3 (abcam ab15093, 1:100), PLZF (Santa Cruz Bio- technology sc-28319, 1:50), γH2AX (Upstate 05–636, 1:100), Fzd4 (abcam ab83042, 1:100) and β-catenin (Cell signaling technology #8480, 1:100). The secondary antibodies were Goat anti- mouse secondary antibody, Alexa Fluor 647 (Invitrogen A-21235 1:500), Goat anti-mouse sec- ondary antibody, Alexa Fluor 568 (Invitrogen A-11031 1:500), Goat anti-rabbit secondary antibody, Alexa Fluor 568 (Invitrogen A-11011 1:500) and Goat anti-rabbit secondary anti- body, Alexa Fluor 488 (Invitrogen A-11034 1:500). Images were acquired under a Leica TCS SP8 confocal microscope. For quantification of β-catenin expression, the Leica Application Suite X program (Leica microsystems, Buffalo Grove, IL, USA) was used for analyzing pixel intensity for β-catenin in a region of interest (ROI) after background subtraction. Mean of pixel intensities in nucleus or cytoplasm of a germ cell was normalized by that of one to three Leydig cells of the same fields. The definition of sub-stages of spermatocytes in prophase I was based on localized patterns of SCP3 [49]. 4 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis Luciferase reporter assay Full length 3’UTR of Fzd4 gene was amplified from mouse genomic DNA by PCR, cloned into an XhoI-NotI site downstream of a luciferase reporter gene in psiCHECK2 vector (Promega C8021). 1–927 or 927–2263 region of Fzd4 3’UTR were amplified from a cloned WT Fzd4 3’UTR. For Fzd4 3’UTR delta, a set of over-lapping oligo-DNA primers were designed around a target site of the miRNA, in which the target sequence itself is deleted, in Fzd4 3’UTR. Fragments of Fzd4 3’UTR up-stream and down-stream to the target site were amplified from a cloned WT Fzd4 3’UTR by using the target site primers and outer primers. PCR products were then annealed in the overlapping primer regions and amplified full length Fzd4 3’UTR in which the target sites were deleted, by using the outer primers. miR-871 and miR-880 genes were amplified from mouse genomic DNA by PCR, cloned into CSII-EF-MCS vector [50] by using In-Fusion cloning kit (Clontech 639648). A luciferase reporter vector and a miRNA expression vector were co-trans- fected in HEK293T human embryonic kidney cells. HEK293T cells were maintained in DMEM (Gibco 11965092) supplemented with 10% FBS. Luciferase activity was measured by using Dual- Luciferase Reporter Assay System (Promega E1910) 48 hour after transfection. The synthetic Renilla luciferase activity was normalized to synthetic firefly luciferase activity for each sample. Total RNA isolation, semi-quantitative RT-PCR of miRNAs and Quantitative RT-PCR Total RNA containing small RNAs was purifies by using the miRNeasy kit (Qiagen 217004), according to the manufacturer’s instructions. For semi-quantitative RT-PCR of miRNAs, total RNA was polyadenylated for 15 min at 37˚C in a 5 μl reaction mixture containing 2 U poly(A) polymerase (New England BioLabs M0276). 5 μl volume of polyadenylated RNA was then incubated at 65˚C for 5 minutes with 3 pmol oligo(dT)-RACE primer and 1μl 10 mM dNTPs (Roche 11969064001) in a reaction volume of 13 μl. Following addition of 200 U SuperScript III (Invitrogen 18080051), 1 μl RNasin Plus RNase Inhibitor (Promega N2611), 1 μl 0.1 M DTT and 4 μl 5X RT buffer, the reaction was incubated at 50˚C for 1 hour followed by 70˚C for 15 minutes. Semi-quantitative RT-PCR reaction was performed with Power SYB Green PCR Master Mix (Applied Biosystems 4367659) in a 20 μl reaction containing 5 pmol forward and reverse primer, 0.1 μl cDNA template and 10 μl Power SYBR Green PCR Master Mix and the cycling conditions; 50˚C for 2 min, 95˚C for 10 min; 40 cycles (95˚C, 15 sec; 60˚C, 1 min). The amplified products were separated on a 3% agarose gel and visualized by ethidium bro- mide staining. U6 snRNA was used as an internal control. For quantitative RT-PCR, 5 μl vol- ume of total RNA was incubated at 65˚C for 5 minutes with 0.3 μl of Random primers (Promega C118A) and 1μl 10 mM dNTPs (Roche 11969064001) in a reaction volume of 13 μl. Following addition of 200 U SuperScript III (Invitrogen 18080051), 1 μl RNasin Plus RNase Inhibitor (Promega N2611), 1 μl 0.1 M DTT and 4 μl 5X RT buffer, the reaction was incubated at 50˚C for 1 hour followed by 70˚C for 15 minutes. PCR reaction were performed with Power SYB Green PCR Master Mix (Applied Biosystems 4367659) in a 20 μl reaction containing 5 pmol forward and reverse primer, 0.1 μl cDNA template and 10 μl of Power SYBR Green PCR Master Mix. PCR signals were detected using CFX Connect (Bio-Rad) and the cycling condi- tions; 50˚C for 2 min, 95˚C for 10 min; 40 cycles (95˚C, 15 sec; 60˚C, 1 min). Arbp was used as an internal control. All primers used in this study were listed in S9 Table. miRNAs function in the spermatogenesis albuginea, testes were incubated for 25 min in 6 ml of Gey’s Balanced Salt Solution (GBSS; Sigma-Aldrich G9779) containing 1.2 mg/ml of Collagenase Type I (Sigma-Aldrich C0130) at 32˚C, and seminiferous tubules were dissociated. Interstitial cells were removed by filtration with a 40 μm Cell strainer (Falcon 352340). Seminiferous tubes retained on the filter were col- lected and incubated at 32˚C for 25 min in the same collagenase-containing buffer as that used for the first step. Cell aggregates were sheared gently by 10 times of pipetting with wide orifice plastic transfer pipet and filtered through 40 μm Cell strainer to remove cell clumps. Cells were washed with GBSS and then resuspended in GBSS containing 1% FBS. Two million cells were diluted in 2 ml of GBSS containing 1% FBS and stained with 5 μg/ml of Hoechst 33342 (Invi- trogen H3570) for 1 h at 32˚C. Cells were kept on ice and protected from light until sorting. Before sorting, 10 μl of propidium iodide (Becton-Dickinson 51-66211E) was added to the stained cells and filtered through 40 μm Cell strainer. Sorting was performed on a Becton- Dickinson FACSAria II cell sorter. Total RNA was purified from 1 x106 cells of round sperma- tids and 1 x 105 cells of other population in testes by using the miRNeasy kit (Qiagen 217004), according to the manufacturer’s instructions. Purification of stage-specific spermatogenic cells by FACS sorting Dissociation of testicular cells and subsequent staining was carried out based on a described method [51]. Testes were dissected from B6 mice at 10 to 12 weeks of age. After removing 5 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Identification of germ cell-specific miRNAs To identify germ cell-specific miRNAs, we compared the expression of miRNAs among somatic tissues, cell lines, and germ cells by using published small RNA-seq data of mouse [40–42]. The proportion of miRNAs to the total reads in germ cells was less than that in somatic tissues (S1 Table). In addition to miRNAs, other small RNAs, such as Piwi-interacting (pi) RNAs that maintain genomic quality were contained in the small RNA-seq data of germ cells [56]. Cluster analysis showed that the expression profiles of these miRNAs were classified into three groups, i.e., those highly expressed in somatic tissues or mouse embryonic fibro- blasts (MEFs), in spermatogenic cells, and in PGCs and embryonic stem (ES) cells (Fig 1A). Of the 20 most abundantly expressed miRNAs in PGCs, many miRNAs were also highly expressed in ES cells (Fig 1B and S2 Table). miR-741-3p, miR-871-3p, and miR-880-3p were highly expressed in PGCs and spermatogonia, but were rarely expressed in ES cells or somatic tissues. The results suggest that these miRNAs have germ cell-specific functions. Interestingly, these miRNA genes were located on the X chromosome in contiguity with other miRNA genes (Fig 1C). Their high expression in testes was confirmed with RT-qPCR (Fig 1D). We named these three miRNAs X chromosome-linked miRNAs (XmiRs). Overexpression of miR-871, miR-880 and stable β-catenin in GSCs Virus particles were collected by centrifuging the cultured medium at 2,330 × g for 30 minutes at 4˚C after incubating with PEG6000 solution [final 2.5% PEG6000 (Wako), 100 mM NaCl, 10 mM HEPES (pH 7.4)] overnight at 4˚C, and they were re-suspended in GS medium [54] (StemPro-34 SFM (Gibco 10640–019) supplemented with StemPro-34 Nutrient Supplement (Gibco 10641–025), 25 μg/ml insulin, 100 μg/ml transferrin, 60 μM putrescine (Sigma P5780), 30 nM sodium selenite (Sigma S9133), 6 mg/ml D-(+)-glucose (Gibco A24940), 30 μg/ml pyruvic acid (Gibco 11360070)), 1 μl/ml DL-lactic acid (Sigma 69785), 5 mg/ml bovine albumin (Sigma A3311), 2 mM L-glutamine, 50 μM 2-mercaptoethanol (Sigma M3148)), 1 x minimal essential medium (MEM) vitamin solution (Gibco 11120052), 1 x MEM nonessential amino acid solution (Gibco 11140050), 100μM ascorbic acid (Sigma A4544), 10 μg/ml d-biotin (Sigma B4639), 30 ng/ml β-estradiol, 60 ng/ml progesterone (Sigma P6149), 20 ng/ml mouse epidermal growth factor, 10 ng/ml human basic fibroblast growth 6 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis factor (Sigma F0291), 10 ng/ml recombinant rat glial cell line-derived neurotrophic factor (GDNF) and 1% fetal calf serum). GSCs (DBAGS) [54] were cultured in GS medium on MEF feeder cells. Infection of the lentivirus vectors to GSCs were carried out as described previously with some modifications. Lentivirus was infected to GS cells at 1 x 104 lentiviral particle/cell [55] and incu- bated at 37˚C with 5% CO2 overnight. The culture medium containing the transfection mixture was discarded and replaced with GS culture medium containing 500 ng/ml puromycin 16–24 h after infection. GSCs were collected by trypsin treatment at day 4, 6, 8 and 10 after the virus infec- tion and cell number were counted. GSCs were collected by trypsin treatment at day 8 and total RNA was purified by using the miRNeasy kit (Qiagen 217004), according to the manufacturer’s instructions. All primers used in this study were listed in S9 Table. Overexpression of miR-871, miR-880 and stable β-catenin in GSCs miR-871 and miR-880 genes were amplified from mouse genomic DNA by PCR, cloned into an AgeI-EcoRI site of pLKO1 vector [52]. For construction of stable β–catenin, amino acid substitu- tions of S33A, S37A, T41A, and S45A to prevent phosphorylation by GSK3 [53] were introduced. A set of over-lapping oligo-DNA primers were designed around the mutation site. Fragments of stable β–catenin up-stream and down-stream to the mutation site were amplified from mouse cDNA by using the target site primers (up-stream: Ctnnb1-AAAA-Rev, down-stream: Ctnnb1-AAAA-Fw) and outer primers (up-stream: Kozak-Ctnnb1-Fw-for-CS2-EF-MCS, down- stream: Ctnnb1-Rev-for-CS2-EF-MCS). An up-stream fragment of stable β–catenin was annealed to Ctnnb1 AAAA AS oligo, which is partially overlapped with Ctnnb1-AAAA-Rev and Ctnnb1-AAAA-Fw. It was then amplified with Kozak-Ctnnb1-Fw-for-CS2-EF-MCS primer, which results in an elongated up-stream fragment covering all mutation sites. The elongated up- stream fragment was then annealed with the downstream fragment in the overlapping regions, and full length stable β–catenin was amplified by using the outer primers (Kozak-Ctnnb1-Fw-for- CS2-EF-MCS and Ctnnb1-Rev-for-CS2-EF-MCS). PCR product was cloned into CSII-EF-MCS vector [50] by using In-Fusion cloning kit (Clontech 639648). Lentivirus particles were produced as described previously. Briefly, pLKO1 (miR-971, miR-880)- or CSII-EF-MCS (stable-β–catenin)- lentivirus vector, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp [50] were co-transfected into HEK293T cells by the calcium phosphate method. After 48 hours, cell supernatant containing lenti- viral particles was collected, and virus concentration was determined by using Lenti-X qRT-PCR Titration Kit (Clontech 631235) according to the manufacturer’s instructions. PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis Fig 1. The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. P < 0.01. Fig 1. The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g001 few seminiferous tubules at 8 and 24 weeks of age (Fig 3E and 3F), suggesting that XmiRs act in a functionally redundant manner. To test this possibility, we examined target mRNAs of each XmiR and other miRNAs nearby shown in Fig 1C by using three web programs (miRDB (http://www.mirdb.org), Tar- getScan (http://www.targetscan.org), and microT-CDS (http://diana.imis.athena-innovation. gr/DianaTools/index.php?r=microT_CDS/)) that predict targets of miRNAs by evaluating each potential miRNA-target mRNA pair based on complementarity of a seed sequence and target sequences [46–48]. Hierarchical clustering analysis showed that the predicted target mRNAs of XmiRs converged on the same clusters (Fig 4A), and a portion of their target mRNAs actually overlapped (Fig 4B and S3 Table). To further investigate the functional redundancy of XmiRs, we generated mutant mice with a large deletion covering all three XmiR genes (ΔXmiRs) (Fig 4C and 4D). We confirmed that ΔXmiRs region did not contain any protein coding genes. We obtained three independent mutant mouse lines (OT84, OT87, and OT100), which showed identical testicular abnormali- ties. Expression of the XmiRs was not detected in the testes of a ΔXmiRs mouse with RT-PCR (Fig 4E). ΔXmiRs mice grew normally, and their fertility was indistinguishable from that of WT or heterozygous littermates (S4 Table). As in mice with deficiency of a single XmiR, testis weight was not affected but seminiferous tubules were thinner in ΔXmiRs compared with those in WT (S1C and S1D Fig). However, in the testes of ΔXmiRs mice, a few abnormal semi- niferous tubules were observed at 8 weeks of age, and the number of defective seminiferous tubules increased with age (Fig 4F and S5 Table). Similar to miR-871/miR-880 doubly mutated mice, fewer germ cells were present in the abnormal seminiferous tubules. In the severely affected seminiferous tubules, germ cells were rarely observed, while a few germ cells were found in the mildly affected tubules (Fig 4F, 30 weeks). To determine the spermatogenic stages affected in ΔXmiRs mice, we performed immunos- taining of testes of WT and ΔXmiRs mice using an antibody against Synaptonemal complex protein 3 (SCP3), a component of the synaptonemal complex [63], and an antibody against Promyelocytic leukemia zinc finger protein (PLZF), which is required for self-renewal of undifferentiated SSCs [64, 65]. Number of SSCs, leptotene, zygotene and pachytene spermato- cytes was not significantly changes in the mildly affected seminiferous tubules of ΔXmiR mice, but diplotene spermatocytes were decreased, and spermatids which are observed as cells with condensed nuclei following DAPI staining as well as spermatozoa were rarely found (Fig 5A and 5B). We also examined the expression of γH2AX (phosphorylated histone H2AX), and found dot-like signals likely representing localization on XY body in pachytene spermatocytes in ΔXmiRs as well as WT testes (S2 Fig). This result suggests that spermatogenesis was arrested in meiosis in the abnormal seminiferous tubules of ΔXmiR mice. Defective spermatogenesis in XmiR-deficient mice To examine the functions of XmiRs in germ cells, we generated knockout mice for each XmiR gene with the CRISPR/Cas9-mediated genome editing system [57]. gRNAs were designed in the Dicer processing site [58], seed sequence [59], and Drosha processing site [60] for miR- 741, miR-871, and miR-880, respectively. In the ΔmiR-741 mouse, a deletion occurred in the Dicer processing site that shortened the predicted terminal loop and stem. Drosha has a strong preference for pri-miRNA hairpin structures with a large (>10 nucleotides) terminal loop, suggesting that this deletion causes a severe defect in miR-741-3p production (Fig 2A) [61]. In the ΔmiR-871 mouse, the seed sequence was completely deleted, suggesting that miR-871-3p cannot recognize its target mRNA (Fig 2B) [59]. In the ΔmiR-880 mouse, the Drosha process- ing site was deleted, and the lower stem that binds to DGCR8 was shortened, suggesting that the ΔmiR-880 mouse cannot generate functional pre-miRNA (Fig 2C) [62]. Testis weight (S1A Fig) and spermatogenesis evaluated by histological analysis (Fig 3A–3D) revealed no differ- ences between wildtype (WT) and mutant testes in which a single XmiR gene was deficient, though seminiferous tubules were thinner in testes of ΔmiR-741 and ΔmiR-871 (S1B Fig). Among these XmiR gene-deficient mice, we obtained two mice (OT15 and OT124) in which miR-871 and miR-880 were doubly mutated by chance due to loss of the seed sequence in miR-871-3p and shortening of the hairpin structure of miR-880-3p due to deletion of the Drosha target site (Fig 2D and 2E). These two mice showed abnormal spermatogenesis in a PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 7 / 26 miRNAs function in the spermatogenesis PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 8 / 26 https://doi.org/10.1371/journal.pone.0211739.g001 Identification of target genes of XmiRs Because ΔXmiRs mice showed similar abnormalities as those in miR-871/miR-880 double mutant mice (Figs 3E and 3F and 4F), miR-871/miR-880 but not miR-741 are likely important for spermatogenesis. Therefore, we attempted to identify common target genes of miR-871-3p and miR-880-3p. We first selected putative target mRNAs of miR-871-3p and miR-880-3p by 9 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis miRNAs function in the spermatogenesis Fig 2. Predicted altered secondary structures of XmiR precursors by genome editing. Secondary structures of predicted miRNA precursors of (A) miR-741 WT (left) and ΔmiR-741 (OT16) (right), (B) miR-871 WT (left) and ΔmiR-871 (OT17) (right), (C) miR-880 WT (left) and ΔmiR-880 (OT49) (right), and (D, E) miR-871 (top) and miR-880 (bottom) in ΔmiR-871/ΔmiR-880 in two different mice, OT15 (D) and OT124 (E). Fig 2. Predicted altered secondary structures of XmiR precursors by genome editing. Secondary structures of predicted miRNA precursors of (A) miR-741 WT (left) and ΔmiR-741 (OT16) (right), (B) miR-871 WT (left) and ΔmiR-871 (OT17) (right), (C) miR-880 WT (left) and ΔmiR-880 (OT49) (right), and (D, E) miR-871 (top) and miR-880 (bottom) in ΔmiR-871/ΔmiR-880 in two different mice, OT15 (D) and OT124 (E). Fig 2. Predicted altered secondary structures of XmiR precursors by genome editing. Secondary structures of predicted miRNA precursors of (A) miR-741 WT (left) and ΔmiR-741 (OT16) (right), (B) miR-871 WT (left) and ΔmiR-871 (OT17) (right), (C) miR-880 WT (left) and ΔmiR-880 (OT49) (right), and (D, E) miR-871 (top) and miR-880 (bottom) in ΔmiR-871/ΔmiR-880 in two different mice, OT15 (D) and OT124 (E). https://doi.org/10.1371/journal.pone.0211739.g002 https://doi.org/10.1371/journal.pone.0211739.g002 https://doi.org/10.1371/journal.pone.0211739.g002 using the same web programs used for the analysis in Fig 4A, and chose mRNAs commonly selected by at least two of the three web programs as promising candidates. We consequently identified 46 common target mRNAs of miR-871-3p and miR-880-3p by comparing their tar- gets (S3 Fig and S6 Table). We next used RT-qPCR to examine the expression of those target genes in the testes of WT and ΔXmiR mice. Among the candidates, the expression of Fzd4, Mllt3, and Stox2 was signifi- cantly increased in the testes of ΔXmiR mice compared with that in WT testes, and Chdh and Magix also tended to be upregulated (Figs 6A and S4). Several studies have reported the contri- bution of the WNT/β-catenin signaling pathway to spermatogenesis [33–38]. Fzd4 encodes a WNT receptor. Because no previous reports have shown roles for Mllt3, Stox2, or Magix in spermatogenesis, and the only known function of Chdh is involvement in sperm motility [66], we focused on Fzd4 for further analysis. Fig 3. Spermatogenesis in mice with mutations in miR-741, miR-871, and miR-880. PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 10 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Hematoxylin-eosin (HE)- stained sections of seminiferous tubules in the testes of (A) WT, (B) ΔmiR-741 (OT16), (C) ΔmiR-871 (OT17), (D) ΔmiR-880 (OT49), and ΔmiR-871/ΔmiR-880 (E) (OT15) mice at 8 weeks of age, and (F) ΔmiR-871/ΔmiR-880 (OT124) mouse at 24 weeks of age. The arrowheads show abnormal seminiferous tubules. Scale bars = 100 μm. https://doi.org/10.1371/journal.pone.0211739.g003 Fig 3. Spermatogenesis in mice with mutations in miR-741, miR-871, and miR-880. Hematoxylin-eosin (HE)- stained sections of seminiferous tubules in the testes of (A) WT, (B) ΔmiR-741 (OT16), (C) ΔmiR-871 (OT17), (D) ΔmiR-880 (OT49), and ΔmiR-871/ΔmiR-880 (E) (OT15) mice at 8 weeks of age, and (F) ΔmiR-871/ΔmiR-880 (OT124) mouse at 24 weeks of age. The arrowheads show abnormal seminiferous tubules. Scale bars = 100 μm. https://doi.org/10.1371/journal.pone.0211739.g003 11 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Inhibition of luciferase (luc)-Fzd4-30-UTR reporter gene expression by XmiRs We performed a luc assay to investigate whether miR-871 and miR-880 directly repressed Fzd4 expression. To predict the miRNA target site, we used DIANA tools in the microT-CDS pro- gram (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/) [48], and found one and two candidate target sites for miR-871-3p and miR-880-3p, respectively (Fig 6C). Expression vectors for miR-871 or miR-880 were co-transfected with a luc reporter containing the Fzd4-30-UTR into HEK293T cells. miR-871 and miR-880 repressed luc activity (Fig 6D and 6E). We then used a luc reporter containing the Fzd4-30-UTR in which a miR- 871-3p target site was deleted (Fzd4-30-UTR ΔmiR-871). In this case, luc activity was not repressed by miR-871 (Fig 6D), indicating that miR-871-3p inhibited the expression of the reporter via its target site. Because the Fzd4-30-UTR has two candidate target sites for miR- 880-3p, we co-transfected miR-880 with luc reporters containing a part of the Fzd4-30-UTR that has each candidate target site. miR-880 significantly repressed luc reporters with each frag- ment of the Fzd4-30-UTR. However, miR-880 also repressed luc activity from reporter vectors with Fzd4-30-UTR fragments in which putative miR-880-3p target sites were deleted (Fzd4-30- UTR 1–927 ΔmiR-880; Fzd4-30-UTR 927–2263 ΔmiR-880) (Fig 6E). The results suggest that miR-880 targets other sequences in the Fzd4-30-UTR that were not predicted by the program. miRNAs function in the spermatogenesis 12 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis Fig 4. Relationship between target mRNAs of XmiRs and generation of ΔXmiRs mice. (A) A dendrogram of hierarchical clustering analysis of target mRNAs of XmiRs and their neighboring miRNAs. (B) Venn diagram showing the relationship among putative target mRNAs of miR-741-3p, miR-871-3p, and miR-880-3p. Corresponding gene lists are shown in S3 Table. (C) A schematic presentation of the WT and ΔXmiRs locus. gR-741 and gR-871 represent positions of guide RNAs used for genome editing. (D) Representative PCR for genotyping of WT and ΔXmiRs (OT84) mice. Arrows in the right panel represent primers used for PCR. (E) Expression of XmiRs in WT and a ΔXmiRs testes (F2 of OT84) determined with semi-quantitative RT-PCR analysis. U6 snRNA was used as an internal control. (F) HE-stained sections of seminiferous tubules in WT (left) and ΔXmiR (F2 of OT100) (right) testes at 8, 12, 16, and 30 weeks of age. The second and fourth panels for 30 weeks show higher magnification views corresponding to the rectangular area in the first and third panels. Lower two panels show mildly affected seminiferous tubules. Arrowheads show abnormal seminiferous tubules. Scale bar = 50 μm (8, 12, 16 weeks), 200 μm (30 weeks, lower magnification), 100 μm (30 weeks, higher magnification). https://doi.org/10.1371/journal.pone.0211739.g004 Because the expression of Fzd4 was increased in ΔXmiRs mouse testes, the expression of downstream genes of WNT/β-catenin signaling is also likely upregulated in ΔXmiRs mouse testes. To investigate this possibility, we used RT-qPCR to examine the expression of previ- ously reported possible downstream genes of WNT/β-catenin signaling in the testes of WT and ΔXmiR mice. Four of the five tested downstream genes were significantly increased, and c- Myc also tended to be upregulated in ΔXmiRs testes (Fig 6B). We also found that the expres- sion of β-catenin protein was increased both in cytoplasm and nucleus in some SSCs and sper- matocytes in ΔXmiRs testes compared with that in WT testes (S5 Fig). It suggests that β- catenin protein is stabilized to transmit signal in ΔXmiR testis. These results suggest that XmiRs repress WNT/β-catenin signaling via repression of Fzd4 expression. The expression of XmiRs and Fzd4 in spermatogenic cells To determine the stages of spermatogenic cells in which miR-871, miR-880, and Fzd4 are expressed, we purified testicular germ cells at different spermatogenic stages with fluorescence- activated cell sorting (FACS) (Fig 7A). We first confirmed the purity of the sorted germ cells by examining the expression of stage-specific germ cell marker genes (Fig 7B). Spermatogonia-spe- cific Gfra1 [67], spermatocyte-specific Scp3 [63] and Rad21l [68], and spermatid-specific Acrv1 [69] showed the expected expression levels in each cell fraction. The expression of Fzd4 mRNA increased from spermatogonia to the preleptotene-zygotene stage and then decreased from the pachytene stage onward (Fig 7C). Meanwhile, FZD4 protein was apparently upregulated in PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 13 / 26 miRNAs function in the spermatogenesis 0 1371/j l 0211739 F b 1 2019 14 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 14 / 26 miRNAs function in the spermatogenesis Fig 5. Spermatogenesis was arrested at meiotic prophase in abnormal seminiferous tubules in ΔXmiR testes. (A) Testis sections were co-stained with anti-SCP3 (green) and anti-PLZF (cyan) antibodies in WT and ΔXmiRs (F2 generation of the OT100 line) mice at 12 weeks of age. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. Mildly affected seminiferous tubules in ΔXmiR testis shown in Fig 4F are presented. Spermatogonia (yellow arrowheads), leptotene spermatocytes (white arrowheads), pachytene spermatocytes (white arrows), and round spermatids (yellow arrows) are indicated. Scale bars = 50 μm (the first and third columns) and 25 μm (the second, and fourth columns). (B) Number of cells at different spermatogenic stages determined by staining for Scp3 and Plzf in WT and mildly affected seminiferous tubules in ΔXmiR testis. Cells in two sections in a single mouse of WT and ΔXmiR testes were counted. Vertical lines in the graphs indicate means. P < 0.05 and P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g005 https://doi.org/10.1371/journal.pone.0211739.g005 pachytene spermatocytes (S6 Fig), suggesting its translational regulation by XmiRs. On the other hand, miR-871-3p was expressed from spermatogonia to leptotene-zygotene spermato- cytes, but its expression decreased after the pachytene stage onward (Fig 7D). miR-880-3p was expressed in spermatogonia, and its expression decreased at the onset of meiosis (Fig 7E). miR- 871-3p and miR-880-3p in spermatogenic cells likely play a role in adjusting Fzd4 expression levels and subsequent WNT/β-catenin signaling levels that are suitable for proper development of spermatogonia and spermatocytes. Overexpression of XmiRs represses growth and/or survival of germ stem cells (GSCs) To examine functions of XmiRs in SSCs, we overexpressed XmiRs in a GSC line in culture [54]. We confirmed that the expression of miR-871 and miR-880 was upregulated at 8 days after infection of lenti-virus vectors (Fig 8A). The expression of Fzd4 was repressed by overex- pression of miR-880 and/or miR-871, and those miRNAs additively repressed Fzd4 expression (Fig 8B). The number of GSCs was unchanged when expression vectors of miR-871-3p and/or miR-880-3p were infected, whereas GSCs increased in number with an empty vector (Fig 8C and 8D). The results suggest that excess miR-871-3p and miR-880-3p repress proliferation and/or survival of GSCs. We then tested whether WNT/β-catenin signaling was under the control of XmiRs in GSCs. We co-transfected an expression vector of stable β-catenin [53] with an expression vec- tor of miR-871 or miR-880. Stable β-catenin alone enhanced GSC number, indicating that WNT/ β-catenin signaling stimulates proliferation and/or survival of GSCs (Fig 8E and 8F). In addition, GSCs also increased in number by stable β-catenin with the expression of miR-871 or miR-880. Although decreased GSCs by miR-871 or miR-880 were not completely recovered by stable β-catenin, ratios of increased GSCs by stable β-catenin with miR-871 or miR-880 was higher compared with those with a control XmiR vector (S7 Table). It suggests that stable β- catenin partially rescues the influence of the XmiRs in GSCs, implying that WNT/β-catenin signaling functions under the control of XmiRs. Discussion In this study, we identified miR-741-3p, miR-871-3p, and miR-880-3p as being highly and preferentially expressed in germ cells. The genes for these three miRNAs are clustered on the X chromosome. In mice, 28.1% of miRNA genes form clusters on various chromosomes [70]. miRNA gene clusters may arise by de novo formation of miRNA-like hairpin structures in existing primary miRNA transcript units or by tandem duplication of a single miRNA gene [71]. XmiRs and additional miRNA gene clusters nearby may also be generated by similar molecular mechanisms. A single mRNA is likely targeted by multiple miRNAs due to the short length of seed sequences. Several studies proposed that miRNAs in the same gene cluster are often PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 15 / 26 miRNAs function in the spermatogenesis Fig 6. Upregulation of WNT/β-catenin signaling genes in testes of ΔXmiRs mice, and repression of Fzd4 by XmiRs. (A, B) Relative expression of the putative common target genes of miR-871-3p and miR-880-3p (A) and of the downstream genes of WNT/β-catenin signaling (B) in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. The expression in WT testis was set as 1. (C) Potential target sites of miR-871-3p and miR-880-3p in Fzd4-30-UTR. (D, E) Relative luciferase activity from the reporter vectors with Fzd4-30-UTR with or Fig 6. Upregulation of WNT/β-catenin signaling genes in testes of ΔXmiRs mice, and repression of Fzd4 by XmiRs. (A, B) Relative expression of the putative common target genes of miR-871-3p and miR-880-3p (A) and of the downstream genes of WNT/β-catenin signaling (B) in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. The expression in WT testis was set as 1. (C) Potential target sites of miR-871-3p and miR-880-3p in Fzd4-30-UTR. (D, E) Relative luciferase activity from the reporter vectors with Fzd4-30-UTR with or Fig 6. Upregulation of WNT/β-catenin signaling genes in testes of ΔXmiRs mice, and repression of Fzd4 by XmiRs. (A, B) Relative expression of the putative common target genes of miR-871-3p and miR-880-3p (A) and of the downstream genes of WNT/β-catenin signaling (B) in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. miRNAs function in the spermatogenesis without expression vectors for miR-871 (D) or miR-880 (E). Fzd4-30-UTR ΔmiR-871; Fzd4-30-UTR with deleted miR-871-3p target site (D). Fzd4-30-UTR 1–927 or 927–2263 with or without ΔmiR-880; 1–927 or 927–2263 bp fragment of Fzd4-30-UTR with or without deleted miR-880-3p target sites (E). Luciferase activity was measured 48 h after transfection. Luciferase activity with an empty expression vector was set as 1. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. without expression vectors for miR-871 (D) or miR-880 (E). Fzd4-30-UTR ΔmiR-871; Fzd4-30-UTR with deleted miR-871-3p target site (D). Fzd4-30-UTR 1–927 or 927–2263 with or without ΔmiR-880; 1–927 or 927–2263 bp fragment of Fzd4-30-UTR with or without deleted miR-880-3p target sites (E). Luciferase activity was measured 48 h after transfection. Luciferase activity with an empty expression vector was set as 1. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g006 functionally correlated, and those miRNAs exert cooperative and/or redundant repressive effects on the same target genes. For example, members of the miR-17-92 gene cluster are highly conserved among vertebrates, and cooperatively regulate transforming growth factor (TGF)-β signaling and cell cycle regulation; miR-17 and miR-20a directly target TGF-β recep- tor II mRNA, whereas miR-18a targets the mRNA of Smad2 and Smad4, two members of the TGF-β signaling pathway [72]. In addition, miR-17 and miR-20a cooperatively modulate the expression of E2F1 [73], a member of the E2F family of transcription factors that play a central Fig 7. Expression of XmiRs and Fzd4 in spermatogenic cells. (A) Scatter plot of FACS for testicular cells stained with Hoechst 33342. Spermatogonia (SPG), pre-leptotene (PreL), leptotene-zygotene (L/Z), pachytene-diplotene (P/D), round spermatids (RS). (B-E) Relative expression of stage-specific germ cell marker genes (B), Fzd4 (C), miR-871-3p (D), and miR-880-3p (E) in FACS-purified spermatogenic cells. Gfra1 for spermatogonia, Scp3 and Rad21l for spermatocytes, Acrv1 for spermatids. Gene expression was determined by RT-qPCR. Error bars represent standard errors of three biological replicates. P < 0.01. h //d i /10 13 1/j l 0211 39 00 Fig 7. Expression of XmiRs and Fzd4 in spermatogenic cells. (A) Scatter plot of FACS for testicular cells stained with Hoechst 33342. Spermatogonia (SPG), pre-leptotene (PreL), leptotene-zygotene (L/Z), pachytene-diplotene (P/D), round spermatids (RS). (B-E) Relative expression of stage-specific germ cell marker genes (B), Fzd4 (C), miR-871-3p (D), and miR-880-3p (E) in FACS-purified spermatogenic cells. without expression vectors for miR-871 (D) or miR-880 (E). Fzd4-30-UTR ΔmiR-871; Fzd4-30-UTR with deleted miR-871-3p target site (D). Fzd4-30-UTR 1–927 or 927–2263 with or without ΔmiR-880; 1–927 or 927–2263 bp fragment of Fzd4-30-UTR with or without deleted miR-880-3p target sites (E). Luciferase activity was measured 48 h after transfection. Luciferase activity with an empty expression vector was set as 1. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. Discussion The expression in WT testis was set as 1. (C) Potential target sites of miR-871-3p and miR-880-3p in Fzd4-30-UTR. (D, E) Relative luciferase activity from the reporter vectors with Fzd4-30-UTR with or PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 16 / 26 Gfra1 for spermatogonia, Scp3 and Rad21l for spermatocytes, Acrv1 for spermatids. Gene expression was determined by RT-qPCR. Error bars represent standard errors of three biological replicates. P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 17 / 26 miRNAs function in the spermatogenesis Fig 8. Roles of miR-871, miR-880 and stable β-catenin in GSCs. (A, B) Relative expression of miR-871 and miR-880 (A) and Fzd4 (B) in GSCs overexpressing XmiRs at 8 days after infection with the lenti-virus vectors. Gene expression was determined with RT-qPCR. (C, D) The effect of XmiRs overexpression in GSCs in culture. The pLKO1 empty vector was used as the control. Cell number at day 4 to day 10 after infection with the lenti-virus vector (C), and ratios of GSC number at day10 compared with that at day4 (D) are shown. (E, F) The effect of co-overexpression of stable β-catenin and miR-871 or miR-880. The pLKO1 empty vector and CSII-EF-MCS vector were used as the control. Cell number at day4 Fig 8. Roles of miR-871, miR-880 and stable β-catenin in GSCs. (A, B) Relative expression of miR-871 and miR-880 (A) and Fzd4 (B) in GSCs overexpressing XmiRs at 8 days after infection with the lenti-virus vectors. Gene expression was determined with RT-qPCR. (C, D) The effect of XmiRs overexpression in GSCs in culture. The pLKO1 empty vector was used as the control. Cell number at day 4 to day 10 after infection with the lenti-virus vector (C), and ratios of GSC number at day10 compared with that at day4 (D) are shown. (E, F) The effect of co-overexpression of stable β-catenin and miR-871 or miR-880. The pLKO1 empty vector and CSII-EF-MCS vector were used as the control. Cell number at day4 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 18 / 26 miRNAs function in the spermatogenesis to day10 after infection with the indicated lenti-virus vectors (E), and ratios of GSC number at day10 compared with that at day4 (F) are shown. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. to day10 after infection with the indicated lenti-virus vectors (E), and ratios of GSC number at day10 compared with that at day4 (F) are shown. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g008 role in the regulation of G1 to S phase progression [74]. miR-20a also targets both E2F2 and E2F3 [75]. In the case of XmiRs, we found that miR-871 and miR-880 had redundant functions in spermatogenesis via repression of the expression of a WNT signaling molecule, FZD4. ΔXmiR mice showed subtle abnormalities in spermatogenesis. Although the expression was upregulated in ΔXmiRs testes, the increase of Fzd4 expression was marginal (Fig 6A). The results together suggest that additional miRNAs redundantly target Fzd4. Consistent with this idea, we found other miRNAs in addition to XmiRs, which are predicted to target the Fzd4 30- UTR, among miRNAs expressed in testes (S7 Fig and S8 Table). WNT/β-catenin signaling is an important pathway involved in proliferation and differenti- ation of stem cells in many different tissue types including testicular germ cells. Regarding the WNT receptor gene in testis, Fzd2, Fzd3, Fzd7, and Fzd8 are expressed in spermatogonia [37], whereas we found that Fzd4 was expressed in spermatogonia, spermatocytes, and spermatids (Fig 7C), suggesting functions for FZD4 in those cells. Previous in vitro studies have shown that WNT3A and WNT5A promote proliferation of SSCs [33, 34]. Consistent with this obser- vation, conditional knockout of the β-catenin gene in testicular germ cells with Axin2-Cre sup- presses proliferation of undifferentiated spermatogonia [35], and we also consistently found that stable β-catenin enhanced GSC number in culture (Fig 8E and 8F). However, WNT/β- catenin signaling-activated SSCs show reduced SSC activity after transplantation into seminif- erous tubules [34]. Constitutive activation of β-catenin expression in testicular germ cells in transgenic mice causes progressive loss of spermatocytes and spermatids and reduction of mei- otic germ cells from the leptotene to pachytene stages [38]. Taken together, the results indicate that WNT/β-catenin signaling enhances proliferation of SSCs, but represses their differentiation. We showed that XmiRs targeted Fzd4 mRNA and downstream genes of WNT/β-catenin signaling were upregulated in ΔXmiR testes (Fig 6A and 6B) in which a few spermatocytes but no spermatids were observed in some seminiferous tubules (Fig 5). The results together sug- gest that deficiency in XmiRs results in abnormal enhancement of WNT/β-catenin signaling in spermatogenic cells, which may cause impaired meiosis. Whether or not X-linked miRNAs escape meiotic sex chromosome inactivation (MSCI) is controversial [76–78], but we found the expression of miR-871-3p was downregulated in pachytene/diplotene spermatocytes (Fig 7D), suggesting that it suffers silencing at least in some extent. Meanwhile, sex bodies associat- ing with MSCI was observed in remaining pachytene spermatocytes in ΔXmiR testes (S2 Fig), suggesting MSCI occurs in ΔXmiR spermatocytes. Although the testicular abnormalities in ΔXmiR mice were subtle, the mice showed progres- sively more severe testicular abnormalities with age (Fig 4F and S5 Table). PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 We found that XmiRs repressed GSCs to increase in number in culture, and repression of a WNT receptor, FZD4 and of possible additional targets by XmiR may be involved. The results together suggest that SSCs are maintained by the functions of miRNAs via various molecular pathways. Previous studies showed functions of miRNAs in SSCs. For instance, proliferation of SSCs is stimulated by miR-20 and miR-106 via repressing the expression of Ccnd1 (Cyclin D1) and Stat3 (signal transducer and activator of transcription 3) [18], and by miR-224 via repressing Dmrt1 (Doublesex and mab-3 related transcription factor 1) [19], while survival of SSCs is sup- ported by miR-146 via repressing Med1 (Mediator of RNA polymerase II transcription subunit 1) encoding a retinoic acid receptor associating protein [20]. In addition, miR-202 [24] and miR-221/222 [25] maintain SSCs in undifferentiated status via repressing Rbfox2 (RNA bind- ing fox-1 homolog 2) and unknown direct targets, respectively. We found that XmiRs repressed GSCs to increase in number in culture, and repression of a WNT receptor, FZD4 and of possible additional targets by XmiR may be involved. The results together suggest that SSCs are maintained by the functions of miRNAs via various molecular pathways. In the normal context, appropriate levels of WNT/β-catenin signaling molecules are crucial for spermatogonia and spermatocytes, and miR871-3p and miR-880-3p in those cells may con- tribute to fine tuning of the levels of WNT/β-catenin signaling activity via regulation of Fzd4 expression. The expression of Fzd4 mRNA was decreased at zygotene-pachytene transition in meiosis (Fig 7C), while FZD4 protein expression was upregulated in pachytene/diplotene sper- matocytes and round spermatids (S6 Fig). With regard to the expression of XmiRs, miR-880- 3p and miR-871-3p was downregulated in preleptotene and pachytene/diplotene spermato- cytes, respectively (Fig 7D and 7E). The results suggest that miR-880-3p is involved in de-stabi- lization of Fzd4 mRNA, while miR-871-3p may repress translation of Fzd4 mRNA. It is likely that Fzd4 mRNA starts to translate immediately after downregulation of miR-871-3p at zygo- tene-pachytene transition, and FZD4 protein may stably persist in pachytene spermatocytes onwards. Identification of downstream molecules of WNT/β-catenin signaling that directly function in spermatogenesis is an important subject for future studies. miRNAs function in the spermatogenesis We also found that overexpression of miR-871 and/or miR-880 in GSCs repressed the increase in number, and Fzd4 expression (Fig 8A–8D). In addition, stable β-catenin rescued decreased GSC number by miR-871 or miR-880 (Fig 8E and 8F and S7 Table). These results suggest that control of the expression levels of Fzd4 and subsequent WNT/β-catenin signaling activity by miR-871-3p and miR-880-3p are critical for proliferation and/or survival of GSCs. Meanwhile, repression of GSCs by XmiRs was not completely rescued by stable β-catenin (Fig 8E and 8F), suggesting that XmiRs repress the expression of additional targets other than WNT/β-catenin signaling related genes to regulate GSCs. In addition, it is also likely that XmiRs repress additional WNT/β-catenin signaling related genes other than Fzd4. According to this prediction, we found that Dixdc1 encoding Disheveled-2-associated protein [80] and Tbl1xr1 encoding β-catenin-associated protein [81] are potential targets of XmiRs (S6 Table). We also found that overexpression of miR-871 and/or miR-880 in GSCs repressed the increase in number, and Fzd4 expression (Fig 8A–8D). In addition, stable β-catenin rescued decreased GSC number by miR-871 or miR-880 (Fig 8E and 8F and S7 Table). These results suggest that control of the expression levels of Fzd4 and subsequent WNT/β-catenin signaling activity by miR-871-3p and miR-880-3p are critical for proliferation and/or survival of GSCs. Meanwhile, repression of GSCs by XmiRs was not completely rescued by stable β-catenin (Fig 8E and 8F), suggesting that XmiRs repress the expression of additional targets other than WNT/β-catenin signaling related genes to regulate GSCs. In addition, it is also likely that XmiRs repress additional WNT/β-catenin signaling related genes other than Fzd4. According to this prediction, we found that Dixdc1 encoding Disheveled-2-associated protein [80] and Tbl1xr1 encoding β-catenin-associated protein [81] are potential targets of XmiRs (S6 Table). Previous studies showed functions of miRNAs in SSCs. For instance, proliferation of SSCs is stimulated by miR-20 and miR-106 via repressing the expression of Ccnd1 (Cyclin D1) and Stat3 (signal transducer and activator of transcription 3) [18], and by miR-224 via repressing Dmrt1 (Doublesex and mab-3 related transcription factor 1) [19], while survival of SSCs is sup- ported by miR-146 via repressing Med1 (Mediator of RNA polymerase II transcription subunit 1) encoding a retinoic acid receptor associating protein [20]. In addition, miR-202 [24] and miR-221/222 [25] maintain SSCs in undifferentiated status via repressing Rbfox2 (RNA bind- ing fox-1 homolog 2) and unknown direct targets, respectively. Constitutive activa- tion of β-catenin in testicular germ cells in transgenic mice results in progressive loss of spermatogenic cells, in which fewer than 5% of seminiferous tubules are defective in spermato- genesis at 13 weeks of age. The ratio of defective tubules increases to more than 40% of total tubules at 75 weeks of age [38]. These data suggest that the influences of abnormal activation of WNT/β-catenin signaling by XmiR deficiency may gradually accumulate in some germ cells with aging. Those germ cells may undergo abnormal meiosis when the influences from acti- vated WNT/β-catenin signaling exceed a threshold. Functions of other miRNAs in meiosis in vivo have not been described well. miR-17-92 knockout mouse showed abnormal spermato- genesis with reduced number of sperm, though detailed mechanisms including its target mRNAs were not reported [79]. 19 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis of OT100) mice. Arrowheads show spermatocytes of the indicated stages. The fourth column shows higher magnification views corresponding to the rectangular area in the pictures in the third columns. Scale bars = 50 μm (the third columns), 25 μm (the first, second and fourth col- umns). (TIF) S3 Fig. Venn diagram showing the relationship of putative target mRNAs of miR-871-3p and miR-880-3p. Corresponding gene lists are shown in S6 Table. (TIF) S3 Fig. Venn diagram showing the relationship of putative target mRNAs of miR-871-3p and miR-880-3p. Corresponding gene lists are shown in S6 Table. (TIF) S4 Fig. The expression of the putative common target genes of miR-871-3p and miR-880- 3p in the testes of WT and ΔXmiRs mice. Relative expression of the putative common target genes of miR-871-3p and miR-880-3p in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. The expression in WT testis was set as 1. Error bars represent standard errors of three biological replicates. (TIF) S5 Fig. The expression of β-catenin in ΔXmiR testes. (A) Sections of WT and ΔXmiR (OT84) testes at 12 weeks of age were co-stained by anti- β-catenin (red) and anti-Plzf (cyan) antibod- ies. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. Arrows and arrowheads show Plzf-positive SSCs with intense and faint fluorescence, respectively, for β-catenin. Scale bars = 50 μm (the first, the third columns), 25 μm (second and fourth columns). (B) Quantita- tive estimation of the expression of β-catenin protein in Plzf-positive SSCs in WT and ΔXmiR testes. Relative signal intensity in nucleus and cytoplasm of SSCs compared with that in Leydig cells is shown. Four and eleven Plzf-positive cells in a single ΔXmiR and WT mouse, respec- tively, were measured. P < 0.01. (TIF) S6 Fig. The expression of FZD4 in WT testes. Testis sections were co-stained by anti-SCP3 (red) and anti-FZD4 (green) antibodies in WT. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. White arrowheads: leptotene spermatocytes, yellow arrowheads: zygotene spermato- cytes, white arrows: pachytene spermatocytes, yellow arrows: diplotene spermatocytes. Scale bars = 50 μm (the first, the third columns), 25 μm (second and fourth columns). (TIF) S6 Fig. The expression of FZD4 in WT testes. Testis sections were co-stained by anti-SCP3 (red) and anti-FZD4 (green) antibodies in WT. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. White arrowheads: leptotene spermatocytes, yellow arrowheads: zygotene spermato- cytes, white arrows: pachytene spermatocytes, yellow arrows: diplotene spermatocytes. Scale bars = 50 μm (the first, the third columns), 25 μm (second and fourth columns). Supporting information S1 Fig. Testis weight and diameter of seminiferous tubules of XmiR-deficient mouse. (A, C) Testis weight / body weight of ΔmiR-741 (OT16; n = 2), ΔmiR-871 (OT17; n = 2) and ΔmiR-880 (OT49; n = 2) (A), of ΔXmiRs (OT84; n = 6) (B), and of their wildtype littermates (n = 6 for A and n = 6 for C) at 8 weeks of age for A and 12 weeks of age for C. (B, D) Diameter of seminiferous tubules of ΔmiR-741 (OT16; n = 1), ΔmiR-871 (OT17; n = 1) and ΔmiR-880 (OT49; n = 1) (B), of ΔXmiRs (OT84; n = 3) (D), and of their wildtype littermates (n = 1 for B and n = 3 for D) at 8 weeks of age for B and 12 weeks of age for D. Fifteen seminiferous tubules in each section and three sections of each mouse were measured. P < 0.05 and P < 0.01. (TIF) S2 Fig. Localization of γH2AX in spermatocytes in ΔXmiR testes. Testis sections were co- stained by anti-SCP3 (red) and anti-γH2AX (cyan) antibodies in WT and ΔXmiRs (F2 generation 20 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Acknowledgments We would like to thank Dr. Takashi Shinohara for sharing GS cells, Asuka Takehara for main- tenance of mouse colonies, all the members of the Cell Resource Center for Biomedical Research for helpful discussions, the Center of Research Instruments in the Institute of Devel- opment, Aging, and Cancer and Biomedical Research Unit of Tohoku University Hospital for use of instruments and technical support. S6 Table. Lists of putative target mRNAs of miR-871-3p and miR-880-3p (corresponding to S3 Fig). (XLSX) S7 Table. Relative number of GSCs with the expression vector of stable β-catenin com- pared with those with control expression vector. Values for GSCs with miR-871, miR-880 and control miR expression vectors based on the data in Fig 8F are shown. (DOCX) S8 Table. miRNAs that are predicted to target the Fzd4 3’-UTR expressed in germ cells (corresponding to S7 Fig). Read counts of each miRNA normalized to reads per million (RPM) were shown. ES: embryonic stem cell, mouse embryonic fibroblasts (MEFs), PGCs: pri- mordial germ cells, SPG: spermatogonia, SPZ: spermatozoa. (DOCX) S9 Table. List of primers used in this study. (DOCX) S9 Table. List of primers used in this study. (DOCX) S3 Fig. Venn diagram showing the relationship of putative target mRNAs of miR-871-3p and miR-880-3p. Corresponding gene lists are shown in S6 Table. (TIF) (TIF) S7 Fig. A heat map of miRNAs highly expressed in testis or spermatogonia. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (TIF) S1 Table. Small RNA-seq data used for this study. ES: embryonic stem cells, MEFs: mouse embryonic fibroblasts, PGCs: primordial germ cells. (DOCX) S2 Table. Top 20 miRNAs highly expressed in PGCs (corresponding to Fig 1B). Read counts of each miRNA normalized to reads per million (RPM) were shown. miR-741-3p, miR- 871-3p, and miR-880-3p were highlighted by yellow. ES: embryonic stem cell, mouse embry- onic fibroblasts (MEFs), PGCs: primordial germ cells, SPG: spermatogonia, SPZ: spermatozoa. (DOCX) 21 / 26 PLOS ONE | https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis S3 Table. Lists of predicted target genes of miR-741-3p, miR-871-3p, and miR-880-3p (cor- responding to Fig 4B). (XLSX) S3 Table. Lists of predicted target genes of miR-741-3p, miR-871-3p, and miR-880-3p (cor- responding to Fig 4B). (XLSX) S4 Table. Fertility of ΔXmiRs mice. Three hemizygous ΔXmiRs F2 males of the OT100 line (#2, 4, 5) and their WT littermates (#1, 3, 6) were mated twice each with MCH females. Three homozygous ΔXmiRs F2 females of the OT84 line (#2, 3, 6) and their heterozygous littermates (#4, 10, 11) were mated once with Oct4-ΔPE-GFP transgenic males. The number of pups is shown. (DOCX) S4 Table. Fertility of ΔXmiRs mice. Three hemizygous ΔXmiRs F2 males of the OT100 line (#2, 4, 5) and their WT littermates (#1, 3, 6) were mated twice each with MCH females. Three homozygous ΔXmiRs F2 females of the OT84 line (#2, 3, 6) and their heterozygous littermates (#4, 10, 11) were mated once with Oct4-ΔPE-GFP transgenic males. The number of pups is shown. (DOCX) S5 Table. Ratios of abnormal seminiferous tubules. Ratios of abnormal seminiferous tubules (% of abnormal seminiferous tubules in total seminiferous tubules) in ΔXmiRs mice at 8, 12, 16, and 30 weeks of age. Abnormal seminiferous tubules were counted in three sections from each mouse. Testis sections were prepared from three WT mice and one mouse of each ΔXmiRs line (OT84, OT97, and OT100). ND: not determined. 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Developmental Considerations in Obsessive Compulsive Disorder: Comparing Pediatric and Adult-Onset Cases

Frontiers in psychiatry

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Developmental Considerations in Obsessive Compulsive Disorder: Comparing Pediatric and Adult-Onset Cases There appear to be two peaks of incidence of Obsessive Compulsive Disorder (OCD), one with a pre-adolescent onset and another in early adulthood. As new cases are added, the cumulative prevalence of OCD increases, but the great majority of cases have an onset in youth. The notion that early onset OCD represents a unique developmental subtype of the disorder has been considered by many researchers based on several specific age-related factors. Ascertainment and early intervention in affected youth is critical to abbreviate the functional impairments associated with untreated illness. In this paper we review the clinical, familial and translational biomarker correlates seen in early onset OCD that support the notion of a developmental subtype and discuss implications for research and treatment aimed at this cohort. The importance of cognitive, academic and social development tasks of childhood and adolescence, illness-specific and familial factors, and immune-mediated inflammatory factors are discussed, with their implications for management. *Correspondence: *Correspondence: Daniel A. Geller [email protected] *Correspondence: Daniel A. Geller [email protected] REVIEW published: 14 June 2021 doi: 10.3389/fpsyt.2021.678538 REVIEW published: 14 June 2021 doi: 10.3389/fpsyt.2021.678538 INTRODUCTION Specialty section: This article was submitted to Mood and Anxiety Disorders, a section of the journal Frontiers in Psychiatry For decades, clinical research has posited a developmental subtype of Obsessive Compulsive Disorder (OCD) that affects youth, and which may be distinct in important ways from the adult- onset form. Evidence for such a developmental subtype draws from multiple lines of observation and investigation at the clinical, translational and basic science levels. Despite this, the latest incarnation of the Diagnostic and Statistical Manual of the American Psychiatric Association, the DSM5 (1) does not specify a developmental subtype, but rather includes two different “specifiers” that apply particularly to children and adolescents. In this review, we will examine the differences between the early- or pediatric-onset form of OCD (these terms are used interchangeably) and the adult-onset form, including epidemiology, symptom presentations, clinical correlates, comorbid disorders, familial and genetic factors, environmental and epigenetic factors, salient neurocircuitry, treatment response, course and outcome. Many of these features are different in youth with OCD compared to adult OCD subjects. Received: 09 March 2021 Accepted: 17 May 2021 Published: 14 June 2021 Keywords: obsessive compulsive disorder, pediatric, child and adolescent, developmental, neuropsychology, immune, inflammation, neuroimaging Edited by: Odile Van Den Heuvel, VU University Medical Center, Netherlands Reviewed by: Reviewed by: Chaim Huyser, Independent Researcher, Amsterdam, Netherlands Luisa Lazaro, Hospital Clínic de Barcelona Spain y Chaim Huyser, Independent Researcher, Amsterdam, Netherlands Luisa Lazaro, Hospital Clínic de Barcelona, Spain Keywords: obsessive compulsive disorder, pediatric, child and adolescent, developmental, neuropsychology, immune, inflammation, neuroimaging *Correspondence: Daniel A. Geller [email protected] TABLE 1 | DSM5 OCD specifiers relevant to pediatric OCD. TABLE 1 | DSM5 OCD specifiers relevant to pediatric OCD. either be unable to articulate their intrusive thoughts, or not recognize them as irrational. Even in those with moderate insight, symptoms are often secretive and hidden due to embarrassment or shame. Sometimes impairments are seen only in more familiar home environments, may be domain specific, and may be masked in more public settings such as school. In addition, some symptoms, such as perfectionism, are unwittingly reinforced by parents and teachers based on good grades. Browne et al. (18) reported a cohort prevalence of 0.84% in a recent epidemiological study using the Danish health registry of more than one million youth. This prevalence is likely the most accurate to date and falls between the earlier estimates of 1–2% (16) and the 0.25% point prevalence reported by Heyman et al. (17). The prevalence of OCD in adult populations has been variously reported between 1 and 3% (19), but these figures may include some subthreshold cases. Although one would expect an increasing cumulative prevalence of cases (and therefore a higher cumulative prevalence) in adults as new cases are added to the affected population, the relatively higher rates of remission (10) (∼one third to one half remit or improve to subthreshold levels) of pediatric cases offsets the new adult incidence so that the overall prevalence does not change much over time. The prevalence of both adult (20) and pediatric OCD (18) has been noted to be remarkedly consistent across different counties and continents. With good or fair insight: The individual recognizes that obsessive-compulsive disorder beliefs are definitely or probably not true or that they may or may not be true. With poor insight: The individual thinks obsessive-compulsive disorder beliefs are probably true. With absent insight/delusional beliefs: The individual is completely convinced that obsessive-compulsive disorder beliefs are true. of concurrent psychopathology (2) and neuropsychological function (5, 6). Familial loading (7–9), and the role of the family (10) are amplified in pediatric cases, and youth may be susceptible to environmental triggers that are notably present in the early years (11, 12), and may display unique biosignatures (13). Outcomes are often more favorable for pediatric OCD and treatment response is robust and more durable (10). Clinical Features Age at Onset It is notable that there are two peaks of incidence (new onsets) of OCD, one early peak with a mean age of 9 to 10 (with an SD of ± 2.5 years) years of age and by definition, pre- pubertal, so that two thirds of affected youth will have an onset between about 7 and 12 years of age and well-before adulthood (21). There is a second peak of incidence in the early 20’s (see Figure 1) (22, 23), but, because of cumulative prevalence as new cases are added to the affected population starting with the early peak, two thirds of current adult cases have their onset before adulthood, a potentially confusing finding (24). TABLE 1 | DSM5 OCD specifiers relevant to pediatric OCD. The recognition and management of OCD in youth generally require specialist knowledge and care, not least because of the numerous and specific developmental tasks and milestones of early life that may be disrupted by illness, with potential long term adverse consequences (14). For this reason, OCD affecting youth and more especially, untreated or inadequately treated illness, is of particular concern to OCD clinicians and researchers. Although the effects of early intervention to mitigate long-term adverse outcomes has not been systematically studied, these considerations strongly support effective early intervention in youth affected by OCD (14). Definition of Early- or Pediatric-Onset OCD Pediatric OCD is defined as onset before age 18 years of age. The first comprehensive description of a case series was published in 1991 in (15) “The boy who couldn’t stop washing: The experience and treatment of obsessive compulsive disorder,” demonstrating that the recognition of this disorder in youth is relatively recent. Definition in DSM 5 While the core diagnostic features of OCD are the same across the lifespan, the DSM5 includes two “specifiers” especially relevant to pediatric cases (Table 1). Core symptoms include intrusive obsessions and compulsions (worries and rituals) that are time- consuming, distressing and functionally impairing, that are not better explained by the physiological effects of a substance or another mental disorder. As noted above, there is often a lag between age at ascertainment and age at onset due to under-recognition (25), a finding that may have clinical consequences. Because age at ascertainment generally lags behind onset by several years, onsets are acquired by anamnestic parental report and may therefore be inexact. We may look to other areas of medicine for an understanding of distinct peaks (but overlapping curves) of illness onset. In diabetes, the phenotype of high blood sugar, glucosuria and ketonuria are well-known, but it was only in the era of modern medicine that the distinction between type 1 diabetes (pancreatic insulin insufficiency resulting from auto-immune islet cell destruction) and type 2 diabetes (peripheral insulin resistance from persistent high circulating insulin in overweight subjects) was understood. Type 1 is known to affect younger people with a typical age at onset that is less than type 2, which more often affects older subjects related to insulin resistance secondary to obesity (26). Citation: Geller DA, Homayoun S and Johnson G (2021) Developmental Considerations in Obsessive Compulsive Disorder: Comparing Pediatric and Adult-Onset Cases. Front. Psychiatry 12:678538. doi: 10.3389/fpsyt.2021.678538 Children and adolescents generally display a pre-pubertal onset of their symptoms, some as young as 6 years of age, and may show a distinct symptom pattern (2–4) as well as distinct array June 2021 | Volume 12 | Article 678538 Frontiers in Psychiatry | www.frontiersin.org 1 Pediatric OCD as a Developmental Subtype Geller et al. TABLE 1 | DSM5 OCD specifiers relevant to pediatric OCD. Specify if: With good or fair insight: The individual recognizes that obsessive-compulsive disorder beliefs are definitely or probably not true or that they may or may not be true. With poor insight: The individual thinks obsessive-compulsive disorder beliefs are probably true. With absent insight/delusional beliefs: The individual is completely convinced that obsessive-compulsive disorder beliefs are true. Specify if: Tic-related: The individual has a current or past history of a tic disorder. Epidemiology OCD in children may go unrecognized for some time and in several of the epidemiological studies, youth identified with the disorder had usually not come to clinical attention or received a formal diagnosis (16, 17). Reasons for under-recognition include the limited verbal skills of younger subjects who may June 2021 | Volume 12 | Article 678538 Frontiers in Psychiatry | www.frontiersin.org 2 Geller et al. Pediatric OCD as a Developmental Subtype FIGURE 1 | A Bimodal distribution of incidence of OCD across the lifespan. *Geller et al. (22). **Rasmuseen et al. (23). FIGURE 1 | A Bimodal distribution of incidence of OCD across the lifespan. *Geller et al. (22). **Rasmuseen et al. (23). Primary Symptoms Normal Development However, the recent increase in childhood obesity in the Western world has led to higher incidence of type 2 diabetes in youth blurring these boundaries (27). Thus, similar phenotype does not imply a single etiology or set of genetic risk factors, even when some common pathophysiological pathways are involved. Typically, preschool-age children engage in ritualistic behavior, for example, routines at bedtime or mealtime, but these provide familiarity and comfort and are not disruptive. These are usually easily managed within normal family structure and are not disruptive to either child or family. However, for some children, insistence on routines or rules shows a high degree of inflexibility and rigidity and when not complied with, can lead to behavioral outbursts and disrupted family function. It has been reported however that these children’s insistence on excessive rituals may be a red flag and indicator of risk for OCD in later childhood (32). Magical thinking, or the belief that a person’s thoughts or actions can somehow influence real outcomes even though there is no causal connection between these, reflects normal development in very young children. This ego-centric world view ascribes influence to (irrational) rituals that are often performed to avert feared bad or unsafe outcomes. Normal cognitive development is associated with more reality-based apprehension of causality by the time most children show an onset of OCD (age 8 years and older), so that persistent magical thinking at this age is not normal. It should be noted that magical thinking can persist and be seen in adults, but there is some overlap with extreme superstitions or those that are heavily culturally based. Collecting and saving of personally meaningful items such as sports cards, coins, stamps, comic books etc., is also normal Frontiers in Psychiatry | www.frontiersin.org Pediatric OCD Symptoms In addition to a diminished capacity to articulate their concerns, some younger subjects may lack the ego function to recognize their obsessions as abnormal. On occasion, obsessions must be inferred by the parents who observe rituals in their children (33). inferred by the parents who observe rituals in their children (33). Obsessional anxiety frequently contains themes that reflect exaggerated developmental concerns at any given age, which may be difficult to dissociate from normal childhood development (22). For example, young children may struggle with increasing autonomy and independence, especially around separations from important parental figures, leading to intrusive fears of harm or loss of attachment figures. While this may appear as more typical separation anxiety, checking behaviors and magical rituals are also common (34). Recurrent worries about catastrophic family events or loss can also appear in youth with OCD with no premorbid history of separation anxiety disorder. Verbal checking and reassurance seeking often inadvertently engage parents in accommodation behaviors. Hoarding, saving and collecting rituals affect up to a quarter of youth with OCD (35) and are excessive, often with items that are unusual for the age, causing clutter and upset if discarded. Youth with hoarding also display other rituals and show increased rates of tic disorders. In adults, hoarding has typically been associated with poorer exposure and response prevention (ERP) outcomes but in a recent study, CBT treatment response in youth who hoarded was not adversely impacted (35). Symmetry and ordering, as well as “just-right” rituals have been reported to be more prevalent in pediatric OCD (36), and may reflect comorbidity with chronic tic disorders. Some children cannot articulate specific cognitions that drive rituals, reporting instead a vague feeling of unease or discomfort until certain actions are performed repeatedly. Without concrete cognitive obsessions, ERP may be less successful because habituation to vague but intense feelings of unease cannot utilize a cognitive strategy to “boss back” the urge to ritualize. For example, discomfort from the sensation that one’s hands are greasy may be more difficult to reason through than the idea that they may have touched an object with dangerous germs. While not a core symptom of OCD, children with this disorder also frequently display irritable behavior, that may in turn be a cause of greater impairment of function, especially in the domain of the family (41). Storch et al. Frontiers in Psychiatry | www.frontiersin.org Pediatric OCD Symptoms (41) also reported that profound irritability and tantrums led to more parental accommodation (in order to manage conflict), which is known to reinforce OCD behaviors. According to Guzick et al. (42), parents often report irritability in their affected children, driven by anxiety when frustrated by a need for perfection or certainty, or by an overestimated assessment of responsibility or threat. These findings in youth also conform to reports of greater distress, impairment and treatment resistance in OCD sufferers in the extant literature (43–46). Treatment protocols may benefit from taking the high levels of irritability into account when designing CBT interventions for some youth (47) requiring close work with families (48). Gender Ratio There is some uncertainty about this issue. Whilst earlier reports generally found a male predominance (25, 28), the more recent Danish epidemiological survey (18) reported a slight female preponderance in youth with OCD (29) similar that that generally reported in adult cohorts (30) Notably, common comorbid conditions that are frequently seen in younger OCD subjects, such as chronic tic disorders and Tourette’s syndrome, ADHD, and autism spectrum disorders (ASD) all show a clear male preponderance. The weight of evidence from clinical samples suggests that pediatric OCD does indeed represent a developmental subtype, with male preponderant cases with concurrent tics, ADHD and ASD-like psychopathology that often remit in adolescence and constitute one of the core hallmarks of such a subtype. Rates of OCD may also be much higher in transgender populations, with up to 9.8% prevalence estimated for transgender women and 7.6% for transgender men (31). June 2021 | Volume 12 | Article 678538 Frontiers in Psychiatry | www.frontiersin.org 3 Pediatric OCD as a Developmental Subtype Geller et al. behavior in youth and should not be confused with hoarding of items of little value such as bits of lint, old bottle tops or pieces of paper that lead to clutter and refusal to discard without family conflict. “category” and gender has not been reported to influence specific symptoms. Finally, while some OCD symptoms tend to persist, their relative presence and interference may change with time showing less stability over time than symptoms in adult patients (38). Factor or cluster analysis has often been used to better identify subtypes of “dimensions” of OCD (3) and there is a “dimensional” form of the Yale- Brown OCD Scale DY-BOCS) (39) but this approach has not shown any consistent benefit for genetic or translational approaches or yielded particular biological signatures. More recent network analysis of symptoms to identify meaningful symptom structures may however prove more useful for subtyping subjects for both treatment trials and further translational investigation (40). Role of the Family y Children are embedded in family units and not surprisingly, parents are often deeply engaged in behavior that accommodates their child’s distress that, by providing relief in the immediate moment, inadvertently reinforces the cycle of obsessions and compulsions (59–62). Verbal reassurance, engaging in back and forth verbal rituals, and performing actions that permit children to avoid feared stimuli are all quite common. Examples include opening doors, excessive laundering of “contaminated” personal items such as clothing or linen, or arranging meals in a highly ritualized fashion (63). Add to this the common occurrence of anxiety or even OCD in a parent, given the highly familial nature of this disorder (64), and the management can become complex. Family members, including siblings (65), therefore play a central role in both the maintenance of OCD symptoms and by extension, the effectiveness of CBT, in order to allow response prevention to occur. Scales to assess and quantify the degree of family accommodation (FA) such as the Family Accommodation Scale for OCD–Interviewer Rated [FAS-IR] (66), may be useful and can show decreasing scores that reflect improvement over time with standard CBT protocols. Some treatment intervention models such as the Pediatric Obsessive Compulsive Treatment Study for Young Children [POTS Jr] (67) specifically incorporate structured approaches for family involvement to address unhelpful accommodation. A recent rigorous randomized controlled multi-site trial found that impairments in social, home, and school life were significantly correlated with the degree of FA at baseline, but notably, in this study, baseline FA did not predict a poorer outcome over the course of treatment. Family accommodation decreased significantly with successful implementation of CBT and treatment response, with gains maintained at 6 months follow-up (68) in youth with OCD. Scalar FA scores fell by more than half over a 10 session CBT protocol to non-clinical levels (69). In this cohort, the relationship between severity of OCD symptoms and functional impairment was mediated by FA. In other words, greater involvement of family members was associated with worse OCD symptoms and worse illness- associated impairment (68). One of the more difficult diagnostic dilemmas occurs when there is some evidence of an autism spectrum disorder (ASD), which shows an infrequent but notable comorbidity with pediatric OCD (51) and is thus distinguished from adult OCD. Insight Insight in youth with OCD may be limited. Selles et al. (49) reported a meta-analysis of 573 children and adolescents enrolled in several North American and international CBT treatment trials and found that only 63% had good or excellent insight. Similarly, adults with the condition have been found to have poor/no insight in 13.8–30.7% of cases (50). The construct of insight may be difficult to measure quantitatively in youth with OCD as shown in a study that found no direct correlation between insight and treatment response, but at the same time, found that youth who had limited appreciation of the impairment from their OCD and greater avoidance behaviors, showed less likelihood of response to ERP (49), an apparent contradiction. Insight likely varies with anxiety levels and cognitive maturation, rather than being a static quantity. Studies of adults with OCD may be more informative regarding the adverse impact of insight on treatment. A number of adult studies (23–26) suggest a correlation between (poorer) insight and (poorer) outcome with standard of care treatment but insight at baseline, and changes in insight with CBT, show contradictory results (27, 28). One pediatric study (29) suggested that poorer treatment response correlated with less insight at baseline across all treatment modalities (30). Frank delusional beliefs and psychosis are very uncommon in pediatric OCD, although schizophreniform illness may first manifest as obsessional anxiety. Adolescents often experience tensions around sexual, moral and religious ideas and these thoughts are more often prevalent in the obsessional content of adolescent patients at an age in normal development when such concerns are more likely to cause anxiety or conflict (36). Scrupulosity is therefore seen more commonly in adolescents and young adults, leading to confessing and apologizing rituals (37). The role of religious authorities in management is to be considered for these patients. Most youth report contamination obsessions at some time, similar to adults, and consequently display washing, cleaning and avoidance rituals, but, similar to adults, the primary associated affect may be disgust (“gross”) rather than fear- based, for example, of germs or harm. Most will also demonstrate obsessions and compulsion from more than one June 2021 | Volume 12 | Article 678538 4 Pediatric OCD as a Developmental Subtype Geller et al. Frontiers in Psychiatry | www.frontiersin.org Comorbidity specifier, “with tics” is a clear acknowledgment of this with a preponderance of “just right” rituals that may be confused with complex tics (recurrent touching or tapping) (56). Furthermore, certain comorbid disorders have been shown to diminish treatment response, both to conventional CBT (57) and also to standard SSRI treatment (58). Geller et al. (58) also reported that relapse of OCD was more common following discontinuation of paroxetine in a placebo-controlled randomized withdrawal trial in those with more comorbid conditions. y All studies, including epidemiological studies of non-referred cases (16), find that most OCD-afflicted youth will have concurrent psychopathology, especially over time. Clinically referred and ascertained cases have even higher rates of comorbidity, as high as 80% (51). There is an ontogeny of comorbid conditions affecting those with pediatric OCD that is distinct for youth compared with adult-onset cases (51). This means that certain comorbid conditions arise at different and specific ages over time. As with adults, mood and other anxiety disorders are very common, but some frequently seen concurrent disorders are classically pediatric-onset disorders, such as Attention Deficit Hyperactivity Disorder (ADHD) and Tourette’s Disorder (TD), and predominate in pediatric cases. The majority of children with ADHD and tic disorders are male, and they typically have an earlier onset (2). If gender ratios averaged across all the pediatric years indeed show equal prevalence, as noted above, it would suggest that older affected youth trend toward a female preponderance with less comorbid ADHD and tic disorders. The triad of OCD, Tourette’s disorder and ADHD is not uncommon in pediatric cases, and reflects an underlying inhibitory deficit affecting thoughts (obsessions), motor behavior (tics) and attention (52). Poor inhibitory control may be identified by deficits in neuropsychological tests of inhibition in affected families (53). Neural maturation often brings improvement or remission of some of these symptoms with diminished tics and improved executive function (2). Comorbid mood and anxiety disorders occur at all ages and may persist into the adult years, sometimes becoming the main concern. As described above, youth with predominant irritable presentations may also meet criteria for oppositional defiant disorder (ODD) or even disruptive mood dysregulation disorder (ICD10 F34.81) (54). Role of the Family This overlap presents challenges for both diagnosis and treatment and has a major impact on treatment and educational interventions, role of the family, and outcome (55). Defining symptoms of ASD such as a restricted and narrow range of interests and activities, and stereotypic and repetitive behaviors can lead to confusion in younger children. It is estimated that perhaps 5% of OCD-affected youth also meet the diagnostic threshold for ASD (51). Helpful considerations are whether symptoms are subjectively experienced as ego-dystonic (OCD) or ego-syntonic (ASD), whether anxiety drives rituals (OCD) or occurs when rituals are impeded (ASD), whether rituals are resisted (OCD) or preferred, self-stimulating, and providing gratification (ASD). When classical OCD symptoms such as washing or checking are present, OCD can be reasonably inferred. The impact of FA can be widely felt within families of both children and adults with OCD, although there tends to be less direct involement in rituals by relatives of adult patients compared to parents of children with the condition (70). Storch et al. (71) reported that family members often took over The presence of comorbid disorders may speak to a developmental subtype of OCD with conditions unique to pediatric cases, but also has relevance to phenotype, treatment and outcome (see Treatments section below). The DSM 5 June 2021 | Volume 12 | Article 678538 5 Pediatric OCD as a Developmental Subtype Geller et al. Adverse Perinatal Risk Factors Lensi et al. (87) reported that boys with OCD had elevated rates of adverse perinatal events, such as breech birth or low Apgar scores suggesting hypoxia. Geller et al. (88) compared perinatal history between 130 youths with OCD to 49 matched controls ascertained in a family genetic study and found maternal pregnancy histories of illness that needed medical attention, (x2 = 8.61, p = 0.003), and higher rates of labor difficulties such as induction, forceps, or prolonged labor (x2 = 7.51, p = 0.006). There was a positive correlation between these adverse labor events and earlier age at onset of OCD, greater symptom severity, and presence of concurrent disorders including chronic tics, anxiety, depression and ADHD. These early clinical observations were supported by a large epidemiological study of more than 2.4 million singleton births using the Swedish birth registry over a 24 year period that identified over 17,000 cases of OCD. Genetics It should be clear from the above text that not all that is familial is also genetic. Disentangling familial environmental effects from genetic contribution requires segregation analyses of twin and family genetic studies, as well as the more recent genome-wide association studies (GWAS) (24, 64, 78). Overall, heritability estimates for OCD are in the range of 0.25–0.28 (78). However, when an index case is a child, that is, an pediatric case, the risk of OCD in a first-degree relative is approximately two-fold (79) and as high as 26% compared to about 12% risk in adult- onset cases (64). This means that a pediatric disorder is likely the result of a higher cumulative genetic loading of many genes of small effect. Recent GWAS studies have lent support to the notion that, like most psychiatric disorders, OCD is a polygenic disorder (80), with genes implicated in serotonin transmission (81) and glutamate pathways (9, 78, 82) at the very least. In a recent report from the cross-disorder group of the psychiatric genomics consortium (80) substantial pleiotropy of genetic loci was identified across eight psychiatric disorders. The strongest correlations with OCD were with anorexia nervosa and Tourette’s disorder (but not ADHD) (80). Copy number variants (CNVs), which describe large mega-base deletions or duplications have also been implicated (13). Genetic studies of pediatric cases have reported specific variants of genes coding for receptors in serotonin, glutamate and dopamine pathways, as well as transcription and neurotrophic factors (13, 83). Adverse Perinatal Risk Factors responsibilities of affected youth, while Peris et al. (61) reported that conflict within families increased with the degree of FA. The ability of youth to tolerate exposures delivered as part of CBT may also be diminished by high levels of FA (72) and the outcome of such treatment may be adversely affected (69, 71, 73, 74). For this reason, effective treatment often underscores the need to recognize and manage FA (51, 67, 75–77). Frontiers in Psychiatry | www.frontiersin.org Role of the Family After controlling for shared familial confounds, a number of adverse perinatal risk factors were associated with OCD including maternal smoking during pregnancy (HR, 1.20; 95% CI, 1.13– 1.28), breech presentation (HR, 1.26; 95% CI, 1.15–1.39), and delivery by cesarean section (HR, 1.09; 95% CI, 1.04–1.15) (11). Additionally, low and high birth weight (1,501–2,500,g and >4,500,g, respectively) were related to a slightly higher risk for OCD (LBW: HR, 1.10; 95% CI, 1–1.21; HBW: HR, 1.17; 95% CI, 1.07–1.27) (11). Such epidemiological approaches provide a powerful method for finding epigenetic triggers with very small effect sizes and, although non-specific, indicate that OCD could have antecedents long before the disorder appears, and during periods of vulnerable neural maturation. These findings are consistent with those of Vasconcelos et al. (89), whose research similarly showed a relationship between clinical expression of OCD and perinatal complications in adult cases of OCD compared to controls, including cesarean delivery (p = 0.005), prolonged labor (p < 0.001), and nuchal cord entanglement (p = 0.05), as well as other postnatal complications. In one study that examined perinatal complications among individuals with chronic tic disorders (age range 3–79 years), the authors found that pregnancy, delivery, and postnatal complications were associated with comorbid OCD (12). Psychosocial Stress Because monozygotic twins (with identical DNA) show at most a 50% concordance for OCD (84), it is clear that epigenetic factors (modification of gene expression without change in DNA sequence) and non-genetic factors are equally or even more important. Indeed, more than half of all new cases of new onset OCD occur without a positive first-degree family history of OCD, so called “sporadic” cases (85). While sporadic cases may still have a genetic cause due to a new mutation (86), the frequent appearance of non-familial cases has led to interest in epigenetic triggers and non-shared environmental factors (85), especially as their occurrence cannot be ascribed to an affected relative. Three areas of investigation of possible environmental etiological influences which may be especially relevant to pediatric OCD include studies documenting higher rates of perinatal injury, acute onsets following infection with presumptive immune and/or inflammatory processes, and life events experienced as traumatic. y Ironically, data that shows an association between traumatic life events and OCD affecting youth is extremely sparse, perhaps because definitive linkage is hard to establish. In contrast, the association between OCD and PTSD has been reported in numerous studies of adults with OCD (90) either with sequential or concurrent onsets, including in military veterans (91, 92). In one of the only pediatric studies to report on this potential association, Lafleur et al. (93) examined a cohort of 263 pediatric cases of OCD, finding child and parental reports of salient traumatic and stressful life events at higher rates than matched controls. Domestic violence, sexual or physical assault and forced home entry were some examples reported by youth and families and in some cases, the thematic content of obsessional fears and rituals mirrored the nature of the trauma (e.g., checking for safety repeatedly following a serious domestic assault on a parent) (93). It may be difficult to determine whether any Frontiers in Psychiatry | www.frontiersin.org June 2021 | Volume 12 | Article 678538 6 Pediatric OCD as a Developmental Subtype Geller et al. TABLE 2 | Comparison of pediatric and adult-onset OCD. Areas of Investigation Pediatric OCD Adult-onset OCD Prevalence 0.84% prevalence (1/3–1/2 remission rate) 1–3% prevalence Age at onset 9–10 (with an SD of ±2.5 years) 22–24 years Gender ratio F > M F > M OCD symptoms Children- Intrusive fears of harm or loss of attachment figures. Hoarding. Symmetry and ‘just right’ phenomena. Fewer concrete cognitive obsessions. Psychosocial Stress Adolescents- sexual, moral and religious themes, scrupulosity. Contamination fears. Contamination, more stable over time and across fewer categories of obsessions/compulsion types. Insight Limited- only 63% have good or excellent insight 13.8–30.7% have poor to no insight Comorbidity Up to 80%- Mood and anxiety conditions, ADHD, Tic disorders, ODD, DMDD, ASD (∼5%) Mood and anxiety disorders Family role Greater family involvement leads to worse OCD symptoms and greater functional impairment Family accommodation also seen in relatives of adult onset OCD but less direct involvement in rituals Genetics 26% risk of OCD in a first degree relative 12% risk of OCD in a first degree relative Adverse perinatal risk factors Increased rates, especially in boys with OCD Associated with an earlier age of OCD onset Psychosocial stress Increased rate of traumatic and stressful life events Association with PTSD Immune and inflammatory factors Possible association with GABHS infections. Link with humeral immunodeficiency Possible basal ganglia inflammation. Link with humeral immunodeficiency Neurocircuitry Similar to adult findings, possible increased assymetry of thalamus and palladium volumes and increase in total brain volume CSTC: OFC, ACC, striatum, thalamus Neuropsychological findings Deficits in working memory, visuospatial test performance and processing speed. Inconsistent- salient domains include attention, executive function, short-term memory and visuospatial function Treatment Response Complicated by prevalence and diversity of co-morbidities, and increased risk of behavioral activation and suicidal ideation accompanying SSRIs in youth SSRIs and CBT Course and Outcome Worse outcomes with co-morbid externalizing conditions and greater degrees of family accommodation. Overall higher rates of remission and symptoms becoming subclinical Few cases of full remission over time TABLE 2 | Comparison of pediatric and adult-onset OCD. Areas of Pediatric OCD 1–3% prevalence 22–24 years F > M Contamination, more stable over time and across fewer categories of obsessions/compulsion types. Inconsistent- salient domains include attention, executive function, short-term memory and visuospatial function SSRIs and CBT Few cases of full remission over time Worse outcomes with co-morbid externalizing conditions and greater degrees of family accommodation. Overall higher rates of remission and symptoms becoming subclinical (PANDAS). This hypothesis posits that an immune response to streptococcal infection may be a causative antecedent of OCD in some youth. Evidence to support such a hypothesis derives from observations of other neurobehavioral sequelae of group A strep, notably Sydenham’s chorea, implicating basal ganglia dysfunction (97). Psychosocial Stress Some level of confirmation for the etiological role of streptococcal infection was also derived from the OCD Genetics Association Collaborative (genome-wide association) Study (OCGAS GWAS), where high rates of infection were seen in pediatric OCD cases. Putative immune factors, for example, cross reactive anti-strep antibodies affecting circuits implicated in OCD and causing inflammation and dysfunction, are thought to cause a range of neurobehavioral symptoms including, but not limited to, OCD. Although over two decades have passed, the academic discussion about the validity of such an etiology remains highly controversial, because such antibodies have yet to be reliably demonstrated and other biomarkers have not been consistently identified. Diagnostic criteria include (1) OCD and/or a tic disorder; (2) prepubertal onset between 3 and 12 years of age, or Tanner stage I or II; (3) episodic course (abrupt given event reaches a threshold considered as “trauma” and further to demonstrate statistical significance across pediatric cohorts but for a subset of children, OCD will sometimes follow severe psychological stress. One interesting study illuminates how trauma may be translated into fear related behaviors at the molecular level. McGregor et al. (94) examined children exposed to trauma using the childhood trauma questionnaire (CTQ) and several polymorphisms in genes encoding mono-amine oxidase A and B (MAO-A, MAO-B) and catechol-O-methyl transferase (COMT). Gene by environment interactions suggested that these haplotypes “interacted” with childhood sexual trauma to increase risk for OCD in youth, providing a potential epigenetic mechanism of action for adverse psychosocial experiences (95). Few cases of full remission over time Immunity, Infection, and Inflammation (98) found an association between OCD/Tourette’s disorder and GABHS while others have refuted this finding (99, 100). Giedd et al. (101) described acute and transient structural abnormalities in the brains of some children with putative PANDAS but such findings have not been reproduced. Some suggest that transient increases in tics and OCD are well- known sequelae of many infections and other physiological stressors and not unique to GABHS (102). The search for specific culpable antibodies that co-localize to brain targets of interest has generally been unsuccessful. While anti-streptococcal antibodies can easily be measured in serum (103), presence of such antibodies or indeed other anti-neuronal antibodies, has not been consistently demonstrated. Two very recent studies demonstrated binding of antibodies from sera of children with putative PANDAS to cholinergic interneurons in the striatum with a subsequent alteration in their function, which represents the first such definitive finding but requires replication (104). In 2010, a scientific “white paper” consensus group at the NIMH child psychiatry branch suggested decoupling the acute onset of neuropsychiatric disorders in children from specific pathogens (GABHS) and expanded the clinical presentations to include avoidant/restrictive food intake disorder (ARFID) (120). Disordered eating behaviors have been described in a population-based prospective cohort study of over half a million young women identified thought the Danish longitudinal health register over a 6-year period (121). Anorexia nervosa, bulimia nervosa and eating disorder not otherwise specified was significantly more prevalent among females previously hospitalized for severe infections as well as those who had received anti-microbial treatment. This expanded constellation of clinical presentations was coined pediatric acute onset neuropsychiatric disorders or PANS. There are advantages to this approach (opening up research to other possible pathogens and pathogenetic mechanisms and perhaps expanding treatment options for a subset of affected youth), as well as disadvantages (linkage between infection and subsequent is inferred from acuity of onset and proximity to an infection but with no specificity). If PANDAS and PANS are conceived as variants of an autoimmune encephalitis, then the anti-N-methyl-d- aspartate (NMDA) receptor antibody mediated neurological disorder (122) may serve as a model. However, in PANS and PANDAS, the presumptive immune trigger is an exogenous infection of some kind, and it is the immune response that causes inflammatory-mediated neurobehavioral change, either through innate or adaptive immune responses, including cross reactivity of antibodies and cytokine activation. Immunity, Infection, and Inflammation Immunity, Infection, and Inflammation In 1997, Swedo et al. (96) described a group of children who developed OCD subsequent to infection with group A beta-hemolytic streptococcal (GABHS or strep) infection and introduced the hypothesis of pediatric autoimmune neuropsychiatric disorder associated with streptococcus June 2021 | Volume 12 | Article 678538 Frontiers in Psychiatry | www.frontiersin.org 7 Pediatric OCD as a Developmental Subtype Geller et al. onset and/or exacerbations); (4) symptom onset/exacerbation temporally linked to documented GABHS infections on two occasions; (5) association with neurological abnormalities (96). These criteria do not operationalize several important elements including the temporal duration between GABHS and onset, the data needed to definitively document GABHS, or the nature of neurological abnormalities (usually considered to be chorea or chorea-like movements or a loss of fine motor skills). The relevance for an pediatric subtype derives from the fact that nearly all youth are exposed to GABHS by early adolescence and develop antibodies. Therefore, new GABHS infections are far less common after puberty due to the heard immunity of the adolescent and their peers. question of whether this finding was true or simply a failed trial (115). Similarly, a recent prospective study failed to show exacerbation of tics following documented streptococcal infections (116). While the clinical studies have provided, at best, contradictory evidence, more convincing evidence comes from epidemiological studies that by definition, are retrospective and agnostic to any pre-existing notions of the validity of immune-mediated neuropsychiatric illness. Mataix-Cols et al. (117) examined the records of over seven million youth from the Swedish birth registry born between 1940 and 2007 (mostly before PANDAS had been described) and showed a significantly higher rate of autoimmune illnesses in families of those youth affected by OCD and Tourette’s disorder. Of course, correlation does not equal causation, and both may share underlying etio-pathological mechanisms, but the link between OCD and immune illness appears well-established and provides an important avenue for future research. Orlovska et al. (118) also analyzed the medical records of more than one million youth between birth and age 17 years from the Danish birth registry and found that all psychiatric disorders were over-represented in those with a history of both streptococcal and non-streptococcal. Of interest, tic and OCD showed the greatest increased risk among disorders studied (119). For all supporting studies there appear to be studies with conflicting findings. For example, Mell et al. Frontiers in Psychiatry | www.frontiersin.org NEUROIMAGING FINDINGS function in youth and throughout development may yield findings relevant to translational investigations in OCD. Structural and functioning imaging research over several decades have shown great concordance across studies regarding the underlying neurocircuitry of OCD (53). OCD is associated with abnormal findings in cortico-striato-thalamo-cortical (CSTC) circuitry which originate in the prefrontal cortex connect to the striatum, pallidum and thalamus and then loop back to cortical areas (124). Cortical areas consistently implicated using structural MRI include the orbitofrontal cortex (OFC), anterior cingulate cortex (ACC) and striatum (125) as well as thalamus (126). A finer grained understanding of the involvement of these regions is provided by fMRI studies that have identified specific roles of the dorsal ACC (dACC), medial and lateral OFC, and connections between amygdala and cortex (127, 128). In one study of 102 youth with OCD and matched controls, a standard battery of tests showed reductions in processing speed and timed visuospatial test performance (139, 140). Working memory evaluation showed a similar pattern with deficits in timed tests. Processing speed weaknesses may therefore be central to deficits in neuropsychological performance in youth. Notably, these were relative weaknesses, inasmuch as the overall scores remained within the “normal” range (5th-95th%) but these may have ecologically important consequences in academic settings (140). Similarly, in adults, deficits in non-verbal memory, planning, processing speed and inhibition has also been reported consistently (141). While a recent imaging study of youth with subclinical OC symptoms showed no morphological abnormalities at all, many reviews (129) of the neurocircuitry in adults and children with OCD show concordant abnormal findings (130–132). Confirmation derives from the recent ENIGMA Consortium meta-analysis of 16 pediatric and 30 adult OCD datasets that found cortical thinning in parietal regions in both age groups. However, some differences between age groups were also noted. The ENIGMA study reported asymmetries in thalamus and pallidum volumes in children that were not seen in the adult studies (133, 134). A recent structural MRI study of 2,551 youth enrolled in the Generation R study (135) showed a significant reduction in total brain volume in probable OCD cases compared to non-OCD healthy controls, and a significant increase in thalamic volume (126). While the same cortical thinning that was seen in ENIGMA was not demonstrated in this study, the mean age at imaging was much younger in the R Generation study so that developmental changes may account for discrepancies. NEUROIMAGING FINDINGS Along with earlier findings by Gilbert et al. (136) cumulative evidence has led to a focus on the thalamus as an area that may distinguish pediatric and adult OCD (126). In contrast, an fMRI study (137) reported involvement of the temporal poles during symptom provocation in children with OCD compared to matched healthy controls, rather than the CSTC loops generally reported. It is important to emphasize that morphology and circuitry will mature considerably throughout childhood and adolescence and at differing and variable rates (138) with implications for both imaging research as well as future non-invasive neuromodulation treatment protocols. Interest in the heritability of these deficits has been explored in some familial studies of neurocognitive performance, and have extended to cognitive flexibility and set shifting, decision making and visuospatial integration (142, 143). In a recent pediatric study of OCD youth and their first-degree unaffected relatives designed to examine neuropsychological endophenotypes, poorer proactive control and initial concept formation, seen in tests of set shifting and inhibitory control were found to be heritable (6). Immunity, Infection, and Inflammation One adult study (105) showed inflammation in nuclei of the basal ganglia in adults with OCD compared with controls, but neuroimaging data in putative PANDAS youth has been sparse. Biomarker studies in other psychiatric disorders have frequently been reported to show evidence of inflammation (106), so that this may not be unique to OCD. Indeed Fullana et al. (107) reviewed this literature and found none to be sufficiently sensitive or specific for OCD. Humoral immunodeficiency has also been linked to OCD onset in children (97) as well as psychiatric disorders and suicide in adults (108) perhaps suggesting increased risk for infections and subsequent pathogenic immune responses. Treatment studies represent another approach to validate the immune-mediated etiology using anti-microbials (109, 110), non-steroidal anti-inflammatory agents such as naproxen sodium (111) and cyclo-oxygenase inhibitors such as celecoxib (112, 113), but these studies involved several psychiatric disorders, suggesting a non-specific effect. However, one study reported an improvement in OCD symptoms specifically (114). Finally, direct immune modulation using intravenous immunoglobulin failed to demonstrate a benefit in a randomized placebo-controlled trial in OCD-affected PANDAS youth although several methodological limitations leave open the In summary, evidence is accumulating incrementally that a subset of cases of pediatric OCD are triggered by infections and mediated by immune and inflammatory processes (123). June 2021 | Volume 12 | Article 678538 8 Pediatric OCD as a Developmental Subtype Geller et al. TREATMENT RESPONSE The very high prevalence of comorbid psychiatric disorders associated with OCD in youth, in clinical and also non- referred epidemiological cohorts, present real challenges in treatment which are not seen in adult cases. For example, high rates of Tourette’s syndrome and chronic tic disorders as well as ADHD (144, 145) mean that a child’s OCD cannot be treated in isolation (146). While CBT is the first recommended intervention for all affected youth, it is notable that in the management of OCD, selective serotonin re-uptake inhibitors (SSRIs) (147) are considered first-line medication treatments. In contrast, ADHD responds best to stimulant medication approaches while tics are most often treated with either alpha agonist medications or dopamine blockers. In other words, pharmacotherapy approaches for each comorbid condition diverge markedly despite the frequent triad of these conditions. Add to this the increased risk of behavioral activation and suicidal ideation accompanying SSRIs in youth (148), the potential for increased anxiety, obsessions and tics with use of stimulants and the risk for adverse mood effects with alpha agonists, and the pharmacological approach in affected youth may require increased complexity compared to adult OCD cases. While CBT is clearly the treatment of choice for youth with OCD (147, 149, 150), more severe illness and concurrent psychopathology are indications for consideration of introduction of medication. Poor insight and low levels of family cohesion may also impede delivery of successful CBT. Youth with OCD who represent putative post-infectious, immune-mediated and/or inflammatory etiology have received a variety of anti- microbial and immune modulating treatments (109, 110, 115, Frontiers in Psychiatry | www.frontiersin.org NEUROPSYCHOLOGICAL FINDINGS Although many studies have examined neuropsychological test performance in OCD subjects, in both adults and in affected youth, the literature is rather inconsistent. Salient domains include attention, executive function, short-term memory and visuospatial function. Academic difficulties, seen frequently in youth with OCD, could simply reflect the intrusive effect of primary obsessions and high anxiety, or some deficits related to abnormalities in the CSTC not due to OCD at all (139). Efforts to identify significant performance deficits in neurocognitive Frontiers in Psychiatry | www.frontiersin.org June 2021 | Volume 12 | Article 678538 9 Pediatric OCD as a Developmental Subtype Geller et al. peaks lend credence to the notion of differing pathophysiological mechanisms rather than simply increased genetic loading leading to earlier onsets. Familial patterns, comorbid disorders, phenotypic presentations, etiologies, neurocognitive findings, treatment and outcome are also different, and the many developmental factors that distinguish pediatric cases have been elucidated. Keeping in mind that development throughout the pediatric years is rapid and that accompanying neuronal maturation occurs with similar rapid synchrony, these factors consequently may greatly affect the presentation and research findings in affected youth and adults. Therefore, while there is substantial evidence to support the notion of a “developmental” pediatric subtype of OCD, clarification must await further translational and genetic studies. 151) but none have yet shown consistent efficacy that permits recommendation for routine use. COURSE AND OUTCOME Again, comorbid externalizing symptoms have been reported to affect quality of life ratings at baseline and also with treatment (152). Many researchers have suggested that poorer treatment outcomes may be due to greater levels of family accommodation (FA) and this is especially relevant to pediatric cases (69, 71, 73, 74). As well, concurrent psychopathology has been shown to reduce response rates, particularly for conditions prevalent in pediatric cases. For example, comorbid tic disorders and ADHD reduced response rates to 53 and 59%, respectively, in a randomized controlled trial of paroxetine (153), and relapse was also higher in comorbid cases. March et al. (154) found the same poor response in youth with OCD who had a comorbid tic disorder, again suggesting that this pediatric subtype may be distinct in important ways. ACKNOWLEDGMENTS In this review, we have detailed the many differences between pediatric or pediatric OCD and OCD that onsets in adults. These numerous distinctions are summarized in Table 2. Distinct age The authors wish to acknowledge the helpful contributions of Dr. Kyle Williams, Dr. Sarah O’Dor, and Dr. Ryan Jacoby. FUNDING This work was supported by the National Institute of Mental Health 1R01MH093402-01A1, NCT01404208: 2/2-D-cycloserine augmentation of CBT for pediatric OCD (PI DG); National Institute of Mental Health R01MH 079489: A genome wide association study of early onset Obsessive Compulsive Disorder (PI DG); and National Institute of Mental Health 1K08MH01481-01A: Juvenile onset Obsessive Compulsive Disorder as a meaningful developmental subtype (PI DG). This work was supported by the National Institute of Mental Health 1R01MH093402-01A1, NCT01404208: 2/2-D-cycloserine augmentation of CBT for pediatric OCD (PI DG); National Institute of Mental Health R01MH 079489: A genome wide association study of early onset Obsessive Compulsive Disorder (PI DG); and National Institute of Mental Health 1K08MH01481-01A: Juvenile onset Obsessive Compulsive Disorder as a meaningful developmental subtype (PI DG). AUTHOR CONTRIBUTIONS DG, GJ, and SH declare that they have contributed substantially to the content and production of this review and agree to be accountable for the content of this work. All authors contributed to the article and approved the submitted version. Remission rates of OCD in youth treated with CBT are fair with partial remission reported in 53% and full remission in 27%, but also with some risk of relapse (155, 156). Outcome in youth appear to be better than in adults with some children becoming subclinical or remitting entirely over time (10, 150), whereas only 16.9% of adults were shown to achieve full remission in a 5 year longitudinal study by Eisen et al. (157). The Nordic Long Term OCD Treatment Study (NordLOTS) that used a stepped treatment protocol showed 90% response and 73% rate of clinical remission at 3 year follow-up (150). Fatori et al. 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Quality of life in children and youth with obsessive compulsive disorder. J Child Adolesc Psychopharmacol. (2017) 28:104–110. doi: 10.1089/cap.2017.0091 Conflict of Interest: DG has received grant or research support from the Eunice Kennedy Shriver National Institute of Child Health and Human Development subcontract with Duke Clinical Research Center Pediatric Trials Network, the National Institute of Mental Health, Biohaven Pharmaceuticals, Boehringer Ingelheim, Eli Lilly and Co., Forest Pharmaceuticals, GlaxoSmithKline, the International OCD Foundation, Neurocrine Biosciences, Nuvelution Pharma, Peace of Mind Foundation, Pfizer, Solvay, Syneos Health, Teva Pharmaceutical Industries, Emalex, the OCD Foundation, and the Tourette Association of America.He has served as a consultant to the Arlington Youth Counseling Center. He has served on the editorial board of the Journal of the American Academy of Child and Adolescent Psychiatry, Comprehensive Psychiatry and Annals of Clinical Psychiatry. He has received honoraria from the Massachusetts Psychiatry Academy and the American Academy of Child and Adolescent Psychiatry. He has previously held stock options/ownership in Assurex Health, Revolution Clinics and CD Services of America. 153. Geller DA, Biederman J, Stewart SE, Mullin B, Martin A, Spencer T, et al. Which SSRI? a meta-analysis of pharmacotherapy trials in pediatric obsessive compulsive disorder. Am J Psychiatry. (2003) 160:1919– 28. doi: 10.1176/appi.ajp.160.11.1919 154. March J, Foa E, Gammon P, Chrisman A, Curry J, Fitzgerald D, et al. Cognitive-behavior therapy, sertraline, and their combination for children and adolescents with obsessive-compulsive disorder: the pediatric ocd treatment study (POTS) randomized controlled trial. JAMA. (2004) 292:1969–76. doi: 10.1001/jama.292.16.1969 155. Højgaard D, Hybel KA, Ivarsson T, Skarphedinsson G, Becker Nissen J, Weidle B, et al. One-year outcome for responders of cognitive-behavioral therapy for pediatric obsessive-compulsive disorder. J Am Acad Child Adolesc Psychiatry. (2017) 56:940–7. doi: 10.1016/j.jaac.2017.09.002 156. Mancebo MC, Boisseau CL, Garnaat SL, Eisen JL, Greenberg BD, Sibrava NJ, et al. Long-term course of pediatric obsessive-compulsive disorder: 3 years of prospective follow-up. Compr Psychiatry. (2014) 55:1498– 504. doi: 10.1016/j.comppsych.2014.04.010 The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 157. Frontiers in Psychiatry | www.frontiersin.org REFERENCES Eisen JL, Sibrava NJ, Boisseau CL, Mancebo MC, Stout RL, Pinto A, et al. Five-year course of obsessive-compulsive disorder: predictors of remission and relapse. J Clin Psychiatry. (2013) 74:233–9. doi: 10.4088/JCP.12m07657 Copyright © 2021 Geller, Homayoun and Johnson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 158. Fatori D, de Braganca Pereira CA, Asbahr FR, Requena G, Alvarenga PG, de Mathis MA, et al. Adaptive treatment strategies for children and adolescents with obsessive-compulsive disorder: a sequential multiple assignment randomized trial. J Anxiety Disord. (2018) 58:42–50. doi: 10.1016/j.janxdis.2018.07.002 June 2021 | Volume 12 | Article 678538 Frontiers in Psychiatry | www.frontiersin.org 15

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ELECTROMECHANICAL TRANSIENT PROCESSES DURING SUPPLY VOLTAGE CHANGING IN THE SYSTEM OF POLYMER INSULATION COVERING OF THE CURRENT-CARRYING CORE OF ULTRA HIGH VOLTAGE CABLES

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ELECTROMECHANICAL TRANSIENT PROCESSES DURING SUPPLY VOLTAGE CHANGING IN THE SYSTEM OF POLYMER INSULATION COVERING OF THE CURRENT-CARRYING CORE OF ULTRA HIGH VOLTAGE CABLES Aim. The article is devoted to the analysis of the electromechanical transient processes in a system of three frequency-controlled electric drives based on asynchronous motors that control current-carrying core motion, as well as to the study of the effect of such processes on the modes applying three-layer polymer insulation to the current-carrying core. Technique. The study was con- ducted based on the concepts of electromechanics, electromagnetic field theory, mathematical physics, mathematical modeling. Results. A mathematical model has been developed to analyze transients in an electromechanical system consisting of three fre- quency-controlled electric drives providing current-carrying core motion of ultra-high voltage cables in an inclined extrusion line. The coordination of the electromechanical parameters of the system drives has been carried out and the permissible changes in the supply voltage at the limiting mass while moving current-carrying core of ultra-high voltage cables with applied polymer insulation have been estimated. Scientific novelty. For the first time it is determined that with the limiting mass of the current- carrying core, the electromechanical system allows to stabilize the current-carrying core speed with the required accuracy at short-term decreases in the supply voltage by no more than 27 % of its amplitude value. It is also shown that this system is resis- tant to short-term increases in voltage by 32 % for 0.2 s. Practical significance. Using the developed model, it is possible to calcu- late the change in the configuration and speed of the slack current-carrying core when applying polymer insulation, depending on the specific mass of the current-carrying core per unit length, its tension at the bottom, the torque of the traction motor and the supply voltage to achieve stable operation of the system and accurate working of the set parameters. References 12, figures 7. Key words: electromechanical transient processes, ultra-high voltage cable, mathematical modeling, frequency-controlled electric drives, polymer insulation covering. Цель. Целью статьи является проведение анализа электромеханических переходных процессов в системе из трех частотно регулируемых электроприводов на базе асинхронных двигателей, которые управляют движением токопро- водящей жилы, а также исследование влияния таких процессов на режимы нанесения на жилу трехслойной поли- мерной изоляции. Методика. Для проведения исследований использовались положения электромеханики, теории электромагнитного поля, математической физики, математического моделирования. Результаты. Разработана математическая модель, позволяющая анализировать переходные процессы в электромеханической системе, со- стоящей из трех частотно регулируемых электроприводов, обеспечивающих движение токопроводящей жилы сверх- высоковольтного кабеля в наклонной экструзионной линии. ELECTROMECHANICAL TRANSIENT PROCESSES DURING SUPPLY VOLTAGE CHANGING IN THE SYSTEM OF POLYMER INSULATION COVERING OF THE CURRENT-CARRYING CORE OF ULTRA HIGH VOLTAGE CABLES Проведено согласование электромеханических параметров приводов системы и выполнена оценка допустимых изменений напряжения питающей сети при предельной массе движущейся жилы сверхвысоковольтного кабеля с нанесённой на нее полимерной изоляцией. Научная новизна. Впер- вые определено, что при предельной массе токопроводящей жилы электромеханическая система позволяет стабили- зировать скорость перемещения жилы с необходимой точностью при кратковременных уменьшениях питающего напряжения не более чем на 27 % от его амплитудного значения. Также показано, что данная система является ус- тойчивой к кратковременному увеличению напряжения на 32 % в течение 0,2 с. Практическое значение. Использова- ние разработанной модели позволяет рассчитывать изменение конфигурации и скорости движения провисающей токопроводящей жилы при нанесении на нее полимерной изоляции, в зависимости от удельной массы жилы на еди- ницу длины, ее натяжения в нижней точке, момента тягового электродвигателя и величины питающего напряже- ния для достижения стабильной работы системы и точной отработки заданных параметров. Библ. 12, рис. 7. Ключевые слова: электромеханические переходные процессы, сверхвысоковольтный кабель, математическое моде- лирование, частотно регулируемые электроприводы, нанесение полимерной изоляции. Introduction. The modern phase of technological development for applying polymer insulation to current- carrying cores of power-driven cables is characterized by the use of adjustable AC electric drives. These drives are made on the basis of asynchronous motors with frequency control, which have high dynamic and energy perform- ance. At the same time, when two or more adjustable drives are used in one system, the solution to the problem of their electrical and mechanical parameters coordination becomes much more complicated. When choosing the optimal structure of the control unit of the whole system, it becomes necessary to model complex electrodynamic processes, which now is most appropriate to implement using the Matlab/Simulink/Sim-PowerSystems package. system regimes for its stability, speed and other indicators [10]. When operating these drives systems, there is also the task of studying their operation stability when chang- ing the supply network parameters, which is especially important in case of maximum mechanical loads of the drives and their power supply from the power supply sys- tem with a limited installed capacity of the servicing sub- station. The use of computer modeling to solve this type of problems makes it possible to significantly reduce the material costs and timing of such systems design. This research provides an estimation of the operation stability of electromechanical system with vector control of frequency-controlled electric drives considering short- term changes in the supply network voltage. UDC 621.365.5 UDC 621.365.5 UDC 621.365.5 doi: 10.20998/2074-272X.2018.2.08 V.M. Zolotaryov, M.A. Shcherba, R.V. Belyanin, R.P. Mygushchenko, I.M. Korzhov V.M. Zolotaryov, M.A. Shcherba, R.V. Belyanin, R.P. Mygushchenko, I.M. Korzhov © V.M. Zolotaryov, M.A. Shcherba, R.V. Belyanin, R.P. Mygushchenko, I.M. Korzhov ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 ELECTROMECHANICAL TRANSIENT PROCESSES DURING SUPPLY VOLTAGE CHANGING IN THE SYSTEM OF POLYMER INSULATION COVERING OF THE CURRENT-CARRYING CORE OF ULTRA HIGH VOLTAGE CABLES The study is carried out to coordinate the electrome- chanical parameters of the system two drives and to eval- uate the permissible level of the voltage failure in the supply network at the limiting mass of the moving cable. The possibility of such technology implementation is provided by a special configuration of the inclined vul- canization pipe. The initial part of the pipe, where the current-carrying core polymer layers are still sufficiently liquid, is practically vertical. Then the pipe bends and in its final part, where the current-carrying core polymer layers are sufficiently hardened, becomes almost horizon- tal. The bending and cross-section of the pipe are selected from the conditions of inadmissibility of touching its in- ternal surface by polymeric layers of the current-carrying core with all changes in its cross-section, mass, polymeric layers thickness and linear displacement speed. The material is based on the computer modeling re- sults of electrical system that includes two induction mo- tors with vector control, using the scientific statements presented in [7, 11]. Synthesis of the virtual model uses the tools of the Matlab / Simulink computer modeling software package [8], which contains special blocks and demonstration samples dealing directly with the elements and systems of the automated electric drive. The princi- ples of constructing and exploring individual blocks of virtual models are presented in [6, 7], and the control sys- tems of electric drives in the monograph [5]. Description of the inclined line. The inclined ex- trusion line (Fig. 1,a) is in the form of a metal vulcaniza- tion pipe, inside of which extrusion and vulcanization (cross-linking) of a polyethylene insulation layer as well as two polymeric semiconductive layers are applied on the current-carrying core of high-voltage and ultrahigh voltage cables. In such a line, the insulation of aluminum and copper current-carrying cores of power cables is made with a cross section of 35-2000 mm2 for a voltage of 10-330 kV. The current-carrying core consists of many twisted and compacted current-carrying cores, which can be divided into 5-7 separately sealed and isolated sectors. A polymeric semiconductive layer 0.4-3 mm thick is ap- plied to the current-carrying core, on which an insulating layer of high-quality polyethylene with a thickness of up to 28 mm and another layer of semiconductive polyethyl- ene with a thickness of 0.4-3.5 mm are applied. ELECTROMECHANICAL TRANSIENT PROCESSES DURING SUPPLY VOLTAGE CHANGING IN THE SYSTEM OF POLYMER INSULATION COVERING OF THE CURRENT-CARRYING CORE OF ULTRA HIGH VOLTAGE CABLES It is known [10] that the vector control advantages are the high accu- racy of diagram optimization at a set speed, the preserva- g y p g This approach helps to investigate the laws of fre- quency regulation and determine the most appropriate 47 The principle of line operation, its scheme is shown in Fig. 1 as follows. The current-carrying core wounded on the feeding device cylinder, is passed through the elec- tric drive No. 1 through a triple extrusion head, into which polyethylene insulation melts and a semiconductive pol- ymer are simultaneously fed. The head has three extruders of different capacities: the first (with the highest capacity) for applying a polyethylene insulation layer, and the sec- ond for forming semiconductive polymer layers. tion of the necessary torque magnitude at low rotational speeds, the smooth operation of the motor and the rapid reaction to load jumps due to the high dynamics of regula- tion. At the same time, the quantitative analysis of the stability and accuracy of the specified parameters optimi- zation - motion speed and the torque shaft of the drive, are currently insufficiently studied. The aim of the paper is to analyze the electrome- chanical transients in a system consisting of three fre- quency-controlled electric drives based on asynchronous motors that control the current-carrying core motion, as well as the effect of such processes on the modes of ap- plying three-layer polymer insulation to the current- carrying core. In order for the liquid layer of molten polyethylene to be less displaced relative to the current-carrying core axis, a twisting mechanism is additionally applied. It twists the current-carrying core in the direction of its wires approximately at a pitch equal to one current- carrying core turn around its axis for 30 linear meters of its length. This makes it possible to obtain a cylindrical item with a crust of solidified polyethylene on its surface and avoid the displacement of the polymeric semiconduct- ing and insulating layers relative to the current-carrying core axis, i.e. avoid the polymer layers eccentricity. The paper studies such freelance regimes as the ap- pearance of short-term voltage failures in the supplying three-phase network and short-term increases in this volt- age. ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 ELECTROMECHANICAL TRANSIENT PROCESSES DURING SUPPLY VOLTAGE CHANGING IN THE SYSTEM OF POLYMER INSULATION COVERING OF THE CURRENT-CARRYING CORE OF ULTRA HIGH VOLTAGE CABLES All three layers are simultaneously applied by extrusion using a triple extrusion head and vulcanized in a vulcanization pipe of continuous vulcanization at 450 °C in a medium compressed to 16 atm. nitrogen in the gaseous state. This is necessary to ensure the insulation quality, namely to exclude the presence of conductive microcircuits larger than 50 μm and groups of closely located microinclusions [4, 12]. A current-carrying core with polymeric layers must move in the central part of the vulcanization pipe and can be considered as a heavy material thread. The angle  between the abscissa axis and the line connecting the be- ginning and the point with coordinates (x, y), as is known from mechanics, can be determined from the expression: H gx tg   , (1) (1) where g is the weight of heavy material thread per unit length and Н is the tension at the lowest point. Since the manufacture of insulated cable standards of different cross-sections and at different voltages the value of g varies, the profile of the sag thread also varies: 2 1 x c у  , (2) (2) where c = H/g is sag constant. where c = H/g is sag constant. where c = H/g is sag constant. From the equations given, it is clear that the sag con- stant c should be unchanged to keep the thread profile. Such profile invariance can be made by adjusting the ten- sion force H and correspondingly adjusting the motor shaft of the drive No. 2 (Fig. 1,b), driving the track-type traction device at a constant technological speed V of the current-carrying core motion in the vulcanization pipe. The invariance of the current-carrying core speed is en- sured by adjusting the torque of the traction motor. These relationships are the basis for the motion control system of the current-carrying core inside the vulcanization pipe, which bend is determined from the sagging equations of the current-carrying core as a material heavy thread. The cable current-carrying core with polyethylene insulation and semi-conductive shielding layers applied must move at a speed of 0.3-50 m/min inside the vulcani- zation pipe 172 m long. The motion is carried out as a result of the effort up to 4.5·104 N electric drive No. 1 (Fig. 1,b). 48 Electric drive No. 2 of track-type device 380 V, 50 Hz 380 V, 50 Hz v = 0.3-50 m/min Feeding device Power supply cable Receiving device Track-type device R=1,5 m 380 V, 50 Hz R=1,5 m Gear unit 1 speed ratio i=233.7 Electric drive No. 1 of feeding device Gear unit 2 speed ratio i=233.7 Electric drive No. 3 of receiving device а b Fig. 1. Inclined extrusion line of applying and vulcanizing a polyethylene insulation layer and two semiconductive layers on current-carrying core of high and ultrahigh voltage cables, a – photo of PJSC «Yuzhcable Works» and b – its block diagram Feeding device Power supply 380 V, 50 Hz R=1,5 m Gear unit 1 speed ratio i=233.7 Electric drive No. 1 of feeding device Power supply cable а v = 0.3-50 m/min Receiving device Track-type device Gear unit 1 speed ratio i=233.7 Electric drive No. 2 of track-type device Electric drive No. 1 of feeding device Electric drive No. 3 of receiving device Electric drive No. 3 of receiving device b Fig. 1. Inclined extrusion line of applying and vulcanizing a polyethylene insulation layer and two semiconductive layers on current-carrying core of high and ultrahigh voltage cables, a – photo of PJSC «Yuzhcable Works» and b – its block diagram Problem statement and the development of math- ematical model of electromechanical system. The elec- tromechanical system is studied. where c = H/g is sag constant. Based on the modeling results, it can be noted that the current in the stator of the receiving device's motor changes in amplitude and frequency during start-up, and at the initial area the frequency is low and gradually in- creases as the motor accelerates. It is this trigger mode that is characterized by low energy consumption. The motor is monotonously accelerated to a set speed of 1200 rpm for a time equal to 1.35 s and then accurately reproduces this predetermined rotation frequency in the subsequent time interval. The asynchronous machine model is used and con- sisted of an electrical part represented by a fourth-order state space model and a mechanical part model in the form of a second-order system. All electric variables and machine parameters were driven to the stator. The initial equations of the electrical part of the ma- chine are recorded for a two-phase coordinate system (the d-q axis) and have the form: ds qs qs s qs dt d i R V      , (3) qs ds ds s ds dt d i R V      , (4)  dr r qr qr r qr dt d i R V              , (5)  qr r dr dr r dr dt d i R V              , (6)   ds qs ds ds e i i T    5,1 , (7) where: qr m qs s qs i L i L     , dr m qs s ds i L i L     , (8, 9) qs m qr r qr i L i L       , ds m dr r dr i L i L      , (10, 11) m ls s L L L   , m lr r L L L     . (12, 13) (3) At the next stage of the research, dynamic processes in the drives were modeled with a short-time increase during 0.4 s of the supply voltage from an amplitude value of 380 V to various values up to 500 V. where c = H/g is sag constant. and rotor; Vqs, iqs and V 'qr, i'qr are the projections of the stator and rotor stresses and currents on the q axis; Vds, ids and V 'dr, i'dr are the projections of the stator and rotor voltages and currents on the d axis; φds, φqs and φ'dr, φ'qr, are the projections of the stator and rotor flux linkages on the d and q axes;  is the angular velocity of the rotor; θ is the angular velocity of the rotor; J is the rotor inertia torque; Te is the motor electromagnetic torque; Tm is the static load moment; F is the friction coefficient. This mathematical model has become the basis for the one developed in the Simulink and for the virtual model of the asynchronous machine used in this paper. A number of parameters were calculated from the machine's passport data on the basis of the method described in [6]. At that, the asyn- chronous motor RA160MA4 (11 kW, 1460 rpm) was used in the drive’s receiving device, and RA132S2 (5.5 kW, 1455 rpm) was used in the drive of the tracked chassis. Each of the two drive units, as in [7], model the elec- tric drive operation based on an induction motor with vec- tor control. This model contains an uncontrolled three- phase rectifier, a three-phase inverter with pulse-width modulated current (PWM), an asynchronous motor, a speed controller and an inverter control unit. In order to avoid overvoltage at the rectifier output when the motor is switched on in the electric power generation mode, a spe- cial chopper block is located between the rectifier and the inverter, which provides the connection of a resistor shunting the storage capacitance when the voltage across the set value exceeds it. The block diagram of the drive realized by the DTC method is given in [2, 3, 8]. The analysis of modeling results. Fig. 3 shows the timing diagrams of the main characteristics of the drive’s receiving device for the studied time period – 2 s, corre- sponding to the start-up mode. The diagrams are in good agreement with the results given in [7]. The Fig. 3 shows the time-dependent current of the stator motor winding, the rotor speed, the electromagnetic torque on the motor shaft, and the reference voltage at the inverter input con- sidering stable parameters of the supply network or net- work with infinitely large power. where c = H/g is sag constant. It is schematically shown in Fig. 1,b and contains three electric drives, based on asynchronous motors with vector control. device and provides the required cable tension H when it moves inside the vulcanization pipe. All drives are built based on direct control of the torque and flow of an asyn- chronous motor (DTC method), described in [1, 3, 9]. In this paper, as in [7], a mathematical model was de- veloped using the Matlab / Simulink software package [8] to study the electromagnetic processes in electromechanical system shown in Fig. 1. This system model with two elec- tric drives with vector control is shown in Fig. 2. Drives No. 1 and No. 3 set in motion the cylinder of the deeding and receiving devices and ensure the cable motion at a constant speed V set by the technological con- ditions. Drive No. 2 sets in motion the track-type traction Set torque Set speed Electric drive No. 2 of track-type device Electric drive No. 3 of receiving device Short-term switched load Fig. 2. Mathematical model of a system with two electric drives with vector control Electric drive No. 2 of track-type device Set speed Electric drive No. 3 of receiving device Fig. 2. Mathematical model of a system with two electric drives with vector control Fig. 2. Mathematical model of a system with two electric drives with vector control ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 49 ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 In this model, the effect of two drives No. 1 and No. 3, providing a set speed of the cable motion, is re- placed by the equivalent action of one drive, so a system consisting of two drives is considered in the modeling. The model has a drive’s receiving device, providing a set speed of shaft rotation of the asynchronous motor, and, consequently, a set speed of cable pulling. Also, there is a drive of the track-type device No. 2, which creates a set torque on the motor shaft, and, consequently, a set tension of the cable. Both drives are connected to a three-phase power supply. To model a short-term voltage failure mode, an additional active three-phase load is connected to this source with a key. To visualize the calculation re- sults, the blocks of virtual oscilloscopes Display of the Simulink package are used, the inputs of which are con- nected to the corresponding communication lines. ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 where c = H/g is sag constant. Dynamic processes in the drive’s receiving device at a short-term increase in the supply voltage Electromagnetic torque, Nm Rotor speed, rpm Stator current, А Inverter input voltage, V Stator current, А Rotor speed, rpm Time, s Time, s g diagrams of the main characteristics of the drive’s receiving device for a period of time corresponding to the start-u mode Time, s agrams of the main characteristics of the drive’s receiving device for a period of time corresponding to the start-up mode Stator current, А Rotor speed, rpm Electromagnetic torque, Nm Inverter input voltage, V Time, s Fig. 4. Dynamic processes in the drive’s receiving device at a short-term increase in the supply voltage Rotor speed, rpm Fig. 4. Dynamic processes in the drive’s receiving device at a short-term increase in the supply voltage ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 Time, s Phase voltage, V Fig. 5. Dependence of the phase voltage network on time Fig. 6 shows that the voltage failure (time int 1.4 – 1.8 s) at the inverter input decreases, current in the stator motor winding decreases, but vector trol system increases the electromagnetic torque i der to work at the set speed. It can be seen that this voltage failure, the electromechanical system spite the torque increase, cannot work at the set speed, which decreases at the end of this time int by an amount Δn ≈ 430 rpm. In order to obtain a quantitative dependence o relative decrease in the rotor speed Δn/n0 on the tive decrease in the network voltage Δu/u0, calcula were carried out for different values of the load tionally connected to the network. These depende are shown in Fig. 7. Phase voltage, V Time, s Phase voltage, V Fig. 5. Dependence of the phase voltage network on time Fig. 6 shows that the voltage failure (time interval 1.4 – 1.8 s) at the inverter input decreases, current level in the stator motor winding decreases, but vector con- trol system increases the electromagnetic torque in or- der to work at the set speed. It can be seen that with this voltage failure, the electromechanical system, de- spite the torque increase, cannot work at the set rotor speed, which decreases at the end of this time interval by an amount Δn ≈ 430 rpm. where c = H/g is sag constant. Such a mode was modeled by connecting the drive to an additional voltage source of increased amplitude in a time point of 1.4 s (after reaching the steady state). The results of calculating these processes for the drive’s receiving device are shown in Fig. 4. (4) Based on calculation results it follows that, although the inverter input voltage increases from 580 V to 800 V, rotor speed does not change. That is, the studied system of the two drives is stable to short-time increases in the input voltage in the wide range. At the next stage, dynamic processes in the drives were modeled for a short-time (0.4 s) network voltage failure from the amplitude value u0 to the value u0 – Δu, with Δu/u0 = 0.54. This mode was modeled by connecting to a power supply with a limited power of an additional three-phase load. The mechanical part of the machine was described by two equations:   m e T F T J dt d      1 ,   dt d . (14, 15) The calculating results of these processes for the drive’s receiving device are shown in Fig. 5 for the phase voltage and in Fig. 6 for the stator current, the rotor speed, the electromagnetic torque and the inverter input voltage. The following symbols are used in the equations: Rs, The following symbols are used in the equations: Rs, Lls and R'r, Llr are the resistance and inductance of stator and rotor scattering; Lm is the inductance of the magnetiz- ing circuit; Ls, L'r are the total inductances of the stator 50 Time, s Electromagnetic torque, Nm Rotor speed, rpm Stator current, А Inverter input voltage, V Fig. 3. Timing diagrams of the main characteristics of the drive’s receiving device for a period of time corresponding to the start-up mode Stator current, А Rotor speed, rpm Electromagnetic torque, Nm Inverter input voltage, V Time, s Fig. 4. where c = H/g is sag constant. In order to obtain a quantitative dependence of the relative decrease in the rotor speed Δn/n0 on the rela- tive decrease in the network voltage Δu/u0, calculations were carried out for different values of the load addi- tionally connected to the network. These dependences are shown in Fig. 7. Time, s Fig. 5. Dependence of the phase voltage network on time Time, s Fig. 5. Dependence of the phase voltage network on time ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 51 Rotor speed, rpm Electromagnetic torque, Nm Inverter input voltage, V Δn/n0 = 0.27 Stator current, А Time, s Fig. 6. Dynamic processes in the drive’s receiving device at a short-term failure of the supply voltage Time, s Fig. 6. Dynamic processes in the drive’s receiving device at a short-term failure of the supply voltage 0 20 40 60 80 100 0 10 20 30 40 50 % 00 / 0 n n n = const at Δu /u0 < 27% n = var at Δu /u0 > 27% Δu/u0 · 100% Δn/n0 · 100% Fig. 7. Dependence of the relative decrease in the rotor speed Δn/n0 of the electromechancal system on the relative decrease in the network voltage Δu/u0 0 20 40 60 80 100 0 10 20 30 40 50 % 00 / 0 n n n = const at Δu /u0 < 27% n = var at Δu /u0 > 27% Δu/u0 · 100% Δn/n0 · 100% 2. The coordination of electrical and mechanical pa- rameters of the system is carried out and the analysis of its dynamic processes is made. It is determined that with a critical mass of the current-carrying core, the electrome- chanical system allows stabilizing the current-carrying core speed with the necessary accuracy at short-time fail- ures in the supply voltage of no more than 27 % of its amplitude value. This is one of the basic requirements for the parameters of power supply systems, taking into ac- count their possible connection to additional power loads. 3. It is also shown that this system is stable to a short- term voltage increase for 0.2 s from a value of 380 V to 500 V. 3. It is also shown that this system is stable to a short- term voltage increase for 0.2 s from a value of 380 V to 500 V. Fig. 7. where c = H/g is sag constant. Dependence of the relative decrease in the rotor speed Δn/n0 of the electromechancal system on the relative decrease in the network voltage Δu/u0 REFERENCES 1. Anuchin A., Shpak D., Aliamkin D., Briz F. Adaptive ob- server for field oriented control systems of induction motors. 57th International Scientific Conference on Power and Electri- cal Engineering of Riga Technical University (RTUCON), 2016, pp. 1-4. doi: 10.1109/RTUCON.2016.7763157. As can be seen from this figure, there is a threshold value for the voltage failure Δu/u0100 % = 27 %, below which the drive ensures stabilization of the set speed with high accuracy. When this value is exceeded, the relative rotor speed Δn/n0 increases, that is, the drive does not provide stabilization of the set speed. The obtained data on the threshold value of the voltage failure allow us to formulate the requirements for the parameters of power supply systems, taking into account their possible connec- tion to additional power loads. 2. Krause P.C., Wasynczuk O., Scott D.S. Analysis of Electric Ma- chinery and Drive Systems. Wiley-IEEE Press, 2013. 680 p. 3. Trzynadlowski A. Control of Induction Motors. Academic Press, 2001. 225 p. 4. Bezprozvannych A.V., Kessaev A.G., Shcherba M.A. Fre- quency dependence of dielectric loss tangent on the degree of hu- midification of polyethylene cable insulation. Technical Electro- dynamics, 2016, no.3, pp. 18-24. (Rus). 5. German-Galkin S.G. Matlab/Simulink. Proektirovanie mek- hatronnykh sistem na PK [Matlab/Simulink. Designing mecha- tronic systems on a PC]. St. Petersburg, Korona-Vek Publ., 2008. 368 p. (Rus). Conclusions. ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 Conclusions. 1. A mathematical model has been developed to study the dynamic processes in the electromechanical system used in the production line for the extrusion coating of polyethylene insulation and semiconductive polymer lay- ers on the current-carrying core of ultrahigh-voltage ca- bles. The investigated system includes electric drives on the basis of frequency-controlled asynchronous motors and their load - moving current-carrying core with neces- sary speed and tension. 6. German-Galkin S.G., Cardonov G.A. Elektricheskie mashiny. Laboratornye raboty na PK [Electric machines. Lab. work on a PC]. St. Petersburg: Korona print Publ., 2003. 256 p. (Rus). 7. Zolotaryov V.M., Shcherba A.A., Podoltsev A.D. Modelling of dynamic processes in electromechanical system for the con- trol of superhigh-voltage cable movement in slant extrusion-type line. Technical Electrodynamics, 2010, no.3, pp. 44-51. (Rus). ISSN 2074-272X. Електротехніка і Електромеханіка. 2018. №2 52 V.M. Zolotaryov1, Doctor of Technical Science, M.A. Shcherba2, Candidate of Technical Science, R.V. Belyanin1, R.P. Mygushchenko3, Doctor of Technical Science, I.M. Korzhov3, 1 Private Joint-stock company Yuzhcable works, 7, Avtogennaya Str., Kharkiv, 61099, Ukraine, phone +380 57 7545228, e-mail: [email protected] 2 The Institute of Electrodynamics of the NAS of Ukraine, 56, prospekt Peremogy, Kiev-57, 03680, Ukraine, phone +380 44 3662460, e-mail: [email protected] 3 National Technical University «Kharkiv Polytechnic Institute», 2, Kyrpychova Str., Kharkiv, 61002, Ukraine, phone +380 57 7076116, e-mail: [email protected] 8. Description of the application SimPowerSystems. Available at: www.mathworks.com (accessed 11 May 2017). V.M. Zolotaryov1, Doctor of Technical Science, M.A. Shcherba2, Candidate of Technical Science, 9. Peresada S.M. Nelineinoe i adaptivnoe upravlenie v elek- tromekhanicheskikh sistemakh s vektorno-upravliaemymi elek- trodvigateliami. Diss. dokt. techn. nauk [Nonlinear and adaptive control in electromechanical systems with vector-controlled electric motors. Doc. tech. sci. diss.]. Kyiv, 2007. 472 p. (Rus). R.P. Mygushchenko3, Doctor of Technical Science, 3 R.P. Mygushchenko3, Doctor of Technical Science, I.M. Korzhov3, 1 10. Pivnyak G.G., Volkov A.V. Sovremennye chastotno- reguliruemye asinkhronnye elektroprivody s chastotno- impul'snoi moduliatsiei [Modern frequency-controlled asyn- chronous electric drives with frequency-pulse modulation]. Dni- propetrovsk, NGU Publ., 2006. 468 p. (Rus). 11. Chermalykh V.M., Chermalykh A.V., Maidansky I.Ya. In- vestigation of the dynamics and energy parameters of an asyn- chronous electric drive with vector control by the virtual simula- tion method. Bulletin of NTU «KhPI», 2008, no.30, pp. 41-45. (Rus). phone +380 57 7076116, e-mail: [email protected] 12. Shcherba M.A., Podoltsev О.D. Electric field and current density distribution near water inclusions of polymer insulation of high-voltage cables in view of its nonlinear properties. 12. Shcherba M.A., Podoltsev О.D. Electric field and current density distribution near water inclusions of polymer insulation of high-voltage cables in view of its nonlinear properties. Tech- nical Electrodynamics, 2016, no.1, pp. 11-19. (Rus). V.M. Zolotaryov1, Doctor of Technical Science, M.A. Shcherba2, Candidate of Technical Science, R.V. Belyanin1, R.P. Mygushchenko3, Doctor of Technical Science, I.M. Korzhov3, 1 Private Joint-stock company Yuzhcable works, 7, Avtogennaya Str., Kharkiv, 61099, Ukraine, phone +380 57 7545228, e-mail: [email protected] 2 The Institute of Electrodynamics of the NAS of Ukraine, 56, prospekt Peremogy, Kiev-57, 03680, Ukraine, phone +380 44 3662460, e-mail: [email protected] 3 National Technical University «Kharkiv Polytechnic Institute», 2, Kyrpychova Str., Kharkiv, 61002, Ukraine, phone +380 57 7076116, e-mail: [email protected] Conclusions. Tech- nical Electrodynamics, 2016, no.1, pp. 11-19. (Rus). 12. Shcherba M.A., Podoltsev О.D. Electric field and current density distribution near water inclusions of polymer insulation of high-voltage cables in view of its nonlinear properties. Tech- nical Electrodynamics, 2016, no.1, pp. 11-19. (Rus). Received 15.11.2017 53 53

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https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-021-04395-y

English

Spatial rank-based multifactor dimensionality reduction to detect gene–gene interactions for multivariate phenotypes

BMC bioinformatics

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© The Author(s), 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate‑ rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​ creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​mmons.​org/​publi​ cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Spatial rank‑based multifactor dimensionality reduction to detect gene–gene interactions for multivariate phenotypes Mira Park1, Hoe‑Bin Jeong2, Jong‑Hyun Lee2 and Taesung Park3* *Correspondence: [email protected] 3 Department of Statistics, Seoul National University, Seoul 08826, Republic of Korea Full list of author information is available at the end of the article Abstract Background:  Identifying interaction effects between genes is one of the main tasks of genome-wide association studies aiming to shed light on the biological mechanisms underlying complex diseases. Multifactor dimensionality reduction (MDR) is a popular approach for detecting gene–gene interactions that has been extended in various forms to handle binary and continuous phenotypes. However, only few multivariate MDR methods are available for multiple related phenotypes. Current approaches use Hotelling’s ­T2 statistic to evaluate interaction models, but it is well known that Hotel‑ ling’s ­T2 statistic is highly sensitive to heavily skewed distributions and outliers. Results:  We propose a robust approach based on nonparametric statistics such as spatial signs and ranks. The new multivariate rank-based MDR (MR-MDR) is mainly suitable for analyzing multiple continuous phenotypes and is less sensitive to skewed distributions and outliers. MR-MDR utilizes fuzzy k-means clustering and classifies multi-locus genotypes into two groups. Then, MR-MDR calculates a spatial rank-sum statistic as an evaluation measure and selects the best interaction model with the larg‑ est statistic. Our novel idea lies in adopting nonparametric statistics as an evaluation measure for robust inference. We adopt tenfold cross-validation to avoid overfitting. Intensive simulation studies were conducted to compare the performance of MR-MDR with current methods. Application of MR-MDR to a real dataset from a Korean genome- wide association study demonstrated that it successfully identified genetic interactions associated with four phenotypes related to kidney function. The R code for conducting MR-MDR is available at https://​github.​com/​statp​ark/​MR-​MDR. Conclusions:  Intensive simulation studies comparing MR-MDR with several current methods showed that the performance of MR-MDR was outstanding for skewed distri‑ butions. Additionally, for symmetric distributions, MR-MDR showed comparable power. Therefore, we conclude that MR-MDR is a useful multivariate non-parametric approach that can be used regardless of the phenotype distribution, the correlations between phenotypes, and sample size. Keywords:  Fuzzy clustering, Gene–gene interaction, Multifactor dimensionality reduction, Spatial rank statistic Open Access Park et al. BMC Bioinformatics (2021) 22:480 https://doi.org/10.1186/s12859-021-04395-y Park et al. BMC Bioinformatics (2021) 22:480 https://doi.org/10.1186/s12859-021-04395-y Background Many attempts have been made to identify susceptible genes that influence the risk of complex diseases such as autism, hypertension, and diabetes [1–3]. Analyzing a single locus is not enough to understand the pathophysiology of complex diseases and results in the so-called missing heritability problem. To overcome this problem, several studies have sought to identify gene–gene interactions (GGIs) or gene-environmental interac- tions [4–6]. As a non-parametric model-free approach, multifactor dimensionality reduction (MDR) has been widely applied for detecting GGIs [5]. For binary phenotypes, such as those analyzed in case–control studies, MDR divides high-dimensional genotype com- binations into a one-dimensional variable with two groups (high-risk and low-risk), according to whether the ratio of cases to controls exceeds a threshold. Then it finds the interaction model that best predicts the disease status. Balanced accuracy can be used for an evaluation measure [7]. To prevent overfitting, k-fold cross-validation (CV) can be applied. Cross-validation consistency (CVC), or the number of times each sin- gle-nucleotide polymorphism (SNP) combination is chosen as best, is obtained during the k-fold CV process. The SNP combination with the highest CVC is defined as the final best interaction model [8]. MDR has several advantages: i) the dimensions of the data are effectively reduced, ii) no specific genetic model is assumed, and iii) high-order interactions can be identified, even if there are no significant main effects [9, 10]. iif Since its introduction, many studies have been conducted to broaden the scope of application of MDR. According to Gola et al. [4], about 800 MDR-related studies were found as of February 2014 when searching PubMed and Google Scholar. For discrete traits, log-linear models MDR and robust MDR have been proposed to handle data with empty or sparse cells [11, 12]. Odds ratio MDR was proposed, replacing the naïve classifier with the odds ratio [9] and optimal MDR replaced the fixed threshold with a data-driven threshold using an ordered combinatorial partitioning algorithm [13]. As a method of dealing with continuous traits, generalized MDR (GMDR) was proposed. GMDR can handle both dichotomous and continuous phenotypes and can adjust for covariates [14]. Quantitative MDR (QMDR) for continuous traits uses the sample mean of each genotype combination as a classifier, reducing the computing time with compa- rable performance [15]. Recently, cluster-based MDR (CL-MDR) has been proposed as a method that is less sensitive to outliers and distributional assumptions [16]. © The Author(s), 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate‑ rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​ creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​mmons.​org/​publi​ cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Park et al. BMC Bioinformatics (2021) 22:480 Page 2 of 21 Background For sur- vival time with censored data, Surv-MDR was proposed, which uses the log-rank test statistic to classify the cells of a multifactor combination [17]. Cox-MDR and accelerated failure time MDR are extended versions of GMDR for the survival phenotype based on Cox regression and the accelerated failure time model, respectively [18, 19]. Recently, Kaplan–Meier MDR was also developed, which uses the median Kaplan–Meier survival time as a classifier [20]. i As described above, many studies have been conducted to identify genetic interac- tions associated with single phenotype, but only a few studies have been done on meth- odologies to treat multiple phenotypes. For complex diseases, several correlated traits are often measured together. For example, weight, the waist-hip ratio, and the body mass index (BMI) can be jointly measured as obesity-related traits. The power to detect associations between genes and these traits is expected to increase if the multivariate Park et al. BMC Bioinformatics (2021) 22:480 Page 3 of 21 approach is adopted [21]. Therefore, more research on multivariate methods detect- ing GGIs is needed. Recently, multivariate generalized MDR (GEE-GMDR) extended GMDR to the multivariate case by constructing generalized estimation equation mod- els [22]. GEE-GMDR provides stable results, but it does not always show higher power than GMDR [23]. Multivariate QMDR (multi-QMDR) extended QMDR using princi- pal component analysis scores and Hotelling’s ­T2 statistic as a classifier and an evalua- tion measure instead [23]. Multi-QMDR gives a high CVC and stable results, but it is prone to outliers or influencing points. More recently, multivariate cluster-based MDR (multi-CMDR) has been proposed [24]. Multi-CMDR applies fuzzy k-means clustering to discriminate between high- and low-risk groups and uses Hotelling’s ­T2 statistic for evaluation. Multi-CMDR is less sensitive to outliers and non-symmetric distributions. While MDR is a nonparametric approach, all these methods use parametric test statis- tics as evaluation measures based on a multivariate normal distribution or exponential family distribution. Instead of using parametric approach, this study considers non-para- metric evaluation measures for testing the equality of two multivariate populations. Var- ious methods based on multivariate ranks or distances between the pairs of individual observations have been studied [25]. Note that signs and ranks in a univariate case are based on the ordering of the data. Unfortunately, however, there is no natural ordering of the data for a multivariate case. Background To extend the concept of rank to a multivariate case, several principles have been considered. First, the methods using interdirection were introduced [26, 27]. Interdirection is a measure based on the angular distance between two observation vectors relative to the rest of the data [28]. Second, the tests based on data depth were proposed [29–31]. A data depth measures how deep a multivariate sam- ple lies in the data cloud [32]. Any function which provides a reasonable central-outward ordering of points in multidimensional space can be considered as a depth function [33]. Third, multivariate extensions using spatial signs and ranks were also studied [34, 35]. Affine invariant methods based on spatial sign and rank vectors for various multivariate problems were proposed [34]. More recently, for high-dimensional data, a nonparamet- ric multivariate test using spatial signs [36] and a spatial ranks test for two samples were proposed [37]. Among various approaches and measures, we chose the spatial rank-sum statistic as the non-parametric evaluation measure in this study, because it is one of the most widely statistics and implemented with R program. We propose a new non-parametric multivariate approach for identifying genetic interactions. We call the proposed method multivariate rank-based MDR (MR-MDR). During classification process, MR-MDR utilizes the fuzzy k-means clustering analysis with a noise cluster as in multi-CMDR. For the evaluation process, MR-MDR calculates the spatial rank-sum statistic as an evaluation measure and selects the best interaction model with the largest statistic. The tenfold CV method is adopted and the final best interaction model is determined by maximum CV consistency. This manuscript is organized as follows. We first start with an introduction of the spatial rank statistic. The algorithm of the proposed MR-MDR method is then described in detail. We then present the results of intensive simulation studies to investigate the performance of the proposed method. Our method is compared with multi-QMDR and multi-CMDR. We applied the proposed MR-MDR method to data on four multivariate phenotypes related to kidney function obtained from the Korean Genome and Epidemiology Study (KoGES): Park et al. BMC Bioinformatics (2021) 22:480 Page 4 of 21 blood urea nitrogen (BUN), serum creatinine, urinary albumin, and urinary red blood cell (RBC) levels. MR-MDR successfully identified genetic interactions associated with these four phenotypes. Nonparametric multivariate rank test We first introduce the multivariate non-parametric test used for evaluation. To detect a two-sample location shift in univariate analysis, the two-sample t-test is popularly used when the response variable is normally distributed. The Mann–Whitney test based on the rank sum is well known as a nonparametric counterpart of the two-sample univariate t-test. Various robust univariate non-parametric tests have been developed for the two-sample location problem [38]. For multivariate analysis, a classical approach such as Hotelling’s ­T2 is a popular paramet- ric approach. ­T2 has optimal power under the assumption of a multivariate normal distri- bution. However, it performs poorly in the case of heavy-tailed distributions and is highly sensitive to outliers [39]. As an alternative, we consider a nonparametric approach based on spatial signs and ranks. We start with the definition of spatial sign and spatial rank. i Let Y = (y1, ..., yn)′ be an n × p data matrix with n observations and p variables. The spa- tial sign function and spatial rank function are defined as follows [28]. S(y) =  ||y||−1y, y = 0 0, y = 0 , R(y) = avej{S(y −yj)} = 1 n n  j=1 {S(y −yj)} where avej means the average taken over all observations for j = 1,…n, ||y|| is the Euclid- ean distance of y from 0, and y and yj are p-variate vectors [28]. The observed spatial signs si and observed centered spatial ranks ri are defined as si = S(yi), and ri = avej{sij} = 1 n n  j=1 {S(yi −yj)}, respectively for i, j = 1, …, n. Here, sij = S(yi −yj) , ave{ri} = 0.fi respectively for i, j = 1, …, n. Here, sij = S(yi −yj) , ave{ri} = 0. To make an affine-invariant test statistic, we can apply the spatial sign function to the transformed data points. The test statistic T(y1, ..., yn) is said to be affine-invariant if T(y1, ..., yn) = T(Dy1, ..., Dyn) for every p × p nonsingular matrix D and for every p-variate dataset y1, ..., yn [34]. Affine-invariant spatial signs and ranks are obtained by transforming yi to Ayyi, s∗ i = S(Ayyi), r∗ i = avej{s∗ ij} = 1 n n  j=1 {S(Ay(yi −yj)} s∗ i = S(Ayyi), r∗ i = avej{s∗ ij} = 1 n n  j=1 {S(Ay(yi −yj)} Park et al. Nonparametric multivariate rank test BMC Bioinformatics (2021) 22:480 Page 5 of 21 where Ay is Tyler’s transformation, which makes the spatial sign covariance matrix pro- portional to the identity matrix, that is, ave{r∗′ i r∗ i } = [c2 y/p]Ip , where c2 y = ave{||r∗ i ||2} . Ay can be obtained during the iterative process and chosen so that trace(A′ yAy) = p [34, 40, 41]. The ranks r∗ i lie in the unit p-sphere. The direction of r∗ i roughly points outward from the center of the data cloud and its length tells how far away this point is from the center of the data cloud [42]. Next, for the two-samples location problem, let Y1 = (y1, ..., yn1)′ and Y2 = (yn1+1, ..., yn1+n2)′ be two independent samples with p variables that have the cumulative distribution functions F(x −µ) and F(x −µ −) , respectively. To test the null hypothesis of no differences in location, H0:Δ:0, the multivariate version of Mann–Whitney test statistic U2 can be used. For the combined sample Y = [Y1:Y2], the affine-transformed spatial signs of the transformed differences s∗ ij = S(Ay(yi −yj)) and spatial ranks r∗ i = avej{S∗ ij} are obtained. The multivariate Mann–Whitney test statistic U2 is given by U2 = p c2y 2  i=1 ni||r∗ i ||2 where r∗ i is the sample-wise mean vector of the spatial ranks r∗ i and c2 y = ave{||r∗ i ||2} [34]. The p-value is obtained by Eη(I(U2 γ ≥U2)) , where Eη(·) represents expectation value where η = (η1, ..., ηN) is uniformly distributed over N! permutations of (1, . . . , N) and U2 γ is the value of the test statistic of a permuted sample. As the dimension p increases and the distribution becomes heavier-tailed, the performance of U2 improves relative to Hotelling’s T2 statistic [34]. We investigated the effect of Tyler’s transformation on yi through an empirical study using a toy example. Two multivariate distributions of the response variables were considered: a bivariate normal distribution and a bivariate gamma distribution. The correlations between two response variables were set to 0.4 and 0.8. The original data were transformed to spatial signs, and then spatial centered ranks were obtained by averaging the spatial signs of differences. Figure 1 shows the spatial signs and ranks with and without Tyler’s transformation. Nonparametric multivariate rank test Spatial signs are represented as directions from the origin with unit length, and thus all the spatial signs lie on a circle of radius 1. The spatial signs with Tyler’s transformation tend to be more evenly arranged around the circumference than the spatial signs without Tyler’s transformation. The spatial ranks tend to spread evenly, as if they are uniformly distributed within a circle, for the Tyler’s transformation case. Note that the spatial ranks before and after Tyler’s transformation appear different when the correlation is high (r = 0.8); however, the change due to the transformation is negligible if the correlation is moderate (r = 0.4). MR‑MDR procedureh There are many variations of MDR methods for finding GGIs. However, most MDR approaches have two core procedures: how to classify the cells into two groups and how to evaluate the interaction models. The proposed MR-MDR adopts fuzzy cluster- ing technique in the classification process as in multi-CMDR [24]. For the evaluation Park et al. BMC Bioinformatics (2021) 22:480 Page 6 of 21 Fig. 1  Examples of spatial signs and ranks for two bivariate distributions and ranks for two bivariate distributions Fig. 1  Examples of spatial signs and ranks for two bivariate distributions process, MR-MDR uses a multivariate spatial rank test statistic. Figure 2 shows the flow of the MR-MDR procedure. The detailed algorithm of MR-MDR is as follows. process, MR-MDR uses a multivariate spatial rank test statistic. Figure 2 shows the flow of the MR-MDR procedure. The detailed algorithm of MR-MDR is as follows. process, MR-MDR uses a multivariate spatial rank test statistic. Figure 2 shows the flow of the MR-MDR procedure. The detailed algorithm of MR-MDR is as follows. Step 0. Data • Suppose there are n* samples, with p SNP data-points and q continuous pheno- types. Park et al. BMC Bioinformatics (2021) 22:480 Page 7 of 21 • Standardize all the phenotypes to have a mean of zero and a unit vari- ance. Let Yi = (yi1, yi2, . . . , yiq)T be the standardized phenotype vector and Xi = (xi1, xi2, . . . , xip)T be the genotype vector for the ith subject, respectively. Step 1. Global process: clustering • Perform fuzzy k-means clustering with k = 2 using phenotype information. We add a noise cluster such that noisy data points may be assigned to the noise class [43]. Remove all the samples in the noise cluster. The remaining samples have member- ship degrees for each of the two clusters. Denote these two clusters as C1 and C2. • Perform fuzzy k-means clustering with k = 2 using phenotype information. We add a noise cluster such that noisy data points may be assigned to the noise class [43]. Remove all the samples in the noise cluster. The remaining samples have member- ship degrees for each of the two clusters. Denote these two clusters as C1 and C2. ship degrees for each of the two clusters. Denote these two clusters as C1 and C2. • Define the global ratio θ as • Define the global ratio θ as • Define the global ratio θ as θ = n  i=1 Di1  n  i=1 Di2, where n is the number of remaining samples after deleting noise samples and Dik is the membership degree of the ith subject in cluster Ck, (k = 1, 2) [24]. • For cross-validation (CV), split the samples randomly into 10 subgroups of equal size. Let nine sets of samples be the training dataset and the remaining dataset be the test dataset used for evaluating the model. • For cross-validation (CV), split the samples randomly into 10 subgroups of equal size. Let nine sets of samples be the training dataset and the remaining dataset be the test dataset used for evaluating the model. Step 3. Local process: evaluation • Obtain spatial ranks of Yi for the combined samples from two clusters for i = 1, 2, …, n. r∗ i = avej{S(Ay(yi −yj)} = 1 n n  j=1 Ay(yi −yj)  (Ay(yi −yj))2 where S(·)is a sign function and Ay is Tyler’s transformation. • Calculate the multivariate Mann–Whitney test statistic: where S(·)is a sign function and Ay is Tyler’s transformation. y • Calculate the multivariate Mann–Whitney test statistic: y • Calculate the multivariate Mann–Whitney test statistic: U2 = p c2y 2  k=1 nk||Rk||2 where p is the number of variables, nk is the number of samples in cluster Ck, Rk is the mean vectors of the spatial ranks for cluster Ck, and c2 y = ave{||Rk||2}. y • Obtain U2 for every m SNP combination. Choose the SNP combination with the largest U2 statistic as the best mth-order interaction model in the training data. y • Obtain U2 for every m SNP combination. Choose the SNP combination with the largest U2 statistic as the best mth-order interaction model in the training data. Step 2. Local process: classification • To find mth-order GGIs, select a set of m SNPs from a pool of SNPs. i • Calculate the local ratio θj for the jth genotype combination in the training set, θj = nj  i=1 Dij1  nj  i=1 Dij2,  j = 1, . . . , 3m , Fig. 2  Overview of the MR-MDR algorithm for tenfold cross-validation and second-order interactions Fig. 2  Overview of the MR-MDR algorithm for tenfold cross-validation and second-order interactions Park et al. BMC Bioinformatics (2021) 22:480 Page 8 of 21 where Dijk is the membership degree of the ith subject with the jth genotype combi- nation in cluster Ck. where Dijk is the membership degree of the ith subject with the jth genotype combi- nation in cluster Ck. • Label each genotype combination either “H” if θj ≥ θ, or “L” if θj < θ. • Label each genotype combination either “H” if θj ≥ θ, or “L” if θj < θ. Step 4. Selection process: best interaction model • Repeat step 2 and 3 for each fold and count the number of specific SNP combina- tions chosen for the best model. We call this cross-validation consistency (CVC). • Select the model with the largest CVC as best final interaction model. i • Derive the final rank sum statistic for the best model by averaging all statistics for the test data. • Calculate the empirical p-value by a permutation test. • Calculate the empirical p-value by a permutation test. FPC first principal component, WPC use weighted Summation of principal component, WSPC use weighted Squared Summation of principal component Simulation analysis To compare the performance of the proposed MR-MDR with other existing methods, we conducted simulations for various situations. We considered 20 SNPs, including two- way disease-causal SNPs. For each of the combinations of seven different heritability val- ues (0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4) and two minor allele frequency (MAF) values (0.2, 0.4), five different interaction models (M1-M5) were considered. Typically, penetrance rate is defined as the proportion of individuals with a given genotype who exhibit the phenotype associated with that genotype. However, it is not appropriate for continuous phenotypes and there is no clear definition of continuous phenotype. For QMDR, the penetrance for continuous phenotypes was defined as a function of mean [15]. Simi- larly, we set the penetrance rate as a function of mean of the response variable in each Park et al. BMC Bioinformatics (2021) 22:480 Page 9 of 21 genotype. Thus, a total of 70 epistatic models with various penetrance functions were generated, as done by Velez et al. [7]. All the models had little marginal effect. For the phenotype distribution, we considered a bivariate normal distribution and a bivariate gamma distribution. The correlations of the bivariate phenotypes also varied (0, 0.25, 0.5). Sample sizes of 100, 200, and 400 were considered. Finally, a total of 1000 replicated data sets were generated for 1260 (= 70 × 2 × 3 × 3) combinations. We used both a Tyler’s-transformed version (MR-MDR_transformed) and an untrans- formed version (MRMDR_ untransformed). For the purpose of comparison, multi- CMDR and multi-QMDR methods were also used. All three summary statistics of the multi-QMDR—the first principal component (FPC), weighted sum of principal com- ponents (WPC), and weighted squared sum of principal components (WSPC)—were included. To compare the data with the univariate approach, QMDR was also performed for each phenotype separately. Ten-fold CV was considered. A summary of the simula- tion scheme is shown in Table 1. Since the epistatic models given by Velez et al. [7] were devised only for the discrete phenotype, we modified them to handle continuous phenotypes. Let SNP1 and SNP2 be the two true causal SNPs, Y = (Y1, Y2)T the continuous bivariate phenotypes, and fij the penetrance function for the ith genotype of SNP1 and jth genotype of SNP2 in [7]. For the bivariate normal distribution, Y = (Y1, Y2)T was generated by where µij =  fij fij  and  =  1 ρ ρ 1   . Simulation analysis Here fk and Fk are the marginal probability density function and cumula- tive distribution function of the kth phenotype, respectively, and Φ−1 is the inverse of the cumulative distribution of the standard normal distribution function. In this sim- ulation, we set yk|(SNP1 = i, SNP2 = j) ∼Gamma(2fij, 0.5) for k = 1, 2. We used the mvdc(), rMvdc(), normalCopula() functions in the copula pack- age in R. for k = 1, 2. We used the mvdc(), rMvdc(), normalCopula() functions in the copula pack- age in R. The power was estimated by the hit ratio, which is the proportion of times that each method correctly chose the causal SNP pairs ( SNP1 and SNP2 ) as the best model among all possible two-way interaction models out of each set of 1000 datasets. Fig- ure  3 shows the hit ratios of the eight different methods for the bivariate normal distribution. The power of the multi-QMDR using FPC (multi-QMDR_FPC) was slightly higher than that of the proposed MR-MDR when there was no correlation between phenotypes. However, the difference between multi-QMDR_FPC and MR- MDR decreased as the correlation became stronger. The performances of the trans- formed one (MR-MDR_transformed) and untransformed (MR-MDR_untransformed) one were almost the same. Multi-QMDR with WSPC (multi-QMDR_WSPC) showed lower power even than the QMDR method. Figure 4 shows the hit ratios for a bivariate gamma distribution. The proposed MR- MDR outperformed the other methods for all ranges of heritability. There was little difference between the performance of the two versions of MR-MDR, and the differ- ences between them were less than 0.01 in all situations. The power of multi-CMDR was the next highest. It should be noted that multi-CMDR uses the fuzzy clustering approach to split data as in MR-MDR. The gap between MR-MDR and other methods became larger as the sample size decreased or the correlation became stronger. Multi- QMDR-FPC and multi-QMDR using the WPC (multi-QMDR-WPC) showed lower power than MR-MDR and multi-CMDR, but higher power than QMDR. The perfor- mance of multi-QMDR-WSPC was still poor, although the difference was less than in bivariate normal distribution. Through these simulation studies, we demonstrated that the proposed MR-MDR outperformed the other existing methods for all ranges of heritability when the phenotypes were asymmetrically distributed. However, when the phenotypes are symmetrically distributed, as in a normal distribution, all methods yielded similar performance. Simulation analysis We used the mvrnorm() function of the MASS package in R. Three different values of ρ (0, 0.025, and 0.5) were considered, as men- tioned above. Y|(SNP1 = i, SNP2 = j) ∼MN(µij, ), where µij =  fij fij  and  =  1 ρ ρ 1   . We used the mvrnorm() function of the MASS package in R. Three different values of ρ (0, 0.025, and 0.5) were considered, as men- tioned above. Y|(SNP1 = i, SNP2 = j) ∼MN(µij, ), Y|(SNP1 = i, SNP2 = j) ∼MN(µij, ), For the skewed asymmetrically distributed phenotypes, we used the copula-based multivariate gamma model [44]. A copula-based bivariate gamma distribution is given by Table 1  Summary of the simulation scheme FPC first principal component, WPC use weighted Summation of principal component, WSPC use weighted Squared Summation of principal component Factor Level Value Ref Genotype Number of SNPs 20 SNP1, …, ­SNP20 Number of causal SNPs 2 SNP1, ­SNP2 Minor allele frequency 2 0.2, 0.4 Heritability 7 0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 Interaction model 5 M1, M2, M3, M4, M5 [7] Phenotype Number of phenotypes 2 Y1, ­Y2 Distributions 2 bivariate normal, bivariate gamma [24, 44] Correlation 3 0, 0.25, 0.5 Sample Sample size 3 100, 200, 400 Analysis Methods MR-MDR (transformed, untransformed) [24] 8 multi-CMDR [23, 24] multi-QMDR (FPC, WPC, WSPC) [15] QMDR ­(Y1, ­Y2) Table 1  Summary of the simulation scheme Page 10 of 21 Park et al. BMC Bioinformatics (2021) 22:480 Park et al. BMC Bioinformatics f (y1, y2) = c(u, ) 2  k=1 fk(yk), where c(u, ) = || 1 2 exp  −˜u′(−1−I)˜u 2  , ˜u = (−1(u1), −1(u2))′ , and uk = Fk(yk) where c(u, ) = || 1 2 exp  −˜u′(−1−I)˜u 2  , ˜u = (−1(u1), −1(u2))′ , and uk = Fk(yk) for k = 1, 2. Here fk and Fk are the marginal probability density function and cumula- tive distribution function of the kth phenotype, respectively, and Φ−1 is the inverse of the cumulative distribution of the standard normal distribution function. In this sim- ulation, we set for k = 1, 2. Here fk and Fk are the marginal probability density function and cumula- tive distribution function of the kth phenotype, respectively, and Φ−1 is the inverse of the cumulative distribution of the standard normal distribution function. In this sim- ulation, we set for k = 1, 2. Simulation analysis Three additional simulations were conducted to find out the further properties of the proposed method. The robustness of fuzzy k-means clustering, the effect of noise cluster size, and the effect of outliers were investigated. First, to explore the robust- ness of the fuzzy k-means clustering in MR-MDR algorithm, we performed a compar- ison study to investigate the effect of log-transformation on phenotypes which were Park et al. BMC Bioinformatics (2021) 22:480 Page 11 of 21 Fig. 3  Hit ratios for a bivariate normal distribution Fi 3 Hit ti f bi i t al distribution Fig. 3  Hit ratios for a bivariate normal distribution Fig. 3  Hit ratios for a bivariate normal distribution generated from the bivariate gamma distribution. The power of MR-MDR after log transformation was obtained for each seven heritabilities. We set the correlation coef- ficient between two phenotypes 0.25 and the sample size 200. The average power of MR-MDR for five interaction model (M1-M5) for 1000 random samples are given in Table 2. The power slightly reduced after log-transformation. Therefore, we can con- clude that the fuzzy k-means clustering is robust to the skewed distribution and does not require any further transformation of original data. Secondly, we investigate the efficiency according to the size of noise cluster since the large size of the noise cluster can be a source of loss of efficiency. The size of noise clus- ter depends on the noise distance δ, which needs to be chosen in advance. If δ is too large, outliers are not removed and are classified as a real cluster. On the other hand, if δ is too small, many observations can be misplaced into the noise cluster. Still, the estima- tion of the optimal value of δ is an open-problem [45]. In our approach, we used an itera- tive procedure to determine the value of δ optimally, which is the default of FKM.noise procedure in R. To check the efficiency, we compared the performance with various values of δ. The results are shown in Table 3. For both bivariate normal and bivariate gamma distribu- tion, the average power of MR-MDR for five interaction models (M1-M5) are given in Park et al. BMC Bioinformatics (2021) 22:480 Page 12 of 21 Table 3. This new simulation result shows that the power using the iterative δ yielded the highest hit ratios for all heritabilities. Simulation analysis BMC Bioinformatics (2021) 22:480 Page 13 of 21 Table 3  Hit ratios of MR-MDR according to the noise distance δ (r = 0.25, n = 200) Distribution Heritability δ = 1.5 δ = 2 δ = 2.5 Iterative δ (default) Hit ratio Noise (%) Hit ratio Noise (%) Hit ratio Noise (%) Hit ratio Noise (%) Bivariate 0.01 0.004 14.78 0.007 5.10 0.005 1.27 0.009 12.02 Normal 0.025 0.003 14.78 0.008 4.96 0.007 1.16 0.006 11.90 0.05 0.024 14.79 0.026 5.04 0.038 1.19 0.041 11.92 0.1 0.073 14.54 0.1 4.86 0.131 1.12 0.131 11.59 0.2 0.255 14.27 0.372 4.69 0.423 1.10 0.452 11.21 0.3 0.430 14.28 0.609 4.70 0.669 1.08 0.693 11.14 0.4 0.593 13.944 0.793 4.54 0.846 1.03 0.865 10.736 Bivariate 0.01 0.302 11.89 0.335 7.47 0.352 4.88 0.35 8.25 Gamma 0.025 0.666 11.734 0.709 7.42 0.718 4.84 0.733 7.98 0.05 0.714 12.67 0.738 7.50 0.758 4.62 0.776 8.93 0.1 0.955 13.09 0.972 7.36 0.978 4.25 0.982 9.25 0.2 0.995 13.00 0.996 6.86 0.998 3.68 0.999 9.17 0.3 0.998 12.47 1 6.67 1 3.59 1 8.65 0.4 1 12.18 1 6.30 1 3.37 1 8.27 Table 3  Hit ratios of MR-MDR according to the noise distance δ (r = 0.25, n = 200) Thirdly, we have conducted an additional simulation to investigate the effects of outliers. The power of MR-MDR for the datasets with or without outliers was obtained for each seven heritabilities. We set the correlation coefficient between two phenotypes 0.25 and the sample size 200 as in Table 3. The power for five interaction models (M1-M5) for 1000 random samples were obtained. For the datasets with out- liers, about 5% of the data were replaced by outliers. For both phenotypes, outliers were generated by adding three times of IQR for the randomly chosen 5% samples. The results are summarized in Table 4. In the presence of outliers, the power tends to decrease for all methods. Simulation analysis Note that in this setting outliers were not generated. When there were outliers, a smaller noise cluster would have been created. Fig. 4  Hit ratios for a bivariate gamma distribution Table 2  Hit ratios of MR-MDR according to log transformation for a bivariate gamma distribution (r = 0.25, n = 200) Heritability Hit ratio Without log transformation With log transformation 0.01 0.350 0.325 0.025 0.733 0.685 0.05 0.776 0.693 0.1 0.982 0.942 0.2 0.999 0.989 0.3 1 0.999 0.4 1 1 Fig. 4  Hit ratios for a bivariate gamma distribution di t ib ti Fig. 4  Hit ratios for a bivariate gamma distribution Table 2  Hit ratios of MR-MDR according to log transformation for a bivariate gamma distribution (r = 0.25, n = 200) Heritability Hit ratio Without log transformation With log transformation 0.01 0.350 0.325 0.025 0.733 0.685 0.05 0.776 0.693 0.1 0.982 0.942 0.2 0.999 0.989 0.3 1 0.999 0.4 1 1 Table 2  Hit ratios of MR-MDR according to log transformation for a bivariate gamma distribution (r = 0.25, n = 200) Table 3. This new simulation result shows that the power using the iterative δ yielded the highest hit ratios for all heritabilities. Note that in this setting outliers were not generated. When there were outliers, a smaller noise cluster would have been created. Park et al. W/O hit ratio for the data without outlier, W/ hit ratio for the data with outlier, Diff W/O – W, Multi-QMDR_FPC multi-QMDR using first principal component Simulation analysis The differences were the smallest in MR-MDR, indicating Table 4  Hit ratios of MR-MDR according to the presence or absence of outliers (r = 0.25, n = 200) W/O hit ratio for the data without outlier, W/ hit ratio for the data with outlier, Diff W/O – W, Multi-QMDR_FPC multi-QMDR using first principal component Heritability MR-MDR Multi-CMDR Multi-QMDR_FPC WO W Diff WO W Diff WO W Diff Bivariate 0.01 0.009 0.006 0.003 0.006 0.006 0 0.009 0.007 0.002 Normal 0.025 0.006 0.006 0 0.007 0.006 0.001 0.014 0.013 0.001 0.05 0.041 0.033 0.008 0.039 0.034 0.005 0.036 0.018 0.018 0.1 0.131 0.09 0.041 0.142 0.092 0.05 0.143 0.038 0.105 0.2 0.452 0.364 0.088 0.465 0.375 0.09 0.489 0.121 0.368 0.3 0.693 0.604 0.089 0.711 0.593 0.118 0.727 0.251 0.476 0.4 0.865 0.781 0.084 0.873 0.79 0.083 0.883 0.353 0.53 Bivariate 0.01 0.35 0.26 0.09 0.135 0.096 0.039 0.094 0.072 0.022 Gamma 0.025 0.733 0.609 0.124 0.468 0.355 0.113 0.347 0.276 0.071 0.05 0.776 0.707 0.069 0.661 0.494 0.167 0.606 0.446 0.16 0.1 0.982 0.947 0.035 0.945 0.748 0.197 0.917 0.692 0.225 0.2 0.999 0.992 0.007 0.994 0.934 0.06 0.993 0.82 0.173 0.3 1 1 0 1 0.989 0.011 0.999 0.929 0.07 0.4 1 1 0 1 0.999 0.001 0.999 0.974 0.025 Table 4  Hit ratios of MR-MDR according to the presence or absence of outliers (r = 0.25, n = 200) Park et al. BMC Bioinformatics (2021) 22:480 Page 14 of 21 the minimum power loss of MR-MDR. As a result, MR-MDR showed much higher power than other methods in the presence of outlying observations. Real data analysis BMC Bioinformatics (2021) 22:480 Page 15 of 21 Table 5  Average of phenotypes for global clusters Cluster 1 (L) Cluster 2 (H) All BUN 13.55 16.27 11.06 Serum creatinine 0.78 0.84 0.72 Albumin 2.66 3.39 1.99 RBC 0.20 0.21 0.20 Number of cases 1410 1225 2635 Table 5  Average of phenotypes for global clusters greatly for BUN and serum creatinine. There seemed to be no difference in RBC lev- els between the two clusters. Since the higher values of BUN and creatinine indicate abnormal kidney function, we can interpret the first cluster as a low-risk group and the second cluster as a high-risk group. We selected the SNP pair with the largest CVC as the best interaction model for each order. The test score was the average of spatial rank sum statistics calculated from the test data set. Empirical p-values were obtained by comparing the test score with the statistic from the permuted dataset generated by shuffle phenotype vec- tors. If the score calculated with the permuted data exceeded our score, that case was counted. Then p-value was calculated as a/b, where a is the number of cases with a permuted score higher than the original score and b is the total number of trials. List of the interaction models that had the highest training score at least once dur- ing the tenfold CV process by MR-MDR is shown in Table 6. For the first-order inter- action, rs41476549 had the highest CVC and was selected as the most relevant to the four phenotypes. rs790410 was selected as the best once during 10 CV processes but showed the highest score. All SNPs except rs17168600 had a p-value lower than 0.05. For the second-order interaction model, the pair of rs41476549 and rs1117105 showed the highest CVC, while the pair of rs790410 and rs961413 yielded the highest test score. However, the CVC value was low in most cases. Among the selected SNPs, rs16862782 on chromosome 3 has been reported to be related to BUN in Korean and to serum urea measurement in European [47, 50]. rs4686914 on chromosome 3 is also known to be related to the kidney function in European and East Asian [47, 51]. Both SNPs are mapped to LINC01991 gene. rs17586294 maps to TUBBP11 gene. To the best of our knowledge, there are no studies that have analyzed kidney-related GGI in a multivariate manner. Real data analysis We illustrate the proposed MR-MDR method by analyzing data from the KoGES [46]. The data were collected from two recruitment areas. Each region is a cohort represent- ing city (Ansan) and rural areas (Anseong). After standard quality control procedures for the subjects and SNPs, a total of 8842 participants and 327,872 SNPs were used for this analysis. We considered four phenotypes related to kidney function: BUN, serum creatinine, urinary albumin levels, and urinary RBC levels. Traditionally, BUN and serum creati- nine levels have been used as surrogate markers of kidney function deterioration [47]. The amounts of albumin and RBC in urine also could be good indicators of kidney dis- ease. Although there have been some studies on associations between genes and kidney- related phenotypes, few studies have performed GGI analyses for these phenotypes [48, 49]. In the case of albumin, the urine test is known to be more accurate than in the case of blood test, so the urine test result is used here. However, urine tests are conducted only on a relatively small number of subjects, which resulted in missing observations in phenotypes. For this high rate of missing data, imputation for phenotypes is not appro- priate. We removed observations with at least one missing phenotype value, and 3267 samples remained. A linear model was separately fit to each phenotype with covariate adjustments for sex, age and recruitment area. Finally, 205 SNPs were selected that had significant mar- ginal effects (p < 1 × ­10−7). Residuals were used for the analysis to control for covariate effects. The largest correlation coefficient was 0.32, which was the correlation between BUN and creatinine. The distributions of the phenotypes were heavily skewed. Figure 5 shows scatter plots and box plots of the phenotypes. We applied the proposed MR-MDR method to identify the best first- and second- order interaction models. Table 5 shows the center of the global clusters from fuzzy k-means clustering during step 1. Cluster centers were determined by the means of each phenotype across samples belonging to the cluster. The clusters differed Fig. 5  Scatterplot and boxplot of four phenotypes after adjustment by sex, age and recruitment area. The numbers in the scatter plot are correlation coefficients Fig. 5  Scatterplot and boxplot of four phenotypes after adjustment by sex, age and recruitment area. The numbers in the scatter plot are correlation coefficients Park et al. Real data analysis Therefore, none of the interactions of the selected pair of SNPs have ever been reported. We also applied multi-CMDR and multi-QMDR methods for comparison. Only multi- QMDR using FPC was considered for comparison, because it showed the highest power among three types of multi-QMDRs. There are some similarities between the results of MR-MDR and multi-CMDR. However, the results are totally different for multi-QMDR, which are expected to be caused by some outlying observations. For example, the pair of rs17014894 and rs10517358 was chosen as the best interaction model. However, this pair suffers from sparsity and outliers. In particular, there are four cells with zero counts and one cell with one count having an outlying observation. Figure 6 shows the box plots of the phenotypes after removing the noise cluster for the genotype combinations of the best model selected by MR-MDR. The distribution Park et al. BMC Bioinformatics (2021) 22:480 Page 16 of 21 Table 6  Best interaction models identified by MR-MDR, multi-CMDR and multi-QMDR Method First order Second order rs ID CVC Test Score p-value rs ID CVC Test Score p-value MR-MDR rs41476549 5 5.05 0.022 rs41476549, rs1117105 2 6.23 0.015 rs790410 1 6.20 0.003 rs790410, rs961413 1 7.31 0.002 rs16862782 1 5.17 0.015 rs11250624, rs17168600 1 7.21 0.003 rs1348637 1 4.99 0.027 rs41476549, rs790410 1 7.01 0.004 rs291877 1 4.94 0.029 rs17168600, rs41476549 1 6.94 0.004 rs17168600 1 4.39 0.064 rs291877, rs790410 1 6.48 0.013 rs7706475, rs41476549 1 6.20 0.016 rs4686914, rs16992376 1 3.38 0.583 rs17586294, rs7016675 1 3.33 0.601 Multi-CMDR rs1348637 3 1.32 0.062 rs790410, rs961413 2 1.87 0.003 rs16862821 2 1.23 0.110 rs291877, rs7612956 1 1.93 0.002 rs790410 1 1.57 0.012 rs291877, rs17168600 1 1.73 0.007 rs291877 1 1.54 0.015 rs291877, rs961413 1 1.65 0.012 rs4686914 1 1.45 0.027 rs291877, rs17643945 1 1.45 0.038 rs41476549 1 1.17 0.162 rs17142042, rs1160759 1 1.19 0.002 rs17142042 1 0.89 0.538 rs17643945, rs291877 1 1.45 0.038 rs12337165, rs9958995 1 1.01 0.364 rs9812308, rs12860002 1 0.78 0.733 Multi-QMDR_FPC rs10517358 6 27.05  < 0.001 rs17014894, rs10517358 6 51.05  < 0.001 rs7265852 3 2.86 0.002 rs41457747, rs10517358 2 27.58  < 0.001 rs17014894 1 24.17  < 0.001 rs41457747, rs17014894 1 24.35  < 0.001 rs1631834, rs10517358 1 2.97  < 0.014 Table 6  Best interaction models identified by MR-MDR, multi-CMDR and multi-QMDR of each phenotype was different depending on the genotype combination, suggesting that there was an interaction effect. Real data analysis of each phenotype was different depending on the genotype combination, suggesting that there was an interaction effect. f To evaluate pure interaction effects for continuous phenotypes, we adopted the clas- sical linear model. For the selected SNP combinations, we fit multivariate analysis of variance (MANOVA) model including two main effects and the interaction effect. The SNP effects were tested for the interaction effect only (H01:β12 = 0) and for the total effects including both main and interaction effects (H02:β1 = β12=0 and H03:β2 = β12=0). The results nine SNP pairs are summarized in Table 7. None of the selected SNP pairs showed significant interaction effects (p > 0.05), while some pairs showed significant total effects. This is because most MDR methods tend to choose a model with a large Park et al. BMC Bioinformatics (2021) 22:480 Page 17 of 21 Fig. 6  Box plots of four phenotypes after removing the noise cluster for the best SNP combination identified by MR-MDR ((i, j): ith genotype for rs1117105 and jth genotype for rs41476549, s creatinine, ALBU albmin) i l f h b SNP bi i id ifi d Fig. 6  Box plots of four phenotypes after removing the noise cluster for the best SNP combination identified by MR-MDR ((i, j): ith genotype for rs1117105 and jth genotype for rs41476549, s creatinine, ALBU albmin) Table 7  p-values from MANOVA test for the SNP combination selected by MR-MDR * H01:β12=0 ** H02:β1 = β12=0 *** H03:β2 = β12=0 rs ID Original data (including noise) Trimmed data by MR-MDR (excluding noise) H01* H02** H03*** H01* H02** H03*** rs41476549, rs1117105 0.6971 0.5841 0.2237 0.9209 0.2201 0.1516 rs790410, rs961413 0.6887 0.00001 0.4374 0.4077 0.0546 0.0140 rs11250624, rs17168600 0.4574 0.0000002 0.5569 0.0388 0.0258 0.0027 rs41476549, rs790410 0.7868 0.2603 0.6852 0.7505 0.1327 0.0970 rs17168600, rs41476549 0.7221 0.7838 0.1680 0.4196 0.0704 0.0477 rs291877, rs790410 0.2365 0.0065 0.0543 0.0107 0.0009 0.0009 rs7706475, rs41476549 0.2289 0.5622 0.0586 0.0211 0.0278 0.0024 rs4686914, rs16992376 0.6290 0.0008 0.8686 0.8155 0.0004 0.7291 rs17586294, rs7016675 0.8552 0.6386 0.8003 0.0003 0.0007 0.0011 Table 7  p-values from MANOVA test for the SNP combination selected by MR-MDR total effect ignoring pure interaction effects. However, our further empirical study showed that the introduction of noise cluster by fuzzy k-means increased the power of detecting interaction effects. The same MANOVA model were fit to the trimmed data Park et al. Real data analysis BMC Bioinformatics (2021) 22:480 Page 18 of 21 after removing the noise cluster by MR-MDR. As expected, several significant interac- tion effects were successfully identified after trimming. Although MANOVA requires a Gaussian assumption, the process removing the noise cluster in the proposed method had the effect of yielding more robust and reliable MANOVA results. Among the nine SNP pairs with non-significant interactions, four pairs showed significant interaction effects. The results are summarized in the last three columns of Table 7. Discussionh The proposed MR-MDR method is a non-parametric approach assuming no particular genetic model. To assign samples to two risk groups, MR-MDR uses the fuzzy clustering technique, as in the multi-CMDR method. MR-MDR uses the spatial rank sum statistic as evaluation measure for comparing two groups whereas Hotelling’s ­T2 statistic is used in multi-CMDR and multi-QMDR. Therefore, robust results can be expected in MR- MDR for skewed distributions or those with outliers. When calculating the spatial rank statistic, a data-driven transformation matrix was needed to make the statistic invariant. It is known that an affine-invariant test performs better than noninvariant angle coordinate-wise sign tests when there is significant devia- tion from spherical symmetry caused by the presence of correlations among observed variables. Moreover, the affine-invariant test outperforms Hotelling’s ­T2-test for heavy- tailed non-normal multivariate distributions [52]. As can be seen in our toy example, the invariant statistic differed from the untransformed statistic, especially in the presence of a high correlation between phenotypes. The problem with using Tyler’s transformation statistic is that it takes much longer to calculate than the untransformed statistic. However, the change of the spatial ranks due to the Tyler’s transformation is trivial even when the correlation is moderate (r = 0.4), as seen in the toy example. Moreover, the simulation results showed that there was little improvement in performance compared to untransformed versions of MR-MDR. This phenomenon was also observed in the case of negative correlation. Therefore, we rec- ommend using the untransformed MR-MDR version if the absolute value of correlation between phenotypes is not too high (e.g., |r|< 0.5). To apply the proposed method for GWAS data, we considered a filtering procedure to reduce the number of SNPs to be investigated. We selected SNPs with significant mar- ginal effects and investigated the interactions. Since MDR is an exhaustive method, this kind of filtering is needed. However, this filtering process can lead to ignoring gene– gene interactions for the SNPs with weak marginal effects. Funding g This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2013M3A9C4078158, NRF-2021R1A2C1007788). Competing interests The authors declare no competing interests. Availability of data and materials y The Korea Association Resource (KARE) project data will be publicly distributed by the Distribution Desk of Korea Biobank Network (https://​korea​bioba​nk.​re.​kr/). The data request should be made directly to Distribution Desk of Korea Biobank Network. Any inquiries should be sent to [email protected]. Conclusions In this paper, we proposed the MR-MDR method to detect the best interaction model for multivariate quantitative traits. MR-MDR is based on the fuzzy clustering technique and spatial rank-sum statistic. Intensive simulation studies comparing MR-MDR with several current methods showed that the performance of MR-MDR was outstanding for skewed distributions. The difference in power between the MR-MDR and other methods increased as the sample size became smaller and the correlation became stronger. Addi- tionally, for symmetric distributions, MR-MDR showed comparable power. Therefore, we conclude that MR-MDR is a useful multivariate non-parametric approach that can Page 19 of 21 Park et al. BMC Bioinformatics (2021) 22:480 be used regardless of the phenotype distribution, the correlations between phenotypes, and sample size. Ethics approval and consent to participate The study was reviewed and approved by the Institutional Review Board of Seoul National University (IRB No. E1908/001004). The patients/participants provided their written informed consent to participate in this study. All meth‑ ods were carried out in accordance with relevant guidelines and regulations (Declaration of Helsinki). Authors’ contributions Conceptualization, M.P.; methodology, M.P. and T.P.; software, H.J. and J.L.; formal analysis, H.J. and J.L.; writing—original draft preparation, M.P.; writing—review and editing, T.P. All authors read and approved the final manuscript. 8. Moore JH, Gilbert JC, Tsai CT, Chiang FT, Holden T, Barney N, White BC. A flexible computational framework for detecting, characterizing, and interpreting statistical patterns of epistasis in genetic studies of human disease susceptibility. J Theor Biol. 2006;241(2):252–61. Abbreviations GGI G GGI: Gene–gene interaction; MDR: Multifactor dimensionality reduction; CV: Cross validation; CVC: Cross validation consistency; SNP: Single nucleotide polymorphism; BMI: Body mass index; BUN: Blood urea nitrogen; RBC: Red blood cell; MAF: Minor allele frequency; FPC: First principal component; WPC: Weight sum of principal components; WSPC: Weighted squared sum of principal components; MANOVA: Multivariate analysis of variance. Author details 1 f 1 Department of Preventive Medicine, Eulji University, Daejeon 34824, Republic of Korea. 2 Department of Statistics, Korea University, Seoul 02841, Republic of Korea. 3 Department of Statistics, Seoul National University, Seoul 08826, Republic of Korea. Received: 19 November 2020 Accepted: 17 September 2021 p y 9. Chung Y, Lee SY, Elston RC, Park T. Odds ratio based multifactor-dimensionality reduction method for detecting gene–gene interactions. Bioinformatics. 2007;23(1):71–6. References 1. 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Gene–gene and gene-environment nephropathy. Clin J Am Soc Nephrol. 2014;9(11):2006–13. Page 21 of 21 Page 21 of 21 Park et al. BMC Bioinformatics (2021) 22:480 49. Tin A, Köttgen A. Genome-wide association studies of CKD and related traits. Clin J Am Soc Nephrol. 2020;6:66. 50. Publisher’s Note Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from:

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COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys

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COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys Davidzon, I.; Jegatheesan, K.; Ilbert, O.; de la Torre, S.; Leslie, S.K.; Laigle, C.; ... ; Weaver, J.R. COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys Davidzon, I.; Jegatheesan, K.; Ilbert, O.; de la Torre, S.; Leslie, S.K.; Laigle, C.; ... ; Weaver, J.R. I. Davidzon1,2 , K. Jegatheesan3,4, O. Ilbert4,5,6, S. de la Torre4, S. K. Leslie7, C. Laigle8, S. Hemmati9, D. C. Masters9, D. Blanquez-Sese1,10, O. B. Kauffmann4, G. E. Magdis1,10,2, K. Małek11,4, H. J. McCracken8, B. Mobasher12, A. Moneti8, D. B. Sanders13,1, M. Shuntov8, S. Toft1,2, and J. R. Weaver1,2 Pasteura 7, 02-093 Warszawa, Poland 12 Physics and Astronomy Department, University of California, 900 University Avenue, Riverside, CA 92521, USA 13 i f A i i f ii 2680 dl i l l 96822 SA 12 Physics and Astronomy Department, University of California, 900 University Avenue, Riverside, CA 92521, USA 13 Instit te for Astronom Uni ersit of Ha aii 2680 Woodla n Dri e Honol l HI 96822 USA 12 Physics and Astronomy Department, University of California, 900 University Avenue, Riverside, CA 92521, USA 13 Institute for Astronomy, University of Hawaii, 2680 Woodlawn Drive, Honolulu, HI 96822, USA Received 2 February 2022 / Accepted 6 June 2022 Received 2 February 2022 / Accepted 6 June 2022 ABSTRACT We present a novel method for estimating galaxy physical properties from spectral energy distributions (SEDs) as an alternative to template fitting techniques and based on self-organizing maps (SOMs) to learn the high-dimensional manifold of a photometric galaxy catalog. The method has previously been tested with hydrodynamical simulations in Davidzon et al. (2019, MNRAS, 489, 4817), however, here it is applied to real data for the first time. It is crucial for its implementation to build the SOM with a high- quality panchromatic data set, thus we selected “COSMOS2020” galaxy catalog for this purpose. After the training and calibration steps with COSMOS2020, other galaxies can be processed through SOMs to obtain an estimate of their stellar mass and star formation rate (SFR). Both quantities resulted in a good agreement with independent measurements derived from more extended photometric baseline and, in addition, their combination (i.e., the SFR vs. stellar mass diagram) shows a main sequence of star-forming galaxies that is consistent with the findings of previous studies. We discuss the advantages of this method compared to traditional SED fitting, highlighting the impact of replacing the usual synthetic templates with a collection of empirical SEDs built by the SOM in a “data- driven” way. Such an approach also allows, even for extremely large data sets, for an efficient visual inspection to identify photometric errors or peculiar galaxy types. While also considering the computational speed of this new estimator, we argue that it will play a valuable role in the analysis of oncoming large-area surveys such as Euclid of the Legacy Survey of Space and Time at the Vera C. Rubin Telescope Key words. galaxies: fundamental parameters – galaxies: star formation – galaxies: stellar content – methods: observational – astronomical databases: miscellaneous Key words. galaxies: fundamental parameters – galaxies: star formation – galaxies: stellar content – methods: observational – astronomical databases: miscellaneous Citation Davidzon, I., Jegatheesan, K., Ilbert, O., De la Torre, S., Leslie, S. K., Laigle, C., … Weaver, J. R. (2022). COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys. Astronomy & Astrophysics, 665. doi:10.1051/0004-6361/202243249 Version: Publisher's Version License: Creative Commons CC BY 4.0 license Downloaded from: https://hdl.handle.net/1887/3561782 License: Note: To cite this publication please use the final published version (if applicable). te: To cite this publication please use the final published version (if applicable Astronomy & Astrophysics Astronomy & Astrophysics Astronomy & Astrophysics A&A 665, A34 (2022) https://doi.org/10.1051/0004-6361/202243249 c⃝I. Davidzon et al. 2022 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article is published in open access under the Subscribe-to-Open model. Subscribe to A&A to support open access publication. A34, page 1 of 22 I. Davidzon1,2 , K. Jegatheesan3,4, O. Ilbert4,5,6, S. de la Torre4, S. K. Leslie7, C. Laigle8, S. Hemmati9, D. C. Masters9, D. Blanquez-Sese1,10, O. B. Kauffmann4, G. E. Magdis1,10,2, K. Małek11,4, H. J. McCracken8, B. Mobasher12, A. Moneti8, D. B. Sanders13,1, M. Shuntov8, S. Toft1,2, and J. R. Weaver1,2 I. Davidzon1,2 , K. Jegatheesan3,4, O. Ilbert4,5,6, S. de la Torre4, S. K. Leslie7, C. Laigle8, S. Hemmati9, D. C. Masters9, D. Blanquez-Sese1,10, O. B. Kauffmann4, G. E. Magdis1,10,2, K. Małek11,4, H. J. McCracken8, B. Mobasher12, A. Moneti8, D. B. Sanders13,1, M. Shuntov8, S. Toft1,2, and J. R. Weaver1,2 1 Cosmic Dawn Center (DAWN), Denmark 1 Cosmic Dawn Center (DAWN), Denmark ( ) 2 Niels Bohr Institute, University of Copenhagen, Jagtvej 128, 2200 Copenhagen N, Denmark 2 Niels Bohr Institute, University of Copenhagen, Jagtvej 128, 2200 Copenhagen N, Denmark il 2 Niels Bohr Institute, University of Copenhagen, Jagtvej 128, 2200 Copenhagen N, Denmark e-mail: [email protected] 2 Niels Bohr Institute, University of Copenhagen, Jagtvej 128, 2200 Copenhagen N, Denmark e-mail: [email protected] 2 Niels Bohr Institute, University of Copenh e-mail: [email protected] 3 Núcleo de Astronomía de la Facultad de Ingeniería y Ciencias, Universidad Diego Portales, Av. Ejército Libertador 441, Santiago, Chile il 3 Núcleo de Astronomía de la Facultad de Ingeniería y Ciencias, Universidad Diego Portales, Av. Ejército Libertador 441, Santiago, Chile e-mail: [email protected] 4 4 Aix Marseille Univ., CNRS, CNES, LAM, Marseille, France 5 California Institute of Technology, Pasadena, CA 91125, US 6 Jet Propulsion Laboratory, California Institute of Technology, Pasad 7 6 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA 7 L id Ob L id U i i PO B 9513 2300 RA L id Th N h l d Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA 7 Leiden Observatory, Leiden University, PO Box 9513, 2300 RA Leiden, The Netherlands 7 Leiden Observatory, Leiden University, PO Box 9513, 2300 RA Leiden, The Netherlands 8 y y Sorbonne Université, CNRS, UMR 7095, Institut d’Astrophysique de Paris, 98 bis Bd Arago, 75014 Paris, France 8 Sorbonne Université, CNRS, UMR 7095, Institut d’Astrophysique de Paris, 98 bis Bd A 9 p y q g 9 Infrared Processing and Analysis Center, California Institute of Technology, Pasadena, CA 91125, USA 9 Infrared Processing and Analysis Center, California Institute of Technology, Pasadena, CA 91125, USA 10 g y , gy, , , 10 DTU-Space, Technical University of Denmark, Elektrovej 327, 2800 Kgs. Lyngby, Denmark p y j g y g y 11 National Centre for Nuclear Research, ul. COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys I. Davidzon1,2 , K. Jegatheesan3,4, O. Ilbert4,5,6, S. de la Torre4, S. K. Leslie7, C. Laigle8, S. Hemmati9, D. C. Masters9, D. Blanquez-Sese1,10, O. B. Kauffmann4, G. E. Magdis1,10,2, K. Małek11,4, H. J. McCracken8, B. Mobasher12, A. Moneti8, D. B. Sanders13,1, M. Shuntov8, S. Toft1,2, and J. R. Weaver1,2 1. Introduction mological simulations (e.g., Henriques et al. 2013; Furlong et al. 2015; Katsianis et al. 2017; Davé et al. 2017). Redshift (z), stellar mass (M), and star formation rate (SFR) are fundamental parameters in studies of galaxy evolution. Measur- ing these quantities has been instrumental to the discovery of the main sequence of star-forming galaxies (Brinchmann et al. 2004; Noeske et al. 2007; Elbaz et al. 2007; Daddi et al. 2007), namely, a tight correlation between M and SFR pointing to a “steady” mechanism of gas-to-stars conversion at work in sys- tems that have gone through different evolutionary paths. Other examples include the SFR and stellar mass functions, two demo- graphics that can be computed for galaxies in different redshift bins and are commonly used to either calibrate or validate cos- In the absence of spectroscopic data, which demand more telescope time than broadband photometry, these three quanti- ties can be derived by fitting galaxy templates to the observed spectral energy distribution (SED). Those templates are built via stellar population synthesis models (e.g., Bruzual & Charlot 2003; Maraston 2005; Conroy et al. 2009), assuming a grid of ages and metallicity values, different star formation histories (SFHs), and one or more options for dust attenuation (for a review, see Conroy 2013). With respect to redshift and stellar mass, SED fitting is a standard practice that produces robust results even when the input photometry is limited to a few bands A&A 665, A34 (2022) potential applications of our SOM-based method are discussed in Sect. 5, along with a few caveats. We summarize our work and draw our conclusions in Sect. 6. in the optical and near-infrared (NIR) regime (for a review, see Salvato et al. 2019). The method is sub-optimal for constraining SFR, unless far-infrared (FIR) data are included (Pannella et al. 2009; Buat et al. 2010; Riccio et al. 2021). However, sky cover- age and depth of FIR ancillary data should match those of sur- veys at shorter wavelengths, a condition that is satisfied in only a few extragalactic fields. Throughout this work we use a flat ΛCDM cosmology with H0 = 70 km s−1 Mpc−1, Ωm = 0.3, ΩΛ = 0.7. All magnitudes are in the AB (Oke 1974) system, and the initial mass function (IMF) assumed here is the one proposed in Chabrier (2003). 2.1. SXDF2018 photometric catalog Published in Mehta et al. (2018), the “SXDF2018” photomet- ric catalog includes optical images from the first data release (DR1) of the Hyper-SuprimeCam Subaru Strategic Program (HSC-SSP, Aihara et al. 2018) and MUSUBI, the MegaCam Ultra-deep Survey with U-Band Imaging1 carried out at the Canada-France-Hawaii Telescope (CFHT). In the NIR regime, images come from the ultra-deep area of the UKIRT Infrared Deep Sky Survey (UKIDSS-UDS, Lawrence et al. 2007) and from the VISTA Deep Extragalactic Observations (VIDEO, Jarvis et al. 2013). The entire area is also covered by the Spitzer Space Telescope, with several IRAC programs documented in Euclid Collaboration (2022). The broadbands used here, along with their 5σ sensitivity limits, are listed in Table 1. We refer to Mehta et al. (2018) for further details. In a nutshell, we used the SOM to analyze a multi- dimensional space made by the combination of galaxy colors in the observer’s frame between 0.3 and 5 µm. This data set is a combination of two photometric catalogs in distinct survey fields, already described in previous work (Mehta et al. 2018; Weaver et al. 2022) and summarized here in Sect. 2. The SOM returns an “unsupervised” classification of the input galaxy sam- ple, namely, the various classes (which will be called “cells”) are formed without making any comparisons to already classi- fied examples. The second step does require supervision, since at least a fraction of the galaxies require a label (from ancillary data, especially Spitzer/MIPS) to determine the physical proper- ties of each SOM cell. This set-up allows us to assign the fol- lowing labels: z, M, SFR, to any new object projected onto the SOM. More details about the method are provided in Sect. 3. 1 W.-H. Wang et al. (in prep.). 2. Data The present study relies on two catalogs built from deep photo- metric surveys in the Subaru XMM/Newton and Cosmic Evo- lution Survey fields (Furusawa et al. 2008; Scoville et al. 2007, SXDF and COSMOS, see respectively). The two extragalactic fields, each of them on the order of a square degree, are partic- ularly suited to our ML application: they can be easily used in tandem because they have in common a subset of galaxy colors spanning from UV to mid-infrared (MIR). In the present analy- sis, we are especially interested in broadband photometry since the galaxy colors derived from it are the features used as input by the SOM. In the last two decades COSMOS has been tar- geted by numerous telescopes, producing a wealth of ancillary data beyond MIR. The observations in the COSMOS field are also extremely deep, and likely to increase depth even further in the near future owing to planned surveys with both existing and oncoming facilities. For these reasons, a catalog of COS- MOS galaxies is ideal for training and calibrating the SOM (Sects. 3.1 and 3.2). On the other hand, SXDF galaxies serve as a test sample to verify the reliability of our method (Sect. 3.3). The advantages of such a strategy, relying on multiple layers of independent data with increasing depth, have been illustrated for instance, in Myles et al. (2021). The present study shows the advantages of a ML-based method built on previous work published in Masters et al. (2015), Hemmati et al. (2019a), and Davidzon et al. (2019) to predict galaxy properties. The method involves dimensionality reduction through self-organizing maps (SOMs, Kohonen 1981) and has been previously tested in Davidzon et al. (2019) based on a mock galaxy catalog (derived from hydrodynamical sim- ulations, see Laigle et al. 2019). Knowing the intrinsic proper- ties of those simulated galaxies, (Davidzon et al. 2019) proved that the SOM can serve as an effective “manifold learning” tool that is able to explore the complex, non-linear galaxy parameter space and reproduce an approximated, but still accurate, version in a low-dimensional space. We are now ready to apply the same method to real data and judge its performance in a practical “user case” situation. 1. Introduction In the quoted scaling relations between SFR and rest-frame lumi- nosities, the latter are in units of L⊙≡3.826 × 1033 erg s−1 for the former to be in M⊙yr−1. Another way to estimate the SFR is to rely on machine learning techniques (ML). For example, a galaxy catalog can be processed through a neural network previously trained with a sample of objects whose physical properties are already known (Davidzon et al. 2019; Surana et al. 2020; Gilda et al. 2021; Simet et al. 2021). This means that the targets can be compared to other observed galaxies, instead of synthetic tem- plates, with the advantage of adhering more coherently to the observational parameter space. In many cases, the training sam- ple is also more accurate than standard templates because its features come directly from high-quality data (e.g., a spectro- scopic sample such as the Sloan Digital Sky Survey, Acquaviva 2016) instead of approximated models. On the other hand, such a data-driven approach provides limited physical insights and may introduce an observational bias if the ML algorithm is not trained on a fully representative sample. A possible solution to this drawback is to use a training sample built from cosmolog- ical simulations (e.g., mock photometry or spectra, Lovell et al. 2019; Simet et al. 2021) even though providing galaxy models with “observational-like” properties, as well as realistic error bars, is a complex task (see discussion in Laigle et al. 2019). 2.3. Physical properties of COSMOS2020 galaxies As the reference sample for SOM training and calibration, COS- MOS2020 galaxies must be provided not only with broad- band colors but also with measurements of key physical properties. Most of this additional information is extracted by fitting the observed SED with galaxy models (also known as templates). Namely, the fitting code LePhare (Arnouts et al. 1999; Ilbert et al. 2006) is used with three different configu- rations to derive: (1) photometric redshifts (zLePh); (2) stellar masses (MLePh) and absolute magnitudes; (3) star formation rates only for sources observed in FIR. Each step is summarized below. Estimates (1) and (2) are described more extensively in Weaver et al. (2022) while we implement the third step follow- ing Ilbert et al. (2015). Since information on the FIR regime – namely, a 24 µm detection with Spitzer/MIPS – is required to constrain star formation, (3) can be applied only to a subsample of galaxies (see Fig. 1). Notes. (a)Median of the transmission curve. (b)Depth at 3σ computed on PSF-homogenized images (except for IRAC images) in empty apertures with 2′′ diameter. Notes. (a)Median of the transmission curve. (b)Depth at 3σ computed on PSF-homogenized images (except for IRAC images) in empty apertures with 2′′ diameter. Fig. 1. Observational properties of the galaxy samples used throughout this work. Upper panel: magnitude-redshift distribution of the COS- MOS2020 galaxy sample used to train the SOM (blue color map with hexagonal pixels) after pre-selection at Ks < 24.8 and log(M/M⊙) > 8.5. Individual sources with a 5σ detection in MIPS are overplotted as orange dots. Lower panel: photometric redshift distribution of the COSMOS2020 training set (blue histogram) and the SXDF2018 target sample (green). These data sets are described in Sects. 2.1 and 2.2. Redshift. In this first run of LePhare, the observed SED is interpolated by a library of 33 galaxy templates (originally pro- posed in Ilbert et al. 2013). The intrinsic spectrum of these tem- plates is modified by a dust screen ranging from E(B −V) = 0 to 0.5, with different options to model the extinction curve (Prevot et al. 1984; Calzetti et al. 2000, and two variations of Calzetti’s law including the 2175 Å bump). A complementary library of stellar templates is used, together with other criteria3, to remove stars from the catalog. After evaluating the χ2 of each fit, the resulting zLePh estimates are defined as the median of the redshift likelihood function, i.e. 3 Namely, g −z vs. z −Ks selection and the stellar locus in half- light radius vs. magnitude in HST/ACS and Subaru/HSC bands (see Weaver et al. 2022). 2 We also obtained the SOM from COSMOS2020-Farmer, and have verified that the ML training in our analysis is stable against different photometric versions. 2.2. COSMOS2020 photometric catalog COSMOS2020 is the latest photometric catalog released by the COSMOS collaboration (Weaver et al. 2022). Two versions of the catalog have been produced through different source extraction methods. Here we use the Classic version based on SourceExtractor (Bertin & Arnouts 1996) and IRACLEAN (Hsieh et al. 2012), since that pipeline is similar to the one In Sect. 4, we verify that SOM-based estimates are more precise than what would result from fitting a standard template library to the same optical-NIR photometry. This section also presents the resulting main sequence of star-forming galaxies up to z = 2, and a comparison with previous studies where SFRs have been derived in different ways. Further extensions and other A34, page 2 of 22 I. Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys Table 1. Optical and NIR data building the observer-frame color space explored by the SOM. Instrument Band Central (a) COSMOS depth (b) SXDF depth (b) λ [Å] 2′′ aper. [mag] 2′′ aper. [mag] MegaCam/CFHT u 3858 27.7 27.93 Subaru/HSC g 4847 28.1 27.39 r 6219 27.8 26.91 i 7699 27.6 26.66 z 8894 27.2 26.07 y 9761 26.5 25.34 VISTA/VIRCAM Y 10 216 25.3/26.6 25.41 J 12 525 25.2/26.4 24.89 H 16 466 24.9/26.1 24.56 Ks 21 557 25.3/25.7 24.23 Spitzer/IRAC ch1 35 686 26.4 25.94 ch2 45 067 26.3 25.68 Notes. (a)Median of the transmission curve. (b)Depth at 3σ computed on PSF-homogenized images (except for IRAC images) in empty apertures with 2′′ diameter. Table 1. Optical and NIR data building the observer-frame color space explored by the SOM. Deep” areas but Shuntov et al. (2022) showed that at low redshift they can be safely combined in a joint analysis, if this is lim- ited to magnitudes brighter than the Deep 3σ limit (see Table 1). Medium- and narrow-band images from Subaru and VISTA tele- scopes are also available in COSMOS, as well as GALEX data from Zamojski et al. (2007). These additional bands are taken into account for template fitting (see below) whereas they are not part of the SOM analysis. VISTA observations in COSMOS have been carried out with a dual-layer strategy; therefore, the table provides two values for Y, J, H, and Ks depths. More details about COSMOS2020 can be found in Weaver et al. (2022). Deep” areas but Shuntov et al. 2.2. COSMOS2020 photometric catalog (2022) showed that at low redshift they can be safely combined in a joint analysis, if this is lim- ited to magnitudes brighter than the Deep 3σ limit (see Table 1). Medium- and narrow-band images from Subaru and VISTA tele- scopes are also available in COSMOS, as well as GALEX data from Zamojski et al. (2007). These additional bands are taken into account for template fitting (see below) whereas they are not part of the SOM analysis. VISTA observations in COSMOS have been carried out with a dual-layer strategy; therefore, the table provides two values for Y, J, H, and Ks depths. More details about COSMOS2020 can be found in Weaver et al. (2022). 2.3. Physical properties of COSMOS2020 galaxies the combined probabilities from the various models (each one ∝e−χ2). Fig. 1. Observational properties of the galaxy samples used throughout this work. Upper panel: magnitude-redshift distribution of the COS- MOS2020 galaxy sample used to train the SOM (blue color map with hexagonal pixels) after pre-selection at Ks < 24.8 and log(M/M⊙) > 8.5. Individual sources with a 5σ detection in MIPS are overplotted as orange dots. Lower panel: photometric redshift distribution of the COSMOS2020 training set (blue histogram) and the SXDF2018 target sample (green). These data sets are described in Sects. 2.1 and 2.2. Stellar mass and other physical properties. After fixing the redshift of each source to zLePh, the LePhare code is used to measure galaxy stellar mass. Templates optimized for such a task are derived from Bruzual & Charlot (2003) models (BC03 hereafter) as done in Laigle et al. (2016), namely, with a grid of 44 time steps for eight star formation histories, two non-evolving metallicity values (Z⊙and 0.2 Z⊙), and a range of dust extinction parameters similar to the one adopted in the first configuration. Absolute magnitudes in different broad-band filters, required to compute rest-frame colors, are an additional output. They are derived from the apparent magnitude that most closely samples the desired rest-frame filter. For example, the observed i band is used to calculate the rest-frame u magnitude for a galaxy at zLePh ≃1. When needed, a k-correction is calculated from the best-fit template, which also provides the apparent magnitude of the object when SourceExtractor cannot measure it in any band. applied to SXDF20182 The COSMOS field is covered by the same instruments and bands mentioned in Sect. 2.1, although here they are generally deeper than in SXDF (see Table 1). In particular, HSC-SSP images come from an updated release (DR2, Aihara et al. 2019) while the NIR is covered by UltraV- ISTA (McCracken et al. 2012) with an exposure time per pixel >150 h (compared to the 10−20 h of VIDEO). The UltraVISTA depth is not homogeneous, that is, there are “Deep” and “Ultra- 2 We also obtained the SOM from COSMOS2020-Farmer, and have verified that the ML training in our analysis is stable against different photometric versions. 4 Since Dale & Helou (2002) models start from 3 µm (rest frame), the SED fitting may include IRAC data point as well. However, we prefer to restrict the photometry to the FIR range for a more homogeneous constraint. 2.3. Physical properties of COSMOS2020 galaxies A34, page 3 of 22 A34, page 3 of 22 A&A 665, A34 (2022) The same run of LePhare would also provide SFRLePh, but we deem the FIR-based estimates more robust for the calibration of our method as will be discussed in Sect. 3.2. Nonetheless, the SFRLePh values are stored in output to compare to the SOM- based estimates derived from the same photometric baseline. require their photometric redshifts to be within ±30% of zLePh. In COSMOS, after converting the two sets of SFRUVIR estimates to a common framework5 we find a scatter of 0.17 dex and a small systematic offset (−0.03 dex) due to a handful of objects over- estimated by Barro et al. (2019) (Fig. B.2). Although the SOM will be calibrated only with LePhare outcomes to ensure con- sistency, these alternate estimates will play an important role for testing (Sect. 4.2). Star formation from infrared continuum. A major role in our analysis is played by the star formation tracer that lever- ages the correlation between SFR and rest-frame emission in UV and infrared (hereafter SFRUVIR). Interstellar dust absorbs stel- lar light from UV to optical and re-emits those photons mostly between 8 and 1000 µm, namely, in the infrared (IR) and sub-mm regime. The IR integrated luminosity (LIR) is therefore closely (albeit indirectly) linked with star formation activity. We derived the LIR for COSMOS2020 galaxies as in Ilbert et al. (2015) using FIR data as an observational constraint. In brief, we assign a 24 µm flux to 28 347 objects over the whole COSMOS area via a cross-correlation between COSMOS2020 sources and the Spitzer/MIPS catalog from Le Floc’h et al. (2009). We cut the parent sample at zLePh < 1.8 and signal-to-noise ratio S/N > 5 in the 24 µm band. The redshift upper limit ensures that the poly- cyclic aromatic hydrocarbons (PAH) complex at a rest-frame of 6.2−7.7 µm does not contaminate the 24 µm measurements. We also removed sources with an X-ray counterpart to mini- mize contamination from their active galactic nucleus (AGN). The remaining 22 571 MIPS galaxies constitute the calibration sample to enable the SFR estimation with the SOM. 2.4. Selection functions The COSMOS2020 training sample is limited to galaxies out- side masked areas of the survey (i.e., avoiding the surround- ings of bright stars6) and with zLePh < 1.8 to be consistent with the MIPS-detected sample. In principle, it would be possible to extend the SOM to higher redshift, but in that case the calibra- tion would require a different SFR proxy (see Sect. 5.3). We also cut the training sample at Ks < 24.8 to remove objects with a low signal-to-noise ratio (S/N). Eventually, the COSMOS train- ing sample includes 174 522 galaxies, with 13% of them being MIPS-detected. We apply a 3σ cut also to the SXDF2018 catalog, corre- sponding to a Ks < 24.2 limit (see Table 1). Since the goal is to replace LePhare physical properties with SOM estimates, and not the photometric redshifts obtained beforehand, we have the latter ones at our disposal to limit the SXDF2018 sample to zLePh < 1.8 and exclude stellar-like objects. As a result, there are 208 404 galaxies in the SXDF field used, as detailed in the following. The LIR estimates are obtained by means of LePhare by fitting Dale & Helou (2002) templates to the MIPS data4, then integrating the best-fit model in the rest frame between 8 and 1000 µm. For 7% of them there is also a 5σ detection in Herschel/PACS (either 100 or 160 µm from Lutz et al. 2011), while 9% have a counterpart in Herschel/SPIRE (250, 350, or 500 µm from Oliver et al. 2012). During this procedure, the red- shift is fixed to the zLePh value from the optical-NIR fitting. No systematic effect is introduced when the 24 µm flux is com- plemented with Herschel measurements up to 500 µm (Fig. 1 of Ilbert et al. 2015). Unobscured star formation is traced by the luminosity in the near-UV band (LNUV), which is obtained from the BC03 template fitting described above. Accounting for both contributions, we eventually compute the total SFR as in Arnouts et al. (2013): 7 Although it would be formally correct to define them as a network of artificial neurons, we decided to use the term “weight”, so as not to mis- lead the majority of readers who commonly associate “neural networks” with a different class of machine learning architectures. 5 Same IMF, luminosity-SFR calibration, etc. (see Appendix B). 6 Masking is done by selecting FLAG_COMBINED==0 in the catalog. 7 Although it would be formally correct to define them as a network of artificial neurons, we decided to use the term “weight”, so as not to mis- lead the majority of readers who commonly associate “neural networks” with a different class of machine learning architectures. 8 Other renderings (3D or even higher dimensions) are also allowed but do not bring the same advantages, e.g., for human eye inspection. More exotic geometries can also be used as long as they are equipped with appropriate metrics. 5 Same IMF, luminosity-SFR calibration, etc. (see Appendix B). 6 Masking is done by selecting FLAG_COMBINED==0 in the catalog. 8 Other renderings (3D or even higher dimensions) are also allowed but do not bring the same advantages, e.g., for human eye inspection. More exotic geometries can also be used as long as they are equipped with appropriate metrics. 3.1. Training the SOM with COSMOS2020 The SOM can identify data patterns in a multi-dimensional fea- ture space by sampling it with an adaptive distribution of points called “weights”7. During a training phase, the coordinates of these weights in the multi-dimensional space are adjusted to move them close to the input data, according to a convergence criterion detailed in Davidzon et al. (2019). After that, the SOM reproduces not only the “shape” of the input manifold but also its “density”, that is, a larger number of test particles is located where data are more clustered. The algorithm is self-organizing because the training is unsupervised. The SOM maps the input data set into a lower-dimensional space: first, its network is arranged in a 2D geometry then data will be re-arranged as well, each entry being associated with a given test particle. Despite the discretization due to the finite number of test particles, the dimensionality reduction preserves most of the topological struc- ture of the original manifold. The 2D geometry we chose8 is a square grid (see Fig. 2); therefore throughout this study we refer to the SOM test particles as “cells”. With the manifold’s topol- ogy mostly preserved, elements that are close to each other in the higher-dimensional space are also neighbors in the grid, namely, SFRUVIR = K(LIR + 2.3LNUV), (1) (1) SFRUVIR = K(LIR + 2.3LNUV), where K = 8.6 × 10−11. This equation traces star formation on a ∼100 Myr time scale. The capability of MIPS 24 µm to provide reliable SFR estimates is also discussed in detail in Rieke et al. (2009). Alternate SFRUVIR estimates for both COSMOS and SXDF galaxies are available from Barro et al. (2019), although only in the sub-region that was also observed by the Cosmic Assem- bly Near-infrared Deep Extragalactic Legacy Survey (CAN- DELS, Grogin et al. 2011; Koekemoer et al. 2011). In addition to ground-based facilities, CANDELS includes data from the Hubble Space Telescope (HST) in both optical and near-IR bands, which may generate some difference in template fitting results (especially photometric redshifts). The ancillary Spitzer and Herschel data in Barro et al. (2019) are similar to the one used here. At z < 1.8 there are 509 (608) matches between Barro et al. (2019) and our COSMOS (SXDF) catalog if we A34, page 4 of 22 I. 3.1. Training the SOM with COSMOS2020 Throughout this work, the dimensions of the 2D grid are arbitrarily labeled D1 and D2 since they have no physical meaning. z Ks 1 0 1 2 3 g z 0 1 2 3 4 5 Ks ch. 1 1 0 1 2 0 1000 2000 3000 4000 5000 6000 SOM cell id Fig. 3. Illustration of the relationship between input features and the SOM classification (made by the so-called “cells”). This simplified example shows only three broadband colors in the observer’s frame, for a random subsample of 10 000 COSMOS2020 galaxies. they belong to the same cell or two contiguous cells (see a ped- agogical illustration in Fig. 3). We refer to the seminal work of Kohonen (1981) for a more extensive explanation. The feature space consists of eleven colors in the observer’s frame, derived from the broadband filters listed in Table 1 and paired in sequential order: u −g, g −r, ..., Ks −ch1, ch1 −ch2. Galaxies are associated with their nearest weight according to their (11-dimensional) Euclidean distance. The ones linked to the same weight are expected to have similar SEDs by construc- tion (see Fig. 1 in Masters et al. 2015) modulo their brightness (i.e., a normalization factor) and the scatter due to photometric errors. The COSMOS2020 SOM is presented in Fig. 2. The weight’s coordinates in the multi-dimensional space are actually a set of eleven colors, which can be regarded as the “pro- totype” SED of the cell. This terminology is the same used in Davidzon et al. (2019, see their Table 1). Similarly to that study, shape and size of the grid are chosen in order to minimize the dispersion of data within a given cell, but at the same time to avoid a large fraction of them being empty. The result is a con- figuration with 80×80 cells, with nearly 94% of them containing at least ten objects at the end of the training (Fig. 2, left panel). Number counts vary across the grid by maximum an order of magnitude and only 14 cells have less than three galaxies (one of them being empty). Fig. 3. Illustration of the relationship between input features and the SOM classification (made by the so-called “cells”). This simplified example shows only three broadband colors in the observer’s frame, for a random subsample of 10 000 COSMOS2020 galaxies. 3.1. Training the SOM with COSMOS2020 Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 3 10 50 100 # of galaxies 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 1 2 3 4 5 average distance from cell's weight 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 1 5 10 50 # of MIPS galaxies Fig. 2. SOM of COSMOS2020 galaxies at z < 1.8, selected as described in Sect. 3.1. The same grid is color-coded in three different ways, to show the number of galaxies per cell (left panel), the similarity between galaxies in a given cell and the correspondent SOM weight (middle panel), and the MIPS-detected objects with MLePh > 1010 M⊙(right panel). In each grid, empty cells are white. Left: handful of cells with only one or two objects are colored in gray. Middle: similarity is quantified by means of Eq. (2). Throughout this work, the dimensions of the 2D grid are arbitrarily labeled D1 and D2 since they have no physical meaning. 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 3 10 50 100 # of galaxies 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 1 5 10 50 # of MIPS galaxies 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 average distance from cell's weight 0 D1 D1 D1 Fig. 2. SOM of COSMOS2020 galaxies at z < 1.8, selected as described in Sect. 3.1. The same grid is color-coded in three different ways, to show the number of galaxies per cell (left panel), the similarity between galaxies in a given cell and the correspondent SOM weight (middle panel), and the MIPS-detected objects with MLePh > 1010 M⊙(right panel). In each grid, empty cells are white. Left: handful of cells with only one or two objects are colored in gray. Middle: similarity is quantified by means of Eq. (2). (3) The key advantage of the SOM is that after the training, other data sets can be “projected” onto the grid in a fast and efficient way. Training is the most time-consuming part (0.3 CPU hour in the COSMOS2020 case), while several thousands galaxies can be subsequently projected in a few seconds. The additional data set used here is the SXDF2018 galaxy catalog described in Sect. 2. For sake of consistency, the properties of the SXDF galaxies are derived as done in COSMOS2020 (Weaver et al. 2022, see also Sect. 2.3). However, the only quantity the SOM method needs from the SXDF2018 catalog, besides the observed colors, is redshift for the pre-selection mentioned above. Other properties derived by LePhare, namely, stellar mass and SFR, will be only used a posteriori to compare the two methods. where mi is the original i-band magnitude of the given galaxy. The median of the Mi=22.5 distribution in a given cell is used as its label (Mlabel). In the upper-right panel of Fig. 4 the SOM is color- coded according to that label set, showing a smooth transition from 108 M⊙in the upper-left corner of the grid, corresponding to z ∼0, up to 1012 M⊙in the bottom right. This trend follows, as expected, the evolution in the z label: the higher the redshift, the larger the stellar mass at the fixed 22.5 mag reference. Regarding galaxy star formation, the calibration is done by using only objects with an available SFRUVIR estimate. A rescal- ing similar to Eq. (3) is applied for the same reason: galaxies in any given cell have similar colors by SOM construction, but this does not prevent them from freely spanning a certain magnitude range. Therefore, we define: The procedure described in the following is not strictly designed for SXDF. In principle, it can be applied to any survey that includes the same features used to build the SOM. How- ever, the mapping can be affected by non-negligible systematics if the new survey has a very different set-up with respect to COS- MOS2020, especially regarding its noise properties. This is not an issue for SXDF2018, which has been built using the same instruments, filters, and pipeline of the training data set. SFRi=22.5 = 10+0.4(mi−22.5)SFRUVIR, (4) (4) and we attach a SFRlabel value to the cell based on the median of the SFRi=22.5 distribution. We disregard cells containing only one MIPS-detected galaxy. 3.1. Training the SOM with COSMOS2020 To quantify the accuracy of the 80×80 SOM grid in describ- ing the COSMOS2020 manifold, we calculate the Euclidean dis- tance between galaxies and the weight of the cell associated with them. Namely: ∆= sX Ndim ( fi −wi)2, (2) SEDs with similar shapes but a low S/N ratio. As an example, a couple of those cells will be inspected in Sect. 5.4. We also ver- ify that galaxies that are close to each other in the feature space turn out to be in the same (or nearby) cells, as partially shown in Fig. 3. Another metric with which to quantify goodness of fit is the reduced χ2 distance used, for instance, in Masters et al. (2015). This is further discussed in Appendix B (see in particular Fig. B.1). SEDs with similar shapes but a low S/N ratio. As an example, a couple of those cells will be inspected in Sect. 5.4. We also ver- ify that galaxies that are close to each other in the feature space turn out to be in the same (or nearby) cells, as partially shown in Fig. 3. Another metric with which to quantify goodness of fit is the reduced χ2 distance used, for instance, in Masters et al. (2015). This is further discussed in Appendix B (see in particular Fig. B.1). ∆= sX Ndim ( fi −wi)2, (2) where fi is a galaxy’s feature in the ith dimension (out of Ndim = 11) and wi is one component of the weight representing the cell where the galaxy lies. Across the grid the average ∆per cell is <1 (Fig. 2, middle panel) as it should be if the scatter of observed SEDs around their weight is dominated by photometric uncer- tainties, which for the chosen broadband colors are typically 0.05−0.1 mag. A few cells, however, present larger ∆values, an indication they contain either a wider variety of galaxy types or To conclude the presentation of the training sample, the third (right-hand) panel of Fig. 2 shows the MIPS-detected galaxies in COSMOS. This subsample is spread over 3561 cells (55.6% of the total) which are mainly located on the right half of the grid A34, page 5 of 22 A&A 665, A34 (2022) where quiescent galaxies have: where quiescent galaxies have: (see Fig. 2, right panel). One third of the cells probed by MIPS galaxies contain only one of them. 9 COSMOS2020 includes a flag to clean the catalog from AGN. 10 Implemented in scikit-learn (Pedregosa et al. 2011). 3.1. Training the SOM with COSMOS2020 with C ≡0.17(t −tz=2) being a cosmic time-dependent cor- rection to maintain consistent criteria across the whole redshift range. From Eq. (5), we derive the fraction of quiescent galaxies per cell ( fQ) and use it as the fourth label of the SOM (Fig. 4, lower-right panel). In most of the cases the NUVrK classifica- tion agrees well with the direct assessment of star formation: 86% of the cells with a large quiescent fraction (fQ > 0.5) have either a specific SFR (sSFR, defined as SFR/M) lower than 10−11 yr−1 or do not contain MIPS-detected objects. Some dis- crepancy between the two indicators (i.e., MIPS-detected objects in cells with low sSFR) can be due to an active galactic nucleus (AGN) which has not been removed9 and is boosting the 24 µm signal (see e.g. Delvecchio et al. 2017). Another possibility is a break of the LIR–SFR correlation for post-starburst galaxies (Hayward & Smith 2015). The 68 cells (out of 496) that have fQ > 0.5 but show significant star formation activity (sSFR > 10−10 yr−1) are poorly sampled by the MIPS galaxies: most of them contain only one MIPS galaxy, which may be an inter- loper in that cell, for instance, owing to the similarity between the SEDs of dusty, star-forming galaxies and the “red and dead” ones (e.g., Arnouts et al. 2013). In the following we will opt for a conservative approach and disregard SFR measurements from cells with fQ > 0.9. 3.2. SOM calibration with physical properties The cells in a trained SOM can be calibrated a posteri- ori with redshift values (see e.g., Geach 2012; Masters et al. 2015) or other physical parameters (Hemmati et al. 2019a; Davidzon et al. 2019). We choose the labels to be redshift, stel- lar mass, SFR, and quiescent versus star-forming galaxy type; these four “layers” of classification are shown with different color codes in Fig. 4. For a given cell, the label for each of these quantities is calcu- lated using galaxies inside it. For redshift and stellar mass, this is defined as the median of the zLePh and the normalized MLePh dis- tribution, respectively (Fig. 4, upper panels). The reason for nor- malizing the mass is discussed in Davidzon et al. (2019). Their simulations show that when the SOM is trained with colors, objects in the same cell have similar mass-to-light ratios while still spanning a range of stellar masses (see Appendix B). We proceed as in Davidzon et al. (2019), namely, by rescaling MLePh estimates to 22.5 mag in the i band: 3.1. Training the SOM with COSMOS2020 Most of these undersampled cells define the boundaries of star-forming regions in the SOM, while others are scattered across the left side of the grid, an area that is scarcely occupied because of the MIPS selection function. In fact, those are cells containing galaxies with i > 23 mag and MLePh < 1010 M⊙(see discussion in Appendix B and Fig. B.3). Most of them are expected to be fainter than the survey sen- sitivity limit at 24 µm, as can be deduced, for instance, from Le Floc’h et al. (2009) and Kokorev et al. (2021). (NUV −r) + C > 2.6 and (NUV −r) + C > 2(r −Ks) + 1.7, (5) (NUV −r) + C > 2.6 d (NUV −r) + C > 2.6 and (5) (NUV −r) + C > 2(r −Ks) + 1.7, (NUV −r) + C > 2(r −Ks) + 1.7, with C ≡0.17(t −tz=2) being a cosmic time-dependent cor- rection to maintain consistent criteria across the whole redshift range. From Eq. (5), we derive the fraction of quiescent galaxies per cell ( fQ) and use it as the fourth label of the SOM (Fig. 4, lower-right panel). In most of the cases the NUVrK classifica- tion agrees well with the direct assessment of star formation: 86% of the cells with a large quiescent fraction (fQ > 0.5) have either a specific SFR (sSFR, defined as SFR/M) lower than 10−11 yr−1 or do not contain MIPS-detected objects. Some dis- crepancy between the two indicators (i.e., MIPS-detected objects in cells with low sSFR) can be due to an active galactic nucleus (AGN) which has not been removed9 and is boosting the 24 µm signal (see e.g. Delvecchio et al. 2017). Another possibility is a break of the LIR–SFR correlation for post-starburst galaxies (Hayward & Smith 2015). The 68 cells (out of 496) that have fQ > 0.5 but show significant star formation activity (sSFR > 10−10 yr−1) are poorly sampled by the MIPS galaxies: most of them contain only one MIPS galaxy, which may be an inter- loper in that cell, for instance, owing to the similarity between the SEDs of dusty, star-forming galaxies and the “red and dead” ones (e.g., Arnouts et al. 2013). In the following we will opt for a conservative approach and disregard SFR measurements from cells with fQ > 0.9. 3.3. Predictions for a new data set Mi=22.5 = 10+0.4(mi−22.5)MLePh, (3) A34, page 6 of 22 (3) The resulting labels are shown in Fig. 4 (lower-left panel). We map the SXDF2018 sample using a k-dimensional tree10 with k = 5 to find the five cells with the closest weight to each galaxy (including the cell that the galaxy is associated with). As in Davidzon et al. (2019), we prefer to work with k nearest neighbors instead of considering only the best matching cell, to take into account observational uncertainties. Physical properties can be predicted for any individual galaxy using the labels of its five nearest cells. For example, the stellar mass estimate MSOM Each COSMOS2020 galaxy can also be classified as quies- cent or active depending on its location in the rest-frame plane NUV−r versus r −Ks (NUVrK, Arnouts et al. 2013). This diag- nostic is one of the most common in the literature, together with U −V versus V −J (UVJ, Williams et al. 2009). Either dia- gram can single out quiescent galaxies from other types as the former ones occupy a delimited locus in the color-color spaces. The advantages of NUV −r with respect to U −V are discussed, for instance, in Siudek et al. (2018) and Leja et al. (2019a). We use the rest-frame NUVrK classification as in Ilbert et al. (2015), A34, page 6 of 22 I. Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy survey I. Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 0.0 0.5 1.0 1.5 2.0 redshift label 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 8 9 10 11 12 log(Mlabel [M ]) 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 1 0 1 2 3 log(SFRlabel [M yr 1]) 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 0.0 0.2 0.4 0.6 0.8 1.0 NUVrK quiescent fraction . 4. Different sets of labels added to the SOM, after training it with COSMOS2020 data. Upper left: redshift labels assigned from the med h of galaxies in the given cell. (3) Upper right: median log(Mlabel/M⊙) per cell, where the Mlabel values are galaxy stellar masses obtained a caling every SED to i = 22.5 (Eq. (3)). Lower left: labels from the median SFRlabel per cell applying the same 22.5 mag rescaling as for ste ss (Eq. (4)); with respect to Fig. 2, a larger portion of the SOM grid is empty because we did not assign a SFRlabel to cells containing only o PS galaxy. Lower right: fraction of quiescent galaxies per cell, according to the NUVrK classification (Eq. (5)). derived from the weighted mean of the Mlabel labels and read- ti it f th f i 22 5 t th it d f where ∆j is defined as in Eq. (2) and corresponds to the i hb th fi t ll (i l di th t h 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 8 9 10 11 12 log(Mlabel [M ]) 0 10 20 30 40 50 60 70 80 D 0 10 20 30 40 50 60 70 80 D2 0.0 0.5 1.0 1.5 2.0 redshift label 0.0 0.2 0.4 0.6 0.8 1.0 NUVrK quiescent fraction 3 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 log(SFRlabel [M yr 1]) log(SFRlabel [M yr 1]) NUVrK quiescent fraction 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 D1 Fig. 4. Different sets of labels added to the SOM, after training it with COSMOS2020 data. Upper left: redshift labels assigned from the median zLePh of galaxies in the given cell. Upper right: median log(Mlabel/M⊙) per cell, where the Mlabel values are galaxy stellar masses obtained after rescaling every SED to i = 22.5 (Eq. (3)). Lower left: labels from the median SFRlabel per cell applying the same 22.5 mag rescaling as for stellar mass (Eq. (4)); with respect to Fig. 2, a larger portion of the SOM grid is empty because we did not assign a SFRlabel to cells containing only one MIPS galaxy. Lower right: fraction of quiescent galaxies per cell, according to the NUVrK classification (Eq. (5)). Fig. 4. Different sets of labels added to the SOM, after training it with COSMOS2020 data. 4. Results In this section, our SOM-based analysis in SXDF is compared to other methods that independently derived stellar masses and SFR for the same objects. The alternate measurements for stel- lar masses are produced by means of LePhare, which fits BC03 templates to SEDs that includes not only the data used in the SOM but also UV photometry from GALEX, medium- and narrow-bands in the optical, and IRAC channel 3 and 4. These estimates are originally published in Mehta et al. (2018), but we re-run LePhare to update some of its parameters to the actual configuration used for COSMOS2020 (the redshift being fixed at the same value used in Mehta et al. 2018). For the SFR com- parison, we first compare to Barro et al. (2019), a study already introduced above which is restricted to the CANDELS ∼200 square arcmin area within SXDF. Like in Mehta et al. (2018), Barro et al. (2019) also exploit a set of observations larger than the 12 broadbands colors used to feed the SOM. Their study assumes the same cosmological parameters and IMF used here, but other adjustments related to Eq. (1) are required to convert their SFR estimates to a common ground. This homogenization procedure is described in Appendix A. Another set of indepen- dent estimates comes from spectroscopic data, where nebular emission lines can be used as a proxy for the SFR. We also derive zSOM even though in principle we could use zLePh, as the latter is required in input. This is intended to avoid inconsistency between the various properties of any given galaxy. Moreover, the choice allows us to generalize the method, which could be used also when high-quality photometric red- shifts are not available: in fact, the z < 1.8 cut could have been implemented with inferior redshift measurements or even a color selection (Guzzo et al. 2014). For validation purposes, we also build ten SOMs trained with COSMOS2020 after removing 3000 MIPS-detected galax- ies randomly extracted each time11. These “out-of-bag” objects belong to the MIPS parent sample, meaning that some of them have S/N < 5 and would not contribute to the calibration phase anyway. By construction, all the out-of-bag objects have a SFRUVIR estimate that can be compared to the SFRSOM obtained by projecting them on the respective SOM, similarly to what is done for the SXDF2018 catalog. 4. Results The log(SFRSOM/SFRUVIR) distribution is the same in the ten realizations, modulo stochastic fluctuations. By fitting them with a Gaussian function we find a standard deviation of 0.205 dex and a negligible offset (−0.005) with respect to the reference MIPS estimates (see Fig. 5). We repeat the Monte Carlo experiment only in the left side of the SOM that is undersampled by MIPS: for the galaxies that do receive a SOM prediction – that is, at least one of their nearest neighbors is labeled – we find that offset and scatter (µ = −0.005, σ = 0.205 respectively) are almost the same as the global exper- (3) 0.4 0.2 0.0 0.2 0.4 log(SFRSOM/SFRUVIR) 0 50 100 150 200 250 # of galaxies =-0.005 =0.205 Error bars on an object-by-object basis can be provided in different ways. Hemmati et al. (2019a) proposed a Monte Carlo re-mapping of the target galaxy, each time perturbing its col- ors within the corresponding 1σ uncertainty; the object is scat- tered in nearby cells and the minimum and maximum (mass or SFR) values it reaches are used as lower and upper limits of the error bar. Speagle et al. (2019) adopt a different approach, which assigns to each cell a probability of hosting the target galaxy proportional to its χ2 (see Eq. (B.1)). We tried both methods and found that they often provide significantly different error bars. A possible explanation is the fact that (i) in the Monte Carlo we perturbed colors independently (i.e., without taking into account covariance) or (ii) the color error bars do not correspond pre- cisely to a 68% uncertainty, resulting in a biased χ2. We pre- ferred to invest more time exploring this issue, postponing the ascription of error bars to MSOM and SFRSOM to a future study. Fig. 5. Validation of the SOM estimates with “out-of-bag” objects. The COSMOS2020 SOM described in Sect. 3 is built again ten times, excluding every time ∼3000 galaxies randomly extracted from those with a SFRUVIR. The figure compares SFRSOM and SFRUVIR for each SOM realization (histograms of different colors). The Gaussian fit to the ensemble of distributions (green line) has mean µ = −0.005 and standard deviation σ = 0.205 dex. with the caveat that one or more cells may not contain MIPS objects and therefore have no SFRlabel, j defined (Fig. 4, lower- left panel). When all the five neighbors are not labeled, a SOM estimate cannot be assigned. The choice of the number of neigh- bors is arbitrary, but increasing to more than five cells does not change the results as the additional neighbors are further away from the target galaxy’s cell and weighted in Eqs. (6) and (7) with a much smaller 1/∆j factor. (3) Upper left: redshift labels assigned from the median zLePh of galaxies in the given cell. Upper right: median log(Mlabel/M⊙) per cell, where the Mlabel values are galaxy stellar masses obtained after rescaling every SED to i = 22.5 (Eq. (3)). Lower left: labels from the median SFRlabel per cell applying the same 22.5 mag rescaling as for stellar mass (Eq. (4)); with respect to Fig. 2, a larger portion of the SOM grid is empty because we did not assign a SFRlabel to cells containing only one MIPS galaxy. Lower right: fraction of quiescent galaxies per cell, according to the NUVrK classification (Eq. (5)). is derived from the weighted mean of the Mlabel labels and read- justing it from the reference i = 22.5 mag to the magnitude of the target galaxy: where ∆j is defined as in Eq. (2) and corresponds to the jth neighbor among the five nearest cells (including the one to which the galaxy is attributed). The same applies to SFRSOM: MSOM = P5 j=1 1 ∆j Mlabel, j P5 j=1 1 ∆j × 10−0.4(mi−22.5), (6) SFRSOM = P5 j=1 1 ∆j SFRlabel,j P5 j=1 1 ∆j × 10−0.4(mi−22.5), (7) (6) SFRSOM = P5 j=1 1 ∆j SFRlabel,j P5 j=1 1 ∆j × 10−0.4(mi−22.5), (7) (7) (6) A34, page 7 of 22 A&A 665, A34 (2022) 0.4 0.2 0.0 0.2 0.4 log(SFRSOM/SFRUVIR) 0 50 100 150 200 250 # of galaxies =-0.005 =0.205 Fig. 5. Validation of the SOM estimates with “out-of-bag” objects. The COSMOS2020 SOM described in Sect. 3 is built again ten times, excluding every time ∼3000 galaxies randomly extracted from those with a SFRUVIR. The figure compares SFRSOM and SFRUVIR for each SOM realization (histograms of different colors). The Gaussian fit to the ensemble of distributions (green line) has mean µ = −0.005 and standard deviation σ = 0.205 dex. iment. The analog procedure is performed to establish the MSOM accuracy, dividing the out-of-bag sample in bins of stellar mass and comparing predictions with MLePh. In this case the uncer- tainties are µ = −0.018 and σ = 0.097 dex. Since they are cal- culated for targets coming from the same parent sample of the training galaxies, these values can be interpreted as lower limits for the MSOM and SFRSOM uncertainties that would result when projecting other data sets. 4.1. Stellar mass estimates The stellar mass comparison (Fig. 6) shows a good agreement between MLePh and MSOM. The systematic offset between the two, measured as log(MSOM/MLePh), is <0.02 dex. The relative scatter of 0.25 dex is nearly symmetric, with the exception of a ∼3% for which MSOM is severely underestimated (i.e., more than a factor 3). The majority of that subsample has 1.2 < zLePh < 1.8 and is located either along the borders of the grid (i.e., they suffer from “boundary effects”) or in the central area characterized by large ∆values (see Sect. 5.4). 11 In general, every training of the SOM results in a different outcome when the weights are initialized at random positions. For this task how- ever, we force the iterative adaptation to start from the same configura- tion of the reference SOM. Moreover, the number of galaxies removed is so small compared to the whole training sample that the ten new SOMs are almost indistinguishable from the original one. At a first glance, such an agreement may seem obvious because the set of Mlabel used to derive individual masses in the SOM is built from LePhare estimates (Eq. (3)). However, A34, page 8 of 22 Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys 8 9 10 11 log(MSOM [M ]) 8 9 10 11 log(MLePh [M ]) 0.5 0.0 0.5 log(MSOM/MLePh) 10 50 500 number of galaxies 8 9 10 11 log(MSOM [M ]) 8 9 10 11 log(MLePh [M ]) 0.5 0.0 0.5 log(MSOM/MLePh) 10 50 500 number of galaxies Fig. 6. Comparison between stellar masses obtained through standard template fitting (MLePh) and the new method presented in this work (MSOM). The figure shows the density map of 208 404 galaxies in the SXDF field with the magnitude cut at Ks < 24.2. Upper panel: a solid line marks the 1:1 relationship. Bottom panel: the logarithmic ratio log(MSOM/MLePh) is shown as a function of log(MLePh/M⊙), the solid line marks the zero offset while the two dotted lines are set at ±0.3 dex. adaptive resolution of neighboring cells, namely, the fact that in dense areas of the parameter space the SOM concentrates more weights. 4.2. SFR estimates First, we compared the SOM estimates to the SFRUVIR ones from Barro et al. (2019). The authors only analyze CANDELS- UDS, which is about one-twentieth of the whole SXDF area. As an additional restriction we select only the CANDELS galax- ies with UV-to-FIR data available. Also given the SOM cut at z < 1.8 and Ks < 24.23, there are only 608 galaxies in common with Barro et al. (2019). For AGN contamination, we consider the classification from Mehta et al. (2018) which is similar to the one applied to the COSMOS2020 training sam- ple. We also discard sources with discrepant redshift estimates, that is, the absolute value of the difference between zLePh and the photometric redshift from Barro et al. (2019) is greater than 0.3(1 + zLePh). The 608 sources are plotted in the left panel of Fig. 7, showing a fairly good agreement: the −0.05 dex off- set in log(SFRSOM/SFRUVIR) is comparable with the −0.03 dex initially found in the CANDELS-COSMOS comparison13. The SFR scatter (i.e., the standard deviation of the distribution in the left panel of Fig. 7) is 0.28 dex, comparable to that of the stellar mass distribution. The log(SFRSOM/SFRUVIR) histogram (inset in the left panel of Fig. 7) features a tail of a few objects that the SOM severely underestimates with respect to Barro et al. (2019), many of them being very dusty (AV > 2 mag) according to the latter. Dust correction is indeed one of the major param- eters responsible for this kind of discrepancy, as discussed, for instance, in Speagle et al. (2014). Fig. 6. Comparison between stellar masses obtained through standard template fitting (MLePh) and the new method presented in this work (MSOM). The figure shows the density map of 208 404 galaxies in the SXDF field with the magnitude cut at Ks < 24.2. Upper panel: a solid line marks the 1:1 relationship. Bottom panel: the logarithmic ratio log(MSOM/MLePh) is shown as a function of log(MLePh/M⊙), the solid line marks the zero offset while the two dotted lines are set at ±0.3 dex. there are several deviations from the SED fitting performed by LePhare that make the SOM method substantially different. First, the smaller number of photometric data points used in the latter, as emphasized above. 12 The precise number of templates that LePhare can use at any given z step depends on how many of them represent galaxies older than the age of the universe at that redshift; in fact, those models are excluded by default. 13 This is the residual offset after removing other sources of discrepancy such as IMF and IR calibration (see Fig. B.2). 4.1. Stellar mass estimates Another distinctive feature of the SOM, which may be regarded as an advantage or a shortcoming depending on the sci- entific goal, is that its collection of “empirical” weights is lim- ited by the survey’s characteristics (e.g., its selection function). On the other hand a synthetic template library, at least theoret- ically, may include models of known galaxy types that are not observed in that survey (e.g., low-mass objects fainter than the detection limit), but it may also miss real objects that have not been correctly simulated as templates. Given these differences, the small fraction of catastrophic errors and the overall tight 1:1 correlation are a remarkable confirmation of the effectiveness of the SOM method. 4.2. SFR estimates In addition, the available “solutions” in the SOM fit are much smaller, given the limited number of cells used to describe the data manifold (6400 in total). On the contrary, the synthetic templates used by LePhare are between five and eight thousands at every fixed z step12 and over two mil- lions in total. Another star formation proxy that can be used for compar- ison is the luminosity of Hα nebular emission line (LHα). We assume SFRHα = 2.1 × 10−8 × LHα (Kennicutt 1998) after cor- recting for dust attenuation in the host galaxy. The formula for such a correction is the same as in Kashino et al. (2013), based on the stellar color excess E(B −V) parametrized by LePhare while measuring the photometric redshift. It is more convenient to perform this test in the COSMOS field instead of SXDF, because we have access to an unparal- leled amount of spectroscopic data in the former. The most rel- evant surveys to our purposes are zCOSMOS (Lilly et al. 1996), 3DHST (Brammer et al. 2012; Momcheva et al. 2016), KROSS (Harrison et al. 2017), and FMOS (Kashino et al. 2019), target- ing different galaxy types from z ∼0.1 up to 1.7. They have been selected and merged into a single spectroscopic catalog in Saito et al. (2020). Because of the overlap between these line emitters and the SOM calibration sample, we prefer to compare SFRHα versus SFRSOM for the out-of-bag galaxies of the SOM bootstrap test (see Sect. 3.3 and Fig. 5). Assuming SFRHα to be We also notice that in the SOM method only the weights in the surroundings of the galaxy target are relevant for the MSOM calculation (see Eq. (6)), whereas there are no mass priors in LePhare. In the library of the latter, the BC03 galaxy models are all defined at 1 M⊙, but each of them can be rescaled to fit the observed SED. This means that each template can span the entire stellar mass range of the target pool, that is, it would be eligi- ble to fit any observed galaxies irrespective of its mass (or other characteristics). 4.2. SFR estimates Right panel: comparison to another star formation estimator, that is, Hα nebular emission line. In this case the comparison is performed in COSMOS, for 3718 galaxies (colored dots). In both panels, the color palette from violet to red indicates the stellar mass of each galaxy according to the SOM method; the solid line is the 1:1 relation from which two dashed lines are set at a distance of ±0.3 dex. Each panel also includes an inset that quantifies the dispersion between SFRSOM and the alternate estimate (red histogram); the vertical dotted line marks the median offset, which is −0.05 dex for log(SFRSOM/SFRUVIR) and 0.07 dex for log(SFRSOM/SFRUVIR). 9.0 9.5 10.0 10.5 11.0 log(MSOM [M ]) 1.0 0.5 0.0 0.5 1.0 1.5 2.0 log(SFRSOM [M yr 1]) t 9.7 Gyr (z 0.3) t 7.7 Gyr (z 0.6) t 5.8 Gyr (z 1.0) t 4.1 Gyr (z 1.5) Fig. 8. Evolution of the main sequence of star forming galaxies resulting from the SOM-based measurement of the SXDF2018 catalog. Redshift bins are chosen to correspond to four time steps separated by ∼2 Gyr, whose color is indicated in the legend along with median redshift and cosmic age. In each bin, the main sequence is identified as the most prominent overdensity in the SFRSOM−MSOM plane (shaded areas repre- senting density contours). Each overdensity peak is fit by Eq. (8) either independently (solid lines) or simultaneously at all t (dashed lines). 9.0 9.5 10.0 10.5 11.0 log(MSOM [M ]) 1.0 0.5 0.0 0.5 1.0 1.5 2.0 log(SFRSOM [M yr 1]) t 9.7 Gyr (z 0.3) t 7.7 Gyr (z 0.6) t 5.8 Gyr (z 1.0) t 4.1 Gyr (z 1.5) the “ground truth”, the comparison is a further validation of the SFRSOM estimates, presented in the right panel of Fig. 7. The resulting scatter (0.4 dex) and systematics (+0.07 dex) are rel- atively contained considering the differences between the two methods, especially the shorter time scale of star formation (on the order of 10 Myr) probed by LHα. We also note that the 0.07 dex offset shown in the figure is mainly due to the most massive galaxies, which may have a biased SFRHα: in fact, their dust content is usually high and susceptible to be underestimated by LePhare, whose templates are limited to E(B −V) ≤0.7 and may not assume the correct extinction law (Ilbert et al. 2010; Walcher et al. 2011). 4.2. SFR estimates Moreover, the choice of either using or disre- garding a SOM weight for a certain galaxy is modulated by the A34, page 9 of 22 A&A 665, A34 (2022) 1 0 1 2 log(SFRUVIR[M yr 1]) Barro+19 1 0 1 2 log(SFRSOM[M yr 1]) 1 0 1 log(SFRSOM/SFRUVIR) 1 0 1 2 log(SFRH [M yr 1]) 1 0 1 2 log(SFRSOM[M yr 1]) 8.5 9.0 9.5 10.0 10.5 11.0 11.5 log(MSOM/M ) 1 0 1 log(SFRSOM/SFRH ) Fig. 7. Quality assessments of the SFRSOM estimates using independent measurements from the literature as “ground truth”. Left panel: comparison between SFRSOM and the template-based estimates from Barro et al. (2019, SFRUVIR) for 608 galaxies at z < 1.8 in the SXDF-CANDELS area (colored dots). For sake of consistency, the Barro et al. (2019) estimates are converted to the same reference system of Ilbert et al. (2015), since the latter has also been used to calibrate the SOM method (see Appendix A). Right panel: comparison to another star formation estimator, that is, Hα nebular emission line. In this case the comparison is performed in COSMOS, for 3718 galaxies (colored dots). In both panels, the color palette from violet to red indicates the stellar mass of each galaxy according to the SOM method; the solid line is the 1:1 relation from which two dashed lines are set at a distance of ±0.3 dex. Each panel also includes an inset that quantifies the dispersion between SFRSOM and the alternate estimate (red histogram); the vertical dotted line marks the median offset, which is −0.05 dex for log(SFRSOM/SFRUVIR) and 0.07 dex for log(SFRSOM/SFRUVIR). 1 0 1 2 log(SFRH [M yr 1]) 1 0 1 2 log(SFRSOM[M yr 1]) 8.5 9.0 9.5 10.0 10.5 11.0 11.5 log(MSOM/M ) 1 0 1 log(SFRSOM/SFRH ) 1 0 1 2 log(SFRUVIR[M yr 1]) Barro+19 1 0 1 2 log(SFRSOM[M yr 1]) 1 0 1 log(SFRSOM/SFRUVIR) log(SFRUVIR[M yr 1]) Barro+19 Fig. 7. Quality assessments of the SFRSOM estimates using independent measurements from the literature as “ground truth”. Left panel: comparison between SFRSOM and the template-based estimates from Barro et al. (2019, SFRUVIR) for 608 galaxies at z < 1.8 in the SXDF-CANDELS area (colored dots). For sake of consistency, the Barro et al. (2019) estimates are converted to the same reference system of Ilbert et al. (2015), since the latter has also been used to calibrate the SOM method (see Appendix A). 4.2. SFR estimates Moreover, in (local) massive galaxies, neb- ular extinction often adds a greater contribution to stellar dust extinction (van der Giessen et al. 2022) and BC03 models do not take this into account. Fig. 8. Evolution of the main sequence of star forming galaxies resulting from the SOM-based measurement of the SXDF2018 catalog. Redshift bins are chosen to correspond to four time steps separated by ∼2 Gyr, whose color is indicated in the legend along with median redshift and cosmic age. In each bin, the main sequence is identified as the most prominent overdensity in the SFRSOM−MSOM plane (shaded areas repre- senting density contours). Each overdensity peak is fit by Eq. (8) either independently (solid lines) or simultaneously at all t (dashed lines). Fig. 8. Evolution of the main sequence of star forming galaxies resulting from the SOM-based measurement of the SXDF2018 catalog. Redshift bins are chosen to correspond to four time steps separated by ∼2 Gyr, whose color is indicated in the legend along with median redshift and cosmic age. In each bin, the main sequence is identified as the most prominent overdensity in the SFRSOM−MSOM plane (shaded areas repre- senting density contours). Each overdensity peak is fit by Eq. (8) either independently (solid lines) or simultaneously at all t (dashed lines). Fig. 8. Evolution of the main sequence of star forming galaxies resulting from the SOM-based measurement of the SXDF2018 catalog. Redshift bins are chosen to correspond to four time steps separated by ∼2 Gyr, whose color is indicated in the legend along with median redshift and cosmic age. In each bin, the main sequence is identified as the most prominent overdensity in the SFRSOM−MSOM plane (shaded areas repre- senting density contours). Each overdensity peak is fit by Eq. (8) either independently (solid lines) or simultaneously at all t (dashed lines). 14 See, in particular, their Fig. 1. 4.3. Main sequence of star-forming galaxies the other two include undetected galaxies by stacking either 3 GHz VLA (Leslie et al. 2020) or Herschel (Schreiber et al. 2015) images. Ilbert et al. (2015) derived the MS from MIPS- detected galaxies but in an indirect way, through the SFR func- tion. All the selected studies identify the MS locus by binning their sample in stellar mass and then computing the median SFR in each bin. Although such an approach is suboptimal from a mathematical point of view (Steinhardt & Jermyn 2018), we decided nonetheless to proceed in the same way as the other authors have done in order to maintain coherence17. z range ⟨z⟩ m0 m1 a0 a1 a2 0.30 < z < 0.40 ⟨0.34⟩ 0.44 0.0 0.7 0.19 0.0 0.50 < z < 0.70 ⟨0.60⟩ 0.44 0.1 1.6 0.20 0.6 0.90 < z < 1.10 ⟨0.99⟩ 0.38 0.0 2.1 0.18 0.0 1.40 < z < 1.80 ⟨1.53⟩ 0.06 0.0 1.3 0.14 0.0 0.30 < z < 1.80 – 0.75 0.0 3.2 0.17 0.0 Notes. Parameters in the bottom row result from the simultaneous fit to all the four redshift bins. The SOM results are in good agreements with the other MS measurements (Fig. 9) showing that SFRSOM and MSOM esti- mates are accurate not only when evaluated separately. Overall, the differences between our MS and previous results are com- parable with the differences between them, and smaller than the 1σ error bars that we defined as the 16th–84th percentile range in each mass bin. There are, however, two visible systematics, at both extremes of the probed mass range, which are worth noticing. At the low-mass end, our median points slightly devi- ate from the linear extrapolation of the MS. This is related to a sample incompleteness in those mass bins and a consequent skewness of the SFR distribution towards higher values. The incompleteness is shown in Fig. 9 by white and orange filled circles, marking bins in which the fraction of galaxies with a defined SFRSOM drops below 50 and 70%, respectively18. The other systematic effect concerns the most massive bin, which at z > 1 is always located above the other studies (although within ∼1σ from them, see Fig. 9). In general, our median SFRs do not show the high-mass turnover observed in Ilbert et al. (2015), Lee et al. (2015), and subsequent studies (see discussion in Sect. 5.2). 4.3. Main sequence of star-forming galaxies Besides that, the overall slope and redshift evolu- tion of our MS agree with previous work confirming the reliabil- ity of the SOM method. Notes. Parameters in the bottom row result from the simultaneous fit to all the four redshift bins. effects because galaxies falling far from the labeled cells do not obtain any SFRSOM estimate by construction. Irrespective of that potential bias, which will be discussed in Sect. 5.2, the SXDF2018 sample includes a wide range of galaxies, from star- bursts above the MS to galaxies with low specific star formation rates. To identify the ridge of the star forming sequence we apply the following procedure in every zSOM bin. First, a Gaussian kernel density estimator15 is used to build a probability distri- bution map of SXDF2018 objects in the log(SFRSOM) versus log(MSOM) space. Each map is shown in Fig. 8 with isoden- sity contours of different colors. Resampling them with a grid of points equally spaced by 0.05 dex we find the ridge of the den- sity peak (i.e., the “backbone” of the MS) and track its evolution over ∼6 Gyr. The ridges in the four redshift bins are interpolated by the same function used in Schreiber et al. (2015): y = m −m0 + a0r −a1[max(0, m −m1 −a2r)]2, (8) y = m −m0 + a0r −a1[max(0, m −m1 −a2r)]2, (8) (8) where r ≡log(1 + z), m is the logarithmic stellar mass in units of 109 M⊙, and y is the logarithmic SFR in M⊙yr−1. The other five parameters (m0, m1, a0, a1, a2) are obtained via non- linear least-square minimization using a trust region reflective algorithm16. The best-fit values for the free parameters, listed in Table 2, result into the solid lines shown in Fig. 8. We also fit the four sequences together (dashed lines) obtaining the following parameters: (m0, m1, a0, a1, a2) = (0.75 ± 0.04, 0.0 ± 0.2, 3.2 ± 0.2, 0.17 ± 0.04, 0.0 ± 0.5). The terms m1 and a2 being consis- tent with zero indicates that there is no need for second-order corrections to describe the MS turnover at high masses. This is the major distinction with respect to the MS of Schreiber et al. (2015), where the bending is more pronounced. In fact, thanks to the stacking of Herschel images, (Schreiber et al. 2015) gain sensitivity to measure also galaxies with very low sSFR. 4.3. Main sequence of star-forming galaxies With the SXDF2018 photometric redshifts, stellar masses, and SFRs obtained through the SOM we can trace the main sequence of star-forming galaxies (MS) and its evolution as a function of cosmic time. The SFRSOM versus MSOM plane at different red- shifts is shown in Fig. 8, where we choose the following bins in order to ensure homogeneous time intervals: 0.3 < z ≤0.4, 0.5 < z ≤0.7, 0.9 < z ≤1.1, 1.4 < z ≤1.8. The four bins are centered around median redshifts of z = 0.3, 0.6, 1.0, and 1.5 cor- responding to t = 9.8, 7.7, 5.7, and 4.2 Gyr after the Big Bang. The time step between the last two bins is the only one shorter than 2 Gyr because of the z < 1.8 upper limit imposed by MIPS. Galaxies at z ≲0.2 are not included since COSMOS probes a small cosmic volume below that redshift, thus losing most of its statistical power. A more conventional binning is used later in this section for the purpose of making a compare with the literature. Similarly to the χ2 cut applied in many template-fitting studies (see e.g., Bowler et al. 2015), we removef the “bad fit” objects having ∆> 5. This threshold rules out nearly 1% of the galaxies whose SED is not well represented by the weight of their own cell. To locate the MS we apply a procedure inspired by the MS definition in Renzini & Peng (2015). In that study14, the MS nat- urally emerges as a peak in the surface formed by all the galax- ies in the 3D space SFR versus mass verus number of objects, without any a priori separation between star forming and quies- cent. In the Renzini & Peng (2015) framework the MS core is the “ridge” of such a 3D peak, a line that is stable whether or not the data set includes, for instance, post-starburst galaxies exiting the MS. A similar approach, not requiring a preselection of the star-forming sample, was recently proposed in Leja et al. (2021). The SOM method, however, is not entirely free from selection A34, page 10 of 22 I. Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys Table 2. Best-fit values of the free parameters in Eq. (8) to describe the main sequence of star-forming galaxies at different redshifts. 15 Namely, the gaussian_kde method (Scott 2015) from the scipy suite (Virtanen et al. 2020). 16 Implemented in scipy Branch et al. (1999). 17 Before the actual comparison, however, we converted the results to the same framework as described in Appendix A; see also Speagle et al. (2014) for a thorough discussion about how to homogenize miscella- neous data from the literature. 18 The fraction is calculated with respect to the total number of objects in the given stellar mass bin and classified as star forming by Eq. (5). 4.3. Main sequence of star-forming galaxies The best-fit parameters they find are (m0, m1, a0, a1, a2) = (0.5 ± 0.07, 0.36 ± 0.03, 1.5 ± 0.15, 0.3 ± 0.08, 2.5 ± 0.06). For sake of completeness, we also try a linear interpolation using the func- tion y = (α0t+α1) log(M/M⊙)+(β0t+β1) as done in Speagle et al. (2014, in Eq. (17)), but the goodness of fit is inferior to Eq. (8) and, therefore, it is not shown in the figure. 5.1. SOM-based estimates versus synthetic templates A&A 665, A34 (2022) 9 10 11 1 0 1 2 0.2 < z 0.4 z = 0.33 9 10 11 0.4 < z 0.6 z = 0.50 9 10 11 0.6 < z 0.8 z = 0.71 9 10 11 0 1 2 log(SFR [M yr 1]) 0.8 < z 1.0 z = 0.90 9 10 11 1.0 < z 1.2 z = 1.12 9 10 11 1.2 < z 1.4 z = 1.29 9 10 11 0 1 2 3 1.4 < z 1.6 z = 1.51 9 10 11 log(M [M ]) 1.6 < z 1.8 z = 1.70 Leslie+20 Schreiber+15 Whitaker+14 This work Ilbert+15 Barro+19 log(SFR [M yr 1]) 9 10 11 9 10 11 1.2 < z 1.4 z = 1.29 11 10 9 10 9 10 11 Leslie+20 Schreiber+15 Whitaker+14 This work Ilbert+15 Barro+19 9 10 11 Leslie+20 Schreiber+15 Whitaker+14 This work Ilbert+15 Barro+19 10 11 11 log(M [M ]) Fig. 9. Redshift evolution of the MS of star forming galaxies (see Sect. 4.3) as determined in the present work (red circles), Ilbert et al. (2015, blue diamonds), Barro et al. (2019, pink squares), Whitaker et al. (2014, blue dotted line), Schreiber et al. (2015, brown solid line), and Leslie et al. (2020, green dashed line). Our estimates stop at the z-dependent threshold for stellar mass completeness defined in Weaver et al. (2022). Their error bars are the 16th–84th percentile range and each symbol is filled with a shade of red according to the completeness of the given mass bin; in particular, dark red symbols represent >90% completeness, orange between 60 and 80%, while white-filled circles have ≲50% completeness (see Sect. 5.2). Results from the literature are shown only when the median redshift of the given study is sufficiently close to ours (which is indicated in the upper-left corner of each panel); the same measure may be repeated in more than one panel: for example, the MS of Barro et al. (2019) at 0.5 < z < 1 has a median redshift ⟨z⟩= 0.8 and it is compared to our data both at 0.6 < z < 0.8 and 0.8 < z < 1.0. licity and E(B −V) values, and there are limited options to add nebular emission line contamination (Pacifici et al. 2015; Yuan et al. 2019). 5.1. SOM-based estimates versus synthetic templates To assess any improvement the SOM may provide over stan- dard template fitting, the latter has to be compared to reference estimates (Barro et al. 2019) as it has been done with SFRSOM (Sect. 4.2). Figure 10 shows such a test in the overlapping part of the SXDF field (see Appendix B for COSMOS). When exactly the same 12-band photometry is used (cf. Table 1), the scatter in log(SFRLePh/SFRUVIR) is significantly larger than what found for the SOM-based method (cyan circles in Fig. 10). The dis- tribution becomes narrower, and less skewed, if additional col- ors (25 filters in total) are provided as input to LePhare (blue squares in Fig. 10). Either way, the fit is performed without data points in the FIR regime. For the 12-band fitting we run LePhare using the same version and set-up of COSMOS2020, while the fit to the extended photometry comes from Mehta et al. (2018). Therefore, the latter estimates not only include ancillary data (medium- and narrow-band filters from Subaru and VISTA tele- scopes) but are also derived with a configuration of LePhare that is optimized for SXDF2018. However, even in that case, the template-based estimates are less precise than SFRSOM. g We also select from the literature some of the most rel- evant studies that have measured the MS between z = 0 and 2, namely, Whitaker et al. (2014), Schreiber et al. (2015), Ilbert et al. (2015), Barro et al. (2019), Leslie et al. (2020). To estimate galaxy SFRs those authors use different methods, all based on FIR data with the exception of Leslie et al. (2020) per- forming a radio analysis. Two of these studies deal with individ- ual sources only (Whitaker et al. 2014; Barro et al. 5.1. SOM-based estimates versus synthetic templates 2019) while A34, page 11 of 22 A34, page 11 of 22 A34, page 11 of 22 A&A 665, A34 (2022) A&A 665, A34 (2022) 9 10 11 1 0 1 2 0.2 < z 0.4 z = 0.33 9 10 11 0.4 < z 0.6 z = 0.50 9 10 11 0.6 < z 0.8 z = 0.71 9 10 11 0 1 2 log(SFR [M yr 1]) 0.8 < z 1.0 z = 0.90 9 10 11 1.0 < z 1.2 z = 1.12 9 10 11 1.2 < z 1.4 z = 1.29 9 10 11 0 1 2 3 1.4 < z 1.6 z = 1.51 9 10 11 log(M [M ]) 1.6 < z 1.8 z = 1.70 Leslie+20 Schreiber+15 Whitaker+14 This work Ilbert+15 Barro+19 Fig. 9. Redshift evolution of the MS of star forming galaxies (see Sect. 4.3) as determined in the present work (red circles), Ilbert et al. (2015, blue diamonds), Barro et al. (2019, pink squares), Whitaker et al. (2014, blue dotted line), Schreiber et al. (2015, brown solid line), and Leslie et al. (2020, green dashed line). Our estimates stop at the z-dependent threshold for stellar mass completeness defined in Weaver et al. (2022). Their error bars are the 16th–84th percentile range and each symbol is filled with a shade of red according to the completeness of the given mass bin; in particular, dark red symbols represent >90% completeness, orange between 60 and 80%, while white-filled circles have ≲50% completeness (see Sect. 5.2). Results from the literature are shown only when the median redshift of the given study is sufficiently close to ours (which is indicated in the upper-left corner of each panel); the same measure may be repeated in more than one panel: for example, the MS of Barro et al. (2019) at 0.5 < z < 1 has a median redshift ⟨z⟩= 0.8 and it is compared to our data both at 0.6 < z < 0.8 and 0.8 < z < 1.0. 5.1. SOM-based estimates versus synthetic templates The SOM is color coded to show their cell occupation, renormalized to the number density of COSMOS2020 galaxies to make this figure directly comparable with the left-hand panel of Fig. 2. colors is built with a flat redshift distribution and no differenti- ation in the abundance of galaxy types. This has an impact on the whole SED fitting process comparable to the bias from miss- ing templates (empty cells in Fig. 11). Especially in codes like LePhare, which do not take into account prior probabilities, if a “family” of (z, M, SFR) models is over- or under-represented in the library then the output quantities will be biased too. the expenses of computational speed. In that regard, the advan- tage of SOM is well illustrated in Hemmati et al. (2019a): the time to process 107 objects is less than 0.3 CPU hours, while a typical Bayesian fitting run (e.g., Mehta et al. 2021) would take a similar amount of time for a single object (V. Mehta, priv. comm.). Such a trade-offmay dramatically change in the future, as cheaper machines (i.e., the possibility to rent or buy more CPU time) and more efficient Bayesian methods become avail- able. A promising example in this direction is the SED infer- ence method proposed by Hahn & Melchior (2022) that employs simulation-trained neural networks to accelerate the estimate posterior probability distributions at the pace of 1 s per galaxy (which despite the dramatic improvement would still exceed the 0.3 CPU hours of the SOM to analyze our sample). The severity of this last issue shall decrease in future studies owing to the expected improvement of telescope surveys, since a higher photometric quality shall result in more robust likelihood functions. In such a scenario, the likelihood would drive the SED solution to eventually make the code less sensitive to the models’ priors, but if a model were completely missing from the library (not just over- or underrepresented), then the problem discussed above would persist in spite of the increased quality of the input data. A treatment of probability distribution functions including probability priors is presented in Benítez (2000) and Tanaka (2015) for redshift measurements only. The same Bayesian for- malism is also the foundation of state-of-the-art SED fitting codes to estimate physical properties, with notable examples including BAGPIPES19 and Prospector20. 5.1. SOM-based estimates versus synthetic templates Part of the parameter space covered by the models might have no correspondence in the observed uni- verse (see the discussion in Marchesini et al. 2010; Muzzin et al. 2013 concerning dusty, passive galaxy templates). Moreover, the grid discretization may introduce severe biases, as investi- gated in Mitchell et al. (2013). Besides these approximations, the simplistic modelling of dust attenuation plays a major role (Chevallard et al. 2013; Chevallard & Charlot 2016; Laigle et al. 2019). The effective performance of our method is mainly due to the empirical collection of galaxy prototypes that the SOM creates during training (see also the “phenotypes” in Sánchez & Bernstein 2019). Similarly to eigenvectors in a prin- cipal component analysis, the SOM weights are adapted to the observed sample so that “by construction” their colors represent realistic SEDs, to which physical properties are attached. These properties can be derived from scaling relations or other empiri- cal recipes, which, despite their underlying assumptions, offer a complementary approach to the use of stellar population synthe- sis models. To better visualize these concepts, we derive observed-frame colors for the BC03 templates in the LePhare library, shifting them from z = 0.2 to 1.8 with a linear increment of ∆z = 0.01. Once projected on the COSMOS2020 SOM, these templates occupy only a fraction of the grid (Fig. 11) even after perturbing their photometry with Gaussian-shaped noise (0.05 mag stan- dard deviation for every band). Their distribution (number of templates per cell) is very different compared to that of COS- MOS2020 galaxies (cf. Fig. 11 and the left-hand panel of Fig. 2). Such a tension is not surprising because the sample of BC03 In standard template fitting, the library is generated from the- oretical models that might not be an accurate description of the observed targets – or even of their parent populations – espe- cially regarding their star formation scenarios. In fact, many tem- plate libraries (including the one in Weaver et al. 2022) are built by using a simplistic SFH parametrization (exponential-τ and delayed-τ models, see Ilbert et al. 2013) with a limited num- ber of time steps, which may be inadequate, for instance, for starburst galaxies (Pacifici et al. 2013). Moreover, the physical parameter space is sampled by a coarse grid of stellar metal- A34, page 12 of 22 I. 5.1. SOM-based estimates versus synthetic templates (2019) and LePhare using the same sample of 608 galaxies also shown in the left panel of Fig. 7. The same BC03 library is fit by LePhare to the full photometric base- line available in the SXDF2018 catalog (filled blue circles) and to the 12 filters only used in the SOM method (open cyan circles). Same colors are used in the inset for the histograms showing the respective log(SFRLePh/SFRUVIR) distributions; the log(SFRSOM/SFRUVIR) distri- bution (red histogram, same as the inset in the left panel of Fig. 7) is also included as reference. 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 10 50 100 # of BC03 templates (renorm.) 100 0 1 2 log(SFRUVIR[M yr 1]) Barro+19 0.0 0.5 1.0 1.5 2.0 2.5 log(SFRLePh[M yr 1]) BC03 fit to 25 bands BC03 fit to 12 bands 1 0 1 log(SFRLePh/SFRUVIR) 10 # of BC03 templates (renorm.) log(SFRUVIR[M yr 1]) Barro+19 Fig. 10. Comparison between Barro et al. (2019) and LePhare using the same sample of 608 galaxies also shown in the left panel of Fig. 7. The same BC03 library is fit by LePhare to the full photometric base- line available in the SXDF2018 catalog (filled blue circles) and to the 12 filters only used in the SOM method (open cyan circles). Same colors are used in the inset for the histograms showing the respective log(SFRLePh/SFRUVIR) distributions; the log(SFRSOM/SFRUVIR) distri- bution (red histogram, same as the inset in the left panel of Fig. 7) is also included as reference. Fig. 10. Comparison between Barro et al. (2019) and LePhare using the same sample of 608 galaxies also shown in the left panel of Fig. 7. The same BC03 library is fit by LePhare to the full photometric base- line available in the SXDF2018 catalog (filled blue circles) and to the 12 filters only used in the SOM method (open cyan circles). Same colors are used in the inset for the histograms showing the respective log(SFRLePh/SFRUVIR) distributions; the log(SFRSOM/SFRUVIR) distri- bution (red histogram, same as the inset in the left panel of Fig. 7) is also included as reference. D1 Fig. 11. BC03 templates from the LePhare library projected into the SOM. The templates are shifted from z = 0.2 to 1.8 with steps of ∆z = 0.01, and perturbed with Gaussian noise to mimic real photometry. 5.1. SOM-based estimates versus synthetic templates Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 10 50 100 # of BC03 templates (renorm.) Fig. 11. BC03 templates from the LePhare library projected into the SOM. The templates are shifted from z = 0.2 to 1.8 with steps of ∆z = 0.01, and perturbed with Gaussian noise to mimic real photometry. The SOM is color coded to show their cell occupation, renormalized to the number density of COSMOS2020 galaxies to make this figure directly comparable with the left-hand panel of Fig. 2. 0 1 2 log(SFRUVIR[M yr 1]) Barro+19 0.0 0.5 1.0 1.5 2.0 2.5 log(SFRLePh[M yr 1]) BC03 fit to 25 bands BC03 fit to 12 bands 1 0 1 log(SFRLePh/SFRUVIR) Fig. 10. Comparison between Barro et al. (2019) and LePhare using the same sample of 608 galaxies also shown in the left panel of Fig. 7. The same BC03 library is fit by LePhare to the full photometric base- line available in the SXDF2018 catalog (filled blue circles) and to the 12 filters only used in the SOM method (open cyan circles). Same colors are used in the inset for the histograms showing the respective log(SFRLePh/SFRUVIR) distributions; the log(SFRSOM/SFRUVIR) distri- bution (red histogram, same as the inset in the left panel of Fig. 7) is also included as reference. colors is built with a flat redshift distribution and no differenti- 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 10 50 100 # of BC03 templates (renorm.) Fig. 11. BC03 templates from the LePhare library projected into the SOM. The templates are shifted from z = 0.2 to 1.8 with steps of ∆z = 0.01, and perturbed with Gaussian noise to mimic real photometry. The SOM is color coded to show their cell occupation, renormalized to the number density of COSMOS2020 galaxies to make this figure directly comparable with the left-hand panel of Fig. 2. 0 1 2 log(SFRUVIR[M yr 1]) Barro+19 0.0 0.5 1.0 1.5 2.0 2.5 log(SFRLePh[M yr 1]) BC03 fit to 25 bands BC03 fit to 12 bands 1 0 1 log(SFRLePh/SFRUVIR) Fig. 10. Comparison between Barro et al. 19 Carnall et al. (2018). 20 Johnson & Leja (2017), Leja et al. (2017), Johnson et al. (2021). 5.2. Caveats in the SOM construction The most entrenched limitations in the SOM method are the ones inherited from training and calibration samples. In Sect. 4, we shared the observation that many galaxies do not have an assigned SFRSOM. The lack of estimates is due to the 24 µm flux limit, which prevents the measurement of most of the low-SFR galaxies with M < 1010 M⊙, leaving several SOM cells without a SFRlabel. As a consequence, the SFR distribution in bins below 1010 M⊙is skewed towards higher SFR values, and the observed MS departs from linearity (Fig. 9). This selection bias would affect any MS measurement derived from the COSMOS MIPS survey, irrespective of the method; with the SOM, the way data are visualized makes easier to identify this kind of issues. Incom- pleteness in the training sample is more difficult to quantify because it is necessary to characterize the parent population from which the sample is drawn. It is therefore challenging to estab- lish, before starting the SOM construction, whether a certain galaxy type has not been included in the training. Template fit- ting does not have the same problem, since that particular galaxy type can always be incorporated in the template library; the con- cern in that case is how accurately that galaxy is modeled. Fig. 12. SFR vs. M plane of COSMOS2015 galaxies (colored circles) as it results from the SED fitting performed by the Prospector code (Leja et al. 2021). Only the objects cross-correlated to COSMOS2020 (0.6′′ searching radius) are shown, both those having a MIPS 24 µm counterpart (in the upper panel) and objects without FIR detection (lower panel). Each data point is color-coded according to the differ- ence in SFR between Prospector and the SOM estimator. A dashed line at constant sSFR = 10−11 yr−1 is plotted in each panel to guide the eye. y g y Another bias, more specific to the SOM method itself, is caused by the mixture of different galaxy types in the same cell. The main reason for such a contamination is the similarity of diverse SEDs after photometric errors are taken into account. This has been already shown in Speagle et al. (2019, particu- larly in their Figs. 3 and 4), albeit, for a five-band data set, which is more susceptible to SED degeneracy. In an ideal, noise- less universe, the SOM classification is extremely accurate, as shown in Davidzon et al. 5.2. Caveats in the SOM construction (2019) using a mock galaxy catalog from hydrodynamical simulations. Hence, the necessity of using deep, state-of-the-art data to minimize observational errors and, consequently, the scatter introduced in the SOM. Cells with a quiescent fraction of 0.25 < fQ < 0.75 (5% of the entire grid, see Fig. 4) are another example of the SED mixing, indicating that some galaxies with different NUV −r, r −Ks colors are located together inside the same cell. This bias may affect more seriously those calibrations relying on smaller samples, like SFR in the present study. In cells occupied only by a handful of MIPS galaxies, even a single interloper may have a strong impact on the resulting SFRlabel. One way to find them is to look at some property that is expected to be similar for all galaxies in the cell (e.g., their sSFR). If the inspected object is an (e.g., 2σ) outlier of the distribution, then it can be flagged as a potential interloper. The suggested approach is only one of the possible solutions and it does not work in some cases, for instance, in cells containing only one or two MIPS galaxies. Nonetheless, we test it by re- calibrating the SOM using sigma-clipped SFRUVIR distributions. This effectively removes 2σ outliers in the cells where sigma clipping is feasible. Since the general results of the analysis do not change, we conclude that contamination bias does not have a strong impact overall. 3DHST and COSMOS2015 galaxies up to z ∼ 3. Unlike the present analysis, their input photometry spans from UV to 24 µm (when available) and the emission in those bands is inter- preted under the assumption of energy balance (similarly to da Cunha et al. 2008). With this caveat in mind, we can com- pare to Leja et al. (2021) by examining the COSMOS galaxies Leja et al. (2021) have in common with our study. Among the ones with a 24 µm detection (S/N > 5) we select 790 targets that have been kept out-of-bag during the validation test in Sect. 3.3. Another 16 729 galaxies in our catalog have no 24 µm counter- part (or <5σ), but they are matched with a source in Leja et al. (2021). For the former subsample, we find no significant system- atics in log(SFRSOM/SFRProspector), along with a standard devia- tion of 0.3 dex that is comparable with the typical SED fitting statistical uncertainties (Fig. 5.1. SOM-based estimates versus synthetic templates 2 1 0 1 2 MIPS 24 m 9 10 11 12 log(MProspector [M ]) 2 1 0 1 2 log(SFRProspector [M yr 1]) no MIPS 0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 log(SFRSOM/SFRProspector) 5.1. SOM-based estimates versus synthetic templates Another distinctive feature of these codes is the capability of using SFHs in flex- ible bins of time (often designated as “non-parametric”, see Leja et al. 2019b) or to include a large variety of SFHs from cos- mological simulations (as in BEAGLE, see Chevallard & Charlot 2016). Also, the implementation of parametric SFHs has reached a level of complexity higher than the previous generation of software (see Carnall et al. 2019). This is however achieved at y p ) In recent work, the SFRs derived from the Bayesian codes mentioned above have been compared with other estimators. In Carnall et al. (2019) galaxies in the GAMA survey (Baldry et al. 2018) are analyzed with BAGPIPES and then compared to SFR measurements based on Hα flux, showing a large scatter (see their Fig. 9) and a mass-dependent offset due to the fact the Hα tracer is sensitive to more recent star formation episodes. The test with GAMA galaxies is performed at z < 0.1, fitting bands up to IRAC channel 4. These differences stand in the way of a straightforward comparison with our SFRHα test (Fig. 7, right panel) which, however, shows more consistency even though it has been performed over a larger z range and without the use of channel 3 and 4. 19 Carnall et al. (2018). 20 Johnson & Leja (2017), Leja et al. (2017), Johnson et al. (2021). Another recent study (Leja et al. 2021) thoroughly inspects physical quantities inferred by means of Prospector for A34, page 13 of 22 A&A 665, A34 (2022) 2 1 0 1 2 MIPS 24 m 9 10 11 12 log(MProspector [M ]) 2 1 0 1 2 log(SFRProspector [M yr 1]) no MIPS 0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 log(SFRSOM/SFRProspector) Fig. 12. SFR vs. M plane of COSMOS2015 galaxies (colored circles) as it results from the SED fitting performed by the Prospector code (Leja et al. 2021). Only the objects cross-correlated to COSMOS2020 (0.6′′ searching radius) are shown, both those having a MIPS 24 µm counterpart (in the upper panel) and objects without FIR detection (lower panel). Each data point is color-coded according to the differ- ence in SFR between Prospector and the SOM estimator. A dashed line at constant sSFR = 10−11 yr−1 is plotted in each panel to guide the eye. 5.2. Caveats in the SOM construction Solid lines show the contour density map for the distribution of SXDF2018 galaxies in the SFR–M diagram; this is fit by Eq. (8) as described in Sect. 4.3 (dashed line). Filled circles are SFR median in running bins of galaxies with SFRSOM/MSOM > 10−11 yr−1 (orange symbols) and fQ < 0.5 (red). In both cases, error bars are calculated with the 16th–84th percentile difference. The red circles are interpolated by a linear function (dotted line). , ) ( ) The most evident feature of Fig. 14 is the additional cover- age provided by spectroscopic data, which gives us the capa- bility of labeling 735 cells missed by the 24 µm calibration. Most of them are in the area populated by low-mass galaxies because of the surveys’ selection function targeting many emit- ters with MLePh ≃109 M⊙. This shows how spectroscopic line detection can be complementary to FIR imaging. The figure of merit is expected to improve after next-generation spectro- graphs will start operations. For example, Hα is inside the wavelength range of the Multi-Object Optical and Near-infrared Spectrograph (MOONS, Cirasuolo et al. 2014; Taylor et al. 2018) in low-resolution mode from z ∼0 to 1.7, with Hβ (pivotal for dust correction) entering at z ∼0.3. The sensitivity and mul- tiplex capability of the instrument22 shall enable a more exhaus- tive labeling of the SOM grid (see discussion in Davidzon et al. 2019). Also, the grism spectrograph on board of Euclid23 could be instrumental not only for filling the empty cells but also to dramatically increase the statistics: between 32 000 and 48 000 galaxies per square degree are expected to be observed at 0.40 < z < 1.8 with an Hα flux >5 × 10−17 erg s−1 cm−2 (Pozzetti et al. 2016). Certain sub-samples of galaxies are, however, affected by an ambiguous SOM classification besides the obvious interloper cases. This is the case of the trend at the high-mass end of the MS, already highlighted in Sect. 4.3. Since there is no clear-cut separation between the star-forming and quiescent population in the SOM, we impose a threshold ( fQ < 0.5) that is somewhat arbitrary. Figure 13 illustrates the issue at 0.6 < z < 0.8: massive galaxies with intermediate to low star formation, responsible for the MS turnover at ∼5 × 1010 M⊙, are excluded by the fQ < 0.5 criterion. 5.2. Caveats in the SOM construction 12, upper panel). With respect to the objects that are not detected in MIPS, we are able to confirm the findings in Leja et al. (2021): when sSFRProspector ≳1010 yr−1 there is a good agreement between the two techniques, whereas below the MS, the SFRSOM values are systematically larger (Fig. 12, lower panel). The average discrepancy increases from 2× to more than 10×, as we move towards lower levels of spe- cific star formation. Such a trend is coherent with Prospector non-parametric SFHs since their impact, compared to analytical descriptions of SFH, is more pronounced for old galaxies that have exited the MS. Instead of relying on galaxy physical properties, like in the example above, we can find catastrophic errors in the SOM clas- sification by inspecting galaxies with suspiciously large ∆or χ2 distance (Eqs. (2) and (B.1), respectively) as done in Sect. 4.3. Another way to mark unreliable SFRSOM estimates is according to the number of cells contributing to Eq. (7): for some galaxies not all the N neighbors may be labeled, potentially introducing a bias. Such a bias, if present, must be of second order because removing objects for which only one or two of the five neighbor cells have SFRlabel defined, the MS locus does not change; the only detectable change is a reduction in the error bars of Fig. 9. Another reason behind the differences among the two meth- ods is that galaxy models in Prospector are composed of a combination of various stellar populations. Such a flexibility cor- responds to a variable scaling factor in the SFR–LIR relationship, which can be adjusted on an object-by-object basis instead of the constant K factor used to calibrate the SOM (Eq. (1)). In fact, the latter comes from studies based on simpler stellar population synthesis models. The discrepancy, however, could be removed by construction using Prospector to label the SOM instead of the procedure summarized in Sect. 2.3. This option is further discussed in Sect. 5.3. 5.2. Caveats in the SOM construction A34, page 14 of 22 Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys 9.5 10.0 10.5 11.0 11.5 log(MSOM [M ]) 0.0 0.5 1.0 1.5 log(SFRSOM [M yr 1]) 0.6 < z < 0.8 (t 7.2 Gyr) fit to density map linear fit to medians M-binned medians (sSFR cut) M-binned medians (fQ cut) 9.5 10.0 10.5 11.0 11.5 log(MSOM [M ]) 0.0 0.5 1.0 1.5 log(SFRSOM [M yr 1]) 0.6 < z < 0.8 (t 7.2 Gyr) fit to density map linear fit to medians M-binned medians (sSFR cut) M-binned medians (fQ cut) Fig. 13. Solid lines show the contour density map for the distribution of SXDF2018 galaxies in the SFR–M diagram; this is fit by Eq. (8) as described in Sect. 4.3 (dashed line). Filled circles are SFR median in running bins of galaxies with SFRSOM/MSOM > 10−11 yr−1 (orange symbols) and fQ < 0.5 (red). In both cases, error bars are calculated with the 16th–84th percentile difference. The red circles are interpolated by a linear function (dotted line). 0.6 < z < 0.8 (t 7.2 Gyr) galaxy star formation (Condon 1992), but we do not calculate SFRradio here since we aim at discussing the availability of data rather than the final estimates. The latest, publicly available21 radio data for almost the entire COSMOS field come from inter- ferometry in the 3 GHz band with the ESO Very Large Array (Smolˇci´c et al. 2017). Instead of extracting individual sources, as done with both MIPS and spectroscopic tracers, we could stack 3 GHz map cutouts centered at the position of SOM galax- ies belonging to the same cell. This is the procedure adopted by Leslie et al. (2020), which is similar to the 1.4 GHz stacking described in Karim et al. (2011). Figure 14 shows the coverage of potential calibrations with either Hα or radio stacking, along with the actual FIR-based cali- bration already presented in Fig. 4 (bottom-left panel). We high- light only the cells that would benefit from a high-confidence calibration, namely, those containing at least one galaxy with Hα flux >2 × 10−17 erg s−1 cm−2, or with S/N > 3 in the 3 GHz median stack. The figure of merit for radio stacking is done by following the recipe (size of the cutouts, calculation of median flux, etc.) in Leslie et al. (2020). Fig. 13. 5.2. Caveats in the SOM construction A less conservative constraint (sSFR > 10−11 yr−1) does not remove those galaxies and would better identify the MS turnover, becoming more in agreement with the MS determined by the “selection-free” definition of Renzini & Peng (2015, see Sect. 4.3). The same is observed in the other redshift bins. Figure 13 also shows the general systematics induced by using the median SFR in bins of mass: they locate the center of the MS slightly below the locus traced by the highest density in galaxy number density. ) The third tracer, based on 3 GHz stacking, provides an SFRlabel for 510 cells, corresponds to ∼20% of the area actu- ally sampled by previous calibrations. Very few cells (red pixels in Fig. 14) are newly labeled by the radio sample, partly due to the conservative S/N > 5 cut we imposed: if instead we also allow stacked images with 3 < S/N < 5, then the number of SOM cell probed by radio data nearly doubles. However, even in that case, the expansion with respect to the original MIPS cali- bration would be limited (only 65 additional SFRlabel) due to the FIR-radio correlation and its strong dependency on stellar mass (Delvecchio et al. 2021). Despite such a marginal expansion, it is still useful to have various tracers superimposed on the SOM for a better understanding of the galaxy parameter space. For example, a difference between multiple SFRlabel values in the same cell may help identify galaxy classes with rapidly vary- ing SFH, leveraging the fact that those star formation tracers probe different time scales (which can be taken into account as in Sparre et al. 2015). 21 https://irsa.ipac.caltech.edu/data/COSMOS/tables/ vla/ 22 See specifications at https://vltmoons.org/ 23 https://www.euclid-ec.org/ 6. Summary and conclusions Compared to the body of work focused on ML methods to measure galaxy redshifts, little progress has been made with respect to other physical parameters, which are equally impor- tant in galaxy evolution studies. Here, we continue the investiga- tions presented in Masters et al. (2015), Hemmati et al. (2019a), and Davidzon et al. (2019) to the point where we are now able to exploit an unsupervised ML algorithm such as the SOM to simultaneously derive galaxy redshift, stellar mass, and SFR. This particular use of the SOM has already been described in Davidzon et al. (2019), but so far has been applied only on mock galaxy catalogs that, despite an advanced level of realism, cannot replace the complexity of a test with actual observations. In Leslie et al. (2020), the stacking technique is instrumental to probe the SFR–M plane beyond the boundaries of the original Smolˇci´c et al. (2017) sample, which is made by individual VLA sources whose counterparts are mostly brighter than 22.5 mag in IRAC channels 1 and 2. Moreover, the choice of adaptive binning allows the authors to always gather several hundreds of objects. In the finer grid of the SOM, galaxies that in Leslie et al. (2020) belonged to a single (z, M) bin are now spread over sev- eral cells, “diluting” the stacking signal. One route to solving this problem involves the use of all SOM cells, each one with an occupation probability weight (as in Speagle et al. 2019), instead of assigning the target galaxy to its best-fit (but low-S/N) cell only. To this purpose, in the present work, we used the new COS- MOS2020 galaxy catalog (Weaver et al. 2022) up to z = 1.8, together with the rich archive of complementary data available for the COSMOS field. Not only the quality, but also the large size of COSMOS observations make them an ideal training sam- ple for the SOM. In our method, after the SOM dimensionality reduction created a grid of 80 × 80 cells, we labeled as many cells as possible with reference values of z, M, and SFR. These values are derived by means of ancillary data, in particular MIPS 24 µm from the S-COSMOS survey to constrain galaxy star for- mation. 5.3. Calibration with other tracers The estimates used as reference in Sect. 2.3 are not the only option for calibrating our method. Relying on the same optical-to-FIR data, we can use alternate fitting codes (e.g., Prospector) instead of LePhare. Besides that, owing to the wealth of ancillary data in COSMOS, other proxies for stellar mass and star formation can provide Mlabel and SFRlabel. This is particularly evident for the latter, which can be derived, for instance, from Hα nebular emission or by the radio continuum. Other possibilities, such as the 21 cm atomic hydrogen or sub- mm molecular gas transitions, are not included in the present discussion for sake of simplicity and we address the reader to Kennicutt & Evans (2012) for an exhaustive bibliography on the various star formation tracers. Our goal in the present section is not to repeat the analysis with another sets of MSOM, SFRSOM estimates – but, rather, to show the capacity of different calibra- tion samples to fill the SOM grid. A technique for deriving SFRHα has been presented in Sect. 4.2. Radio continuum emission is also correlated with A34, page 15 of 22 A34, page 15 of 22 A&A 665, A34 (2022) 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 3 GHz stacking H flux MIPS 24 m Fig. 14. Different calibration samples that may be used to label SFR on the SOM. Cells containing galaxies from the original MIPS sample (cf. Fig. 4) are colored in blue; cells containing spectroscopic galax- ies with an Hα measurement and radio galaxies with a 3 GHz stacking S/N > 5 are colored in green and red respectively. Colors combine by following the standard RGB rules, e.g. a cell that can be calibrated by both MIPS and Hα (MIPS and radio) is colored in cyan (magenta). A handful of yellow pixels are cells that can be calibrated by only radio stacking and Hα, while white pixels are cells in which all the three star formation tracers are available. As the discussion in the main text is only about coverage and not the actual SFRlabel values, these are not shown in the present figure. for peculiar SEDs (see also Hovis-Afflerbach et al. 2021, where they apply dimensionality reduction to isolate catastrophic red- shift errors). The bottom panel of Fig. 24 Note that central wavelength and shape of these two Y bands are different (see Fig. 2 in Weaver et al. 2022). 5.3. Calibration with other tracers a cell that can be calibrated by both MIPS and Hα (MIPS and radio) is colored in cyan (magenta). A handful of yellow pixels are cells that can be calibrated by only radio stacking and Hα, while white pixels are cells in which all the three star formation tracers are available. As the discussion in the main text is only about coverage and not the actual SFRlabel values, these are not shown in the present figure. Fig. 14. Different calibration samples that may be used to label SFR on the SOM. Cells containing galaxies from the original MIPS sample (cf. Fig. 4) are colored in blue; cells containing spectroscopic galax- ies with an Hα measurement and radio galaxies with a 3 GHz stacking S/N > 5 are colored in green and red respectively. Colors combine by following the standard RGB rules, e.g. a cell that can be calibrated by both MIPS and Hα (MIPS and radio) is colored in cyan (magenta). A handful of yellow pixels are cells that can be calibrated by only radio stacking and Hα, while white pixels are cells in which all the three star formation tracers are available. As the discussion in the main text is only about coverage and not the actual SFRlabel values, these are not shown in the present figure. 6. Summary and conclusions Once the SOM was calibrated, we projected other galax- ies onto the grid, obtaining an estimate of their zSOM, MSOM, and SFRSOM depending on the cell to which each target has been associated. We used SXDF galaxies, but any other galaxy sam- ple can be measured as long as the targets have the requisite As an aside, we notice that a stacking strategy would be more beneficial for the SFRUVIR calibration. The MIPS sample used in Sect. 3.2 was pre-selected at S/N > 5 in the 24 µm band. A pre- liminary inspection of sources below that threshold suggests that the number of labeled cells may increase by ∼30% (compared to Fig. 4) by using median SFRs from 24 µm stacking (calculated as in Magdis et al. 2010). 5.3. Calibration with other tracers 15 illustrates this point through the galaxy sample inside one of those problematic cells (the one with coordinates D1, D2 = 46, 34). The sample includes 56 objects at z ∼1.2 with similar colors. When the SEDs are superimposed to their LePhare best-fit templates, we observe that the UltraVISTA H band is systematically below the flux pre- dicted by LePhare (see lower panel of Fig. 15). The correspond- ing photometric uncertainty is also significantly larger than the other UltraVISTA bands, always resulting in a S/N smaller than 2. Interestingly, Chartab et al. (in prep.) also reached the same conclusion by analyzing H-band photometry with a different ML technique, where these objects had escaped standard reliability checks in Weaver et al. (2022). 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 3 GHz stacking H flux MIPS 24 m Another problematic region according to Fig. 2 is the vertical stripe of cells with D1 = 0 and 48 < D2 < 56, from which we randomly select (D1, D2) = (0, 50) for inspection (middle panel of Fig. 15). Galaxy SEDs inside that cell have a much larger dis- persion than non-pathological cells (as the one displayed in the upper panel of Fig. 15 for comparison). Despite the scatter, these objects share a common characteristic, namely, an extremely red yHSC −YVISTA color24 and a blue YVISTA −JVISTA. That color excess can be attributed to nebular emission line contamination in the YVISTA band. Indeed, galaxies in (D1, D2) = (0, 50) are concentrated around two redshifts: z ∼1.15 and z ∼1.7, which approximately correspond to a Y-band flux enhancement due to Hβ (λ4863) and O[ii] (λλ3727, 3729) respectively. Hence, the SOM diagnostic power can serve at the same time to identify peculiar galaxy types like strong line emitters, but also to reveal technical issues such as photometric redshift degeneracy. D1 Fig. 14. Different calibration samples that may be used to label SFR on the SOM. Cells containing galaxies from the original MIPS sample (cf. Fig. 4) are colored in blue; cells containing spectroscopic galax- ies with an Hα measurement and radio galaxies with a 3 GHz stacking S/N > 5 are colored in green and red respectively. Colors combine by following the standard RGB rules, e.g. 5.4. Soundness checks using SOM features (in this case, broadband colors) to be projected into the COSMOS-calibrated SOM. In our case, we also assumed photo- metric redshift are known in advance to preselect galaxies below z < 1.8. This requirement can be disregarded, as the SOM proved to be efficient for redshift measurement too (Masters et al. 2015). However, the other techniques also fix the redshift of their targets before measuring the other physical parameters, therefore we did the same here for sake of comparison. 5000 10000 50000 wavelength [Å] 100 101 flux [ Jy] galaxies in cell (D1, D2) = (30, 30) flux [ Jy] p The SOM is made by a fixed number of cells, each one representing a specific combination of broadband colors (i.e., a galaxy SED prototype). The galaxy properties in output are not strongly discretized because they result from averaging several cells (Eqs. (6) and (7)). Nevertheless, the SOM resolving power is lower than other ML methods (in particular deep neural net- works, see Simet et al. 2021) that describe physical parameters with continuous functions. The SOM “gridding effects”, how- ever, are counter-balanced by the advantage of building such a grid in an unsupervised fashion, and the flexibility of assigning different labels to it a posteriori. As an aside, we note that other methods such as variational autoencoders (see Cheng et al. 2021, for an application in the astronomical domain) may represent a synthesis between the two approaches by offering both an unsu- pervised (or semi-supervised) training and a regression analysis with continuous functions. 5000 10000 50000 wavelength [Å] 100 101 flux [ Jy] galaxies in cell (D1, D2) = (0, 50) We assessed the reliability of our procedure both by way of comparisons to independent estimates and by means of a bootstrap technique (i.e., out-of-bag targets from our COSMOS sample). Most notably the galaxy SFR, generally difficult to recover from optical-NIR fitting, can be measured with good precision (<0.3 dex scatter, see Figs. 5 and 7) and is consis- tent with SFR indicators based on FIR (only 0.05 dex offset from Barro et al. 2019); even though in our SOM method, the input photometry stops at IRAC channel 2. 5.4. Soundness checks using SOM In each panel, the solid line is the median of the BC03 spectral models providing the best fit to each COSMOS2020 galaxy in the LePhare analysis; before calculating the median, all the BC03 spectra (which include only the stellar component, not the nebu- lar emission) have been rescaled to match 22.5 mag in the i band. Such a rescaling also makes easier to compare spectral shapes from the differ- ent panels. The gray shaded area encloses the 16th and 84th percentiles of their distribution. The y-axis range in the upper panel is shorter to zoom in the tight 16th–84th dispersion, which would be barely visi- ble otherwise. Filled circles represent the observed photometry, color- coded for the different bands (see legend); for sake of clarity they have been horizontally shifted from the central wavelength of their band by a random offset within ±3%. In the bottom part of each panel we report the average error bars in the same color of their corresponding pho- tometric band. Black crosses are the fluxes that would result from the cell’s weight after training. All symbols are renormalized to the refer- ence i = 22.5 mag. Fig. 15. Inspection of three SOM cells with coordinates (D1, D2) = [(30, 30), (0, 50), (46, 34)] on the 80 × 80 gird. The first cell (upper panel) has been randomly selected among those with a small galaxy- weight distance ∆(cf. Fig. 2) while the middle and lower panels display two cells located in areas with large ∆values, to provide an insight on SOM shortcomings (see Sect. 5.4). The number of galaxies in these cells is respectively 16, 39, 56. In each panel, the solid line is the median of the BC03 spectral models providing the best fit to each COSMOS2020 galaxy in the LePhare analysis; before calculating the median, all the BC03 spectra (which include only the stellar component, not the nebu- lar emission) have been rescaled to match 22.5 mag in the i band. Such a rescaling also makes easier to compare spectral shapes from the differ- ent panels. The gray shaded area encloses the 16th and 84th percentiles of their distribution. The y-axis range in the upper panel is shorter to zoom in the tight 16th–84th dispersion, which would be barely visi- ble otherwise. 5.4. Soundness checks using SOM None of the three tracers we considered are able to fill the cen- tral area of the SOM (around coordinates D1, D2 = 50, 40), where the average intra-cell distance is particularly large (Fig. 2, middle panel). This is another use of the SOM as a diagnostic A34, page 16 of 22 Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys I. Davidzon et al.: COSMOS2020: Manifold learning 5000 10000 50000 wavelength [Å] 100 101 flux [ Jy] galaxies in cell (D1, D2) = (30, 30) 5000 10000 50000 wavelength [Å] 100 101 flux [ Jy] galaxies in cell (D1, D2) = (0, 50) 5000 10000 50000 wavelength [Å] 100 101 flux [ Jy] galaxies in cell (D1, D2) = (46, 34) Fig. 15. Inspection of three SOM cells with coordinates (D1, D2) = [(30, 30), (0, 50), (46, 34)] on the 80 × 80 gird. The first cell (upper panel) has been randomly selected among those with a small galaxy- weight distance ∆(cf. Fig. 2) while the middle and lower panels display two cells located in areas with large ∆values, to provide an insight on SOM shortcomings (see Sect. 5.4). The number of galaxies in these cells is respectively 16, 39, 56. In each panel, the solid line is the median of the BC03 spectral models providing the best fit to each COSMOS2020 galaxy in the LePhare analysis; before calculating the median, all the BC03 spectra (which include only the stellar component, not the nebu- lar emission) have been rescaled to match 22.5 mag in the i band. Such a rescaling also makes easier to compare spectral shapes from the differ- ent panels. The gray shaded area encloses the 16th and 84th percentiles of their distribution. The y-axis range in the upper panel is shorter to zoom in the tight 16th–84th dispersion, which would be barely visi- ble otherwise. Filled circles represent the observed photometry, color- coded for the different bands (see legend); for sake of clarity they have been horizontally shifted from the central wavelength of their band by a random offset within ±3%. In the bottom part of each panel we report the average error bars in the same color of their corresponding pho- tometric band. Black crosses are the fluxes that would result from the cell’s weight after training. All symbols are renormalized to the refer- ence i = 22.5 mag. 5.4. Soundness checks using SOM We also found a good agreement between the SOM main sequence of star for- mation and other MS studies from the literature, an indication that the (zSOM, MSOM, SFRSOM) estimates provided by the SOM are simultaneously accurate for most of the targeted objects. g [ ] 5000 10000 50000 wavelength [Å] 100 101 flux [ Jy] galaxies in cell (D1, D2) = (46, 34) The computation of physical parameters for 208 404 SXDF galaxies took about 0.1 CPU hours. In addition to the com- putational speed our method offers, other advantages shall be emphasized: – The quality of SFRSOM estimates is better than the results obtained with LePhare, when compared to SFR val- ues independently measured from UV-to-FIR photometry (Barro et al. 2019). Concerning stellar mass estimates, we found that MSOM and MLePh are in overall good agreement; small discrepancies between the two could not be solved because of the lack of a third set of M estimates to be used as “ground truth”. Without advocating that LePhare (or template fitting in general) should be superseded by the novel method, our study demonstrates the benefits of a dual approach with ML and standard template fitting working in a complementary fashion. Indeed, as LePhare was instrumen- tal in preliminary tests on the SOM (here, but also e.g. in Hemmati et al. 2019b) the latter can help to better understand caveats and limitations in template fitting (Sect. 5.1). The key factor making our method competitive with respect to other state-of-the-art fitting codes is that it does not rely on the tra- ditional library of synthetic templates. Such a library can be biased by the theoretical modeling assumptions (stellar pop- ulation synthesis, dust prescriptions, etc.) whereas this new fitting procedure is more data-driven, based on a set of galaxy prototypes coming from the SOM classification of observed SEDs (i.e., the cells and their weights). Fig. 15. Inspection of three SOM cells with coordinates (D1, D2) = [(30, 30), (0, 50), (46, 34)] on the 80 × 80 gird. The first cell (upper panel) has been randomly selected among those with a small galaxy- weight distance ∆(cf. Fig. 2) while the middle and lower panels display two cells located in areas with large ∆values, to provide an insight on SOM shortcomings (see Sect. 5.4). The number of galaxies in these cells is respectively 16, 39, 56. 5.4. Soundness checks using SOM Filled circles represent the observed photometry, color- coded for the different bands (see legend); for sake of clarity they have been horizontally shifted from the central wavelength of their band by a random offset within ±3%. In the bottom part of each panel we report the average error bars in the same color of their corresponding pho- tometric band. Black crosses are the fluxes that would result from the cell’s weight after training. All symbols are renormalized to the refer- ence i = 22.5 mag. features (in this case, broadband colors) to be projected into the COSMOS-calibrated SOM. In our case, we also assumed photo- metric redshift are known in advance to preselect galaxies below The robust performance of SOM does not imply that its out- come is bias-free, that is, the systematics in the training sam- ple may propagate to the target galaxy estimates and also the A34, page 17 of 22 A34, page 17 of 22 A&A 665, A34 (2022) more efficiently (see Speagle & Eisenstein 2017; Gilda et al. 2021). Moreover, galaxy models for measuring photometric red- shift differ significantly from the ones used to derive stellar mass and SFR. Attempts to realize a comprehensive set of templates, able to estimate simultaneously the aforementioned properties, have been recently pursued without exploiting ML algorithms (e.g., Battisti et al. 2019). This issue is beyond the scope of the present study, as we assumed a known redshift for each galaxy – not necessarily a high-precision estimate, but we were able to select z < 2 targets with sufficient confidence (see also Hemmati et al. 2019b). Nonetheless, the SOM can also be used without zLePh preselection by expanding the training to a larger redshift range (up to z = 4−6, as in Masters et al. 2015, 2019; Davidzon et al. 2019) and including a stellar locus (e.g., Kim et al. 2015; Davidzon et al. 2017). In such a framework in future studies, the number of high-z interlopers would be negli- gible in most of the statistical applications. SOM labels have underlying assumptions (see e.g., Eq. (1) for producing SFRlabel). However, the 2D rendering of the SOM put us in a convenient position to solve the problem, as it allows an effective visual inspection of the parame- ter space. Section 5.4 showed an example of such applica- tion, where we flag suspicious cells according to the average galaxy-weight distance inside them. 25 MIRI specifications are from the official documentation at https://jwst-docs.stsci.edu/ 5.4. Soundness checks using SOM We were able to identify not only issues due to corrupted photometry, but also galaxy types whose SED is intrinsically peculiar (e.g., because of nebular emission contamination). – Moreover, a deeper insight into the galaxy parameter space may result from the combination of multiple calibration sam- ples. In fact, the labels used in the present analysis are not the only option in COSMOS. In Sect. 5.3 we discussed two alter- natives to SFRUVIR, since the star formation labels can also be derived from either direct spectroscopic measurements (SFRHα) or VLA 3 GHz image stacking (SFRradio). Having more than one tracer helps, at least to some extent, to cali- brate a larger fraction of the SOM grid, but the most impor- tant advantage is the overlap of the different tracers: in a cell with multiple layers of physical information, any similarity (or tension) between them is informative about the galaxy population inside that cell. Acknowledgements. The authors are grateful to the referee for the thorough and constructive comments they provided. I.D. would like to thank Eric Bell, Micol Bolzonella, Rebecca Bowler, Ivan Delvecchio, Joel Leja, Vihang Mehta for useful discussions. The authors are grateful to Joel Leja also for provid- ing Prospector data in a convenient digital format. This work started dur- ing a meeting in Marseille, founded by the French Agence Nationale de la Recherche for the project “SAGACE”. This research is also partly supported by the Centre National d’Etudes Spatiales (CNES). I.D., J.R.W., K.J., and O.I. made use of the CANDIDE Cluster at the Institut d’Astrophysique de Paris and made possible by grants from the PNCG, CNES and the DIM-ACAV. The Cosmic Dawn Center is funded by the Danish National Research Foundation under grant No. 140. I.D. has received funding from the European Union’s Hori- zon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 896225. J.R.W. acknowledges support from the European Research Council (ERC) Consolidator Grant funding scheme (project ConTExt, grant No. 648179). K.M. is grateful for support from the Polish National Sci- ence Centre via grant UMO-2018/30/E/ST9/00082. G.E.M. acknowledges the Villum Fonden research grant 13160 “Gas to stars, stars to dust: tracing star for- mation across cosmic time” and grant 37440 “The Hidden Cosmos”. D.B.S. is grateful to Danmarks Nationalbank for their generous hospitality in the Nyhavn 18 residence during his extended visit to Copenhagen. 5.4. Soundness checks using SOM We warmly acknowl- edge the contributions of the entire COSMOS collaboration consisting of more than 100 scientists. The HST-COSMOS program was supported through NASA grant HST-GO-09822. More information on the COSMOS survey is available at https://cosmos.astro.caltech.edu. A significant portion of this project took place during the COVID-19 global pandemic; the authors would like to thank all those who made sacrifices in order for us to safely continue our research. While discussing multiple calibration samples, we also take note of the role that next-generation spectrographs such as MOONS or the Prime Focus Spectrograph (PFS, Takada et al. 2014) will play in the near future. With those facilities, an ambitious pro- gramme such as the Complete Calibration of the Color-Redshift Relation (C3R2, Masters et al. 2017) may extend its scope to include the calibration of stellar mass and SFR over the entire area of the SOM grid. Nonetheless, 24 µm calibration shall remain a compelling option as the Mid-IR Instrument (MIRI25) on board James Webb Space Telescope will supersede MIPS images, also offering a choice among more broadbands (four of them between 15 and 25 µm). The field of view of MIRI is only 74′′ × 113′′, but in spite of that it should be possible to assemble a calibration sample of comparable size to the COSMOS one. In fact, owing to the small cosmic variance of independent line of sights (Moster et al. 2011), a hundred of well-separated point- ings should be sufficient (see Trenti et al. 2011, for a similar strategy with HST). gy Irrespective of the preferred calibration strategy, the present work is intended as a blueprint to develop ML-based software for upcoming large-area photometric surveys. Scanning a few thou- sands square degrees (as planned by the Euclid mission) or even the entire sky (like the Rubin Observatory), those surveys will decrease dramatically the (already small) ratio between well- characterized spectroscopic galaxies and objects only described by their broadband colors. In the SOM, the former ones would only be needed for a training sample, proportionally much smaller than the number of targets. 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(2008). 27 http://www2.iap.fr/users/fioc/PEGASE.html 28 https://www.stsci.edu/science/starburst99/docs/ default.htm 29 Kennicutt & Evans explain in section 3.8 that their conversion factor, namely ˙M⋆/ ˙M⋆(K98), takes into account the difference between each reviewed study and K98 in both the IMF and the star formation models. Unfortunately, this detail is not noticed in table 1 (which is located at the end of their article) and inattentive readers may misinterpret the values reported in table 1. Appendix A: SFR-LIR relation in the literature Appendix A: SFR-LIR relation in the literature This may not be appropriate for LIR, as the mean age of stars con- tributing to that emission is 5 Myr (Kennicutt & Evans 2012). In that case, the conversion factor from Salpeter to a bottom-lighter IMF should be smaller. In the present study, and comparison papers, infrared luminosity is used as a proxy of galaxy SFR. 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S., et al. 2021, ArXiv e-print [arXiv:2110.04314] Leslie, S. K., Schinnerer, E., Liu, D., et al. 2020, ApJ, 899, 58 Zibetti, S., Charlot, S., & Rix, H.-W. 2009, MNRAS, 400, 1181 Leslie, S. K., Schinnerer, E., Liu, D., et al. 2020, ApJ, 899, 58 A34, page 19 of 22 A34, page 19 of 22 A&A 665, A34 (2022) Appendix B: Additional tests for SOM implementation Our goal is to set a common ground for the comparison: results from the literature obtained through either the K98 or M11 cal- ibration shall be converted to B05, which is the one used in the SOM method (Eq. 1). In our analysis, KB05 = 0.86 × 10−10, because we convert the original value to Chabrier IMF follow- ing Arnouts et al. (2013). In Sect. 3.1, we computed ∆distances to quantify how well the SOM weights conform to the galaxy data manifold (see Eq. 2). A variation of is a χ2 distance is: χ2 SOM = X Ndim ( fi −wi)2 ϵ2 i , (B.1) Inconsistencies between studies may persist if information about the scaling relation and/or IMF conversion is lacking in one of the articles. For example in Whitaker et al. (2012) the authors cite K98 as the reference for the value 0.98 × 10−10 they use, which is actually KB05. In Barro et al. (2019) the authors use K = 1.09 × 10−10 (Chabrier IMF) and quote K98 as the source. The same value is attributed to BC05 elsewhere (e.g., Straatman et al. 2016) creating some confusion. We deem Barro et al. to be correct, as 1.09 × 10−10 is indeed equal to KK98 ×0.63, with 0.63 being the conversion factor from Salpeter to Chabrier IMF provided in Madau & Dickinson (2014). (B.1) which takes into account not only the difference between observed color ( fi) and SOM weight (wi) but also the photo- metric error of the former (ϵi). This alleviates some of the ten- sion highlighted in Fig. 2 (middle panel). In fact, the average χ2 SOM/Ndim per cell (Fig. B.1) has a narrower spread and regions (discussed in Sect. 5.4) as ∆outliers are not as prominently high- lighted here in Fig. B.1 because the large ( fi−wi)2 values in those cells are compensated by the comparably large uncertainty in the fi photometric measurement. which takes into account not only the difference between observed color ( fi) and SOM weight (wi) but also the photo- metric error of the former (ϵi). This alleviates some of the ten- sion highlighted in Fig. 2 (middle panel). In fact, the average χ2 SOM/Ndim per cell (Fig. B.1) has a narrower spread and regions (discussed in Sect. 5.4) as ∆outliers are not as prominently high- lighted here in Fig. Appendix B: Additional tests for SOM implementation B.1 because the large ( fi−wi)2 values in those cells are compensated by the comparably large uncertainty in the fi photometric measurement. p ( ) The IMF conversion itself is another source of discrepancy. Using the prescription from Madau & Dickinson (2014), KB05 would become either 1.46×10−10 or 0.92×10−10 with Salpeter or Chabrier IMF respectively. The latter can be directly compared to the value provided in Arnouts et al. (2013), which is ∼10% smaller. However, there is not a unique prescription: in the lit- erature one can find other IMF conversions resulting e.g. into KB05 = 1.33 (Cowie & Barger 2008) or 1.56 × 1010 (Franx et al. 2008), both for a Kroupa to Salpeter IMF conversion. It is important to underline that every published value of KB05 (with Salpeter IMF) we found in the literature remains ≲30% smaller than KK98, as also noted in B05. Also noteworthy is the fact that when KM11 is retrieved from the review of Kennicutt & Evans (2012, table 1) the KK98 conversion quoted along with that scal- ing relation is potentially misleading29. 80 0 1 2 3 4 average 2 SOM/Ndim 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 0 1 2 3 4 average 2 SOM/Ndim Fig. B.1. Similarly to middle panel of Fig. 2, this plot show the SOM grid color-coded according to the average χ2 SOM (see Eq. B.1) of galaxies in a given cell, normalized by the number of dimension of the manifold (Ndim = 11 colors). 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 average 2 SOM/Ndim average 2 SOM/Ndim The IMF conversion factor is usually derived from stellar population synthesis models, taking the ratio between a model assuming Salpeter and its analog with Chabrier (or Kroupa) IMF. Different conversion factors are originated from different assumptions to build these stellar populations. For example, they are often calculated after 100 Myr of stellar evolution “under the assumption of a constant SFH” (e.g., Madau & Dickinson 2014). D1 Fig. B.1. Similarly to middle panel of Fig. 2, this plot show the SOM grid color-coded according to the average χ2 SOM (see Eq. B.1) of galaxies in a given cell, normalized by the number of dimension of the manifold (Ndim = 11 colors). Appendix B: Additional tests for SOM implementation (2015) and is summarized in Sect. 3.2 (see Eq. 1). The SFRUVIR estimates on the y axis are described in Barro et al. (2019). A solid line shows the 1:1 relationship. The inset in the top-left cor- ner shows a histogram of the difference between Barro et al. (labeled SFRy) and our estimates (SFRx), and a vertical dashed line that marks the median offset at −0.03 dex. 0.1 0.2 0.3 0.4 0.5 std. dev. of log(MLePh/M ) in cell std. dev. of log(MLePh/M ) in cell Thus, a combination of ∆and χ2 SOM metrics is an efficient way to separate issues related to observational (statistical) errors versus intrinsic failures of the SOM when the weight is not close enough to data despite the precision of the latter. Besides the average in each cell, we can also inspect the reduced χ2 (i.e., χ2 SOM/Ndim, assuming Ndim degrees of freedom) of individual galaxies to find which ones are misrepresented by their weight. This happens at edges of the grid, for instance, where the SOM is forced to pack together sparse objects from the outskirts of the data distribution. A bad match between certain galaxies and the weight linked to them can also concern cells in the interior of the grid (see the cells marked in yellow in Fig. B.1) since a non-linear manifold such as our 0 < z < 1.8 galaxy sample is hard to describe with a relatively limited network of weights. It is nonetheless reassuring that only 4.4% of the COSMOS2020 galaxies overall have χ2 SOM > 24.7; this should be the 99th per- centile threshold of a χ2 distribution with Ndim = 11 degrees of freedom: the fact that more than 1% of the sample exceeds the threshold can be ascribed partly to the SOM modeling, which is not expected to be a perfect description, but also to the fact photometric errors may not be entirely accurate in the COS- MOS2020 catalog30. 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 Another test we implemented concerns the Barro et al. (2019) catalog, which is used in this work as an independent source of information to verify the validity of SOM-based esti- mates. Since the SOM method is calibrated with SFRUVIR mea- Fig. B.3. 30 In fact, the native error bars from SourceExtractor were consid- ered underestimated by Weaver et al. (2022), and needed an adjustment with heuristic boosting factors. Appendix B: Additional tests for SOM implementation 0 10 20 30 0 10 20 30 40 50 60 70 80 D2 9 average log( 0.1 0.2 1 0 1 2 log(SFRUVIR/M /yr) Barro+19 1 0 1 2 log(SFRUVIR/M /yr) this work 0.5 0.0 0.5 log(SFRy/SFRx) Fig. B.2. Comparison between two methods deriving SFR from rest- frame IR, both applied to 509 galaxies at z < 1.8 in the COSMOS- CANDELS area (red dots). The method used in the present work fol- lows Ilbert et al. (2015) and is summarized in Sect. 3.2 (see Eq. 1). The SFRUVIR estimates on the y axis are described in Barro et al. (2019). A solid line shows the 1:1 relationship. The inset in the top-left cor- ner shows a histogram of the difference between Barro et al. (labeled SFRy) and our estimates (SFRx), and a vertical dashed line that marks the median offset at −0.03 dex. 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 9 10 11 average log(M /M ) in cell 80 0.1 0.2 0.3 0.4 0.5 std. dev. of log(MLePh/M ) in cell 11 1 0 1 2 log(SFRUVIR/M /yr) Barro+19 1 0 1 2 log(SFRUVIR/M /yr) this work 0.5 0.0 0.5 log(SFRy/SFRx) log(SFRUVIR/M /yr) this work average log(M /M ) in cell 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 1 0 1 2 log(SFRUVIR/M /yr) Barro+19 1 0 1 2 log(SFRUVIR/M /yr) Barro+19 Fig. B.2. Comparison between two methods deriving SFR from rest- frame IR, both applied to 509 galaxies at z < 1.8 in the COSMOS- CANDELS area (red dots). The method used in the present work fol- lows Ilbert et al. (2015) and is summarized in Sect. 3.2 (see Eq. 1). The SFRUVIR estimates on the y axis are described in Barro et al. (2019). A solid line shows the 1:1 relationship. The inset in the top-left cor- ner shows a histogram of the difference between Barro et al. (labeled SFRy) and our estimates (SFRx), and a vertical dashed line that marks the median offset at −0.03 dex. Fig. B.2. Comparison between two methods deriving SFR from rest- frame IR, both applied to 509 galaxies at z < 1.8 in the COSMOS- CANDELS area (red dots). The method used in the present work fol- lows Ilbert et al. Appendix B: Additional tests for SOM implementation It reassuring that only 4.4% of the COSMOS2020 l have χ2 SOM > 24.7; this should be the 99th per- d of a χ2 distribution with Ndim = 11 degrees of act that more than 1% of the sample exceeds the be ascribed partly to the SOM modeling, which to be a perfect description, but also to the fact rors may not be entirely accurate in the COS- log30. st we implemented concerns the Barro et al. which is used in this work as an independent mation to verify the validity of SOM-based esti- e SOM method is calibrated with SFRUVIR mea- tive error bars from SourceExtractor were consid- ted by Weaver et al. (2022), and needed an adjustment osting factors. 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 9 10 11 average log(M /M ) in cell 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 0.1 0.2 0.3 0.4 0.5 std. dev. of log(MLePh/M ) in cell Fig. B.3. A more detailed description of how galaxies with different stellar masses are distributed across the SOM. Upper panel: Aver- age log(MLePh/M⊙) of COSMOS2020 galaxies within the same cell, coded according to the color bar above the SOM grid (white cells are empty). Upper panel: Standard deviation of the same log(MLePh/M⊙) distribution per individual cell, coded according to the color bar above the SOM grid (also in this case, empty cells are colored in white). 1 0 1 2 log(SFRUVIR/M /yr) Barro+19 1 0 1 2 log(SFRUVIR/M /yr) this work 0.5 0.0 0.5 log(SFRy/SFRx) Fig. B.2. Comparison between two methods deriving SFR from rest- frame IR, both applied to 509 galaxies at z < 1.8 in the COSMOS- CANDELS area (red dots). The method used in the present work fol- lows Ilbert et al. (2015) and is summarized in Sect. 3.2 (see Eq. 1). The SFRUVIR estimates on the y axis are described in Barro et al. (2019). A solid line shows the 1:1 relationship. The inset in the top-left cor- ner shows a histogram of the difference between Barro et al. (labeled SFRy) and our estimates (SFRx), and a vertical dashed line that marks the median offset at −0.03 dex. Appendix B: Additional tests for SOM implementation A34, page 20 of 22 I. Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys estimate physical parameters in large galaxy surveys 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 9 10 11 average log(M /M ) in cell 0 10 20 30 40 50 60 70 80 D1 0 10 20 30 40 50 60 70 80 D2 0.1 0.2 0.3 0.4 0.5 std. dev. of log(MLePh/M ) in cell Fig. B.3. A more detailed description of how galaxies with different stellar masses are distributed across the SOM. Upper panel: Aver- age log(MLePh/M⊙) of COSMOS2020 galaxies within the same cell, coded according to the color bar above the SOM grid (white cells are empty). Upper panel: Standard deviation of the same log(MLePh/M⊙) distribution per individual cell, coded according to the color bar above the SOM grid (also in this case, empty cells are colored in white). I. Davidzon et al.: COSMOS2020: Manifold learning to estimate physical parameters in large galaxy surveys 0 1 2 og(SFRUVIR/M /yr) Barro+19 0.5 0.0 0.5 log(SFRy/SFRx) arison between two methods deriving SFR from rest- applied to 509 galaxies at z < 1.8 in the COSMOS- (red dots). The method used in the present work fol- (2015) and is summarized in Sect. 3.2 (see Eq. 1). The es on the y axis are described in Barro et al. (2019). ws the 1:1 relationship. The inset in the top-left cor- ogram of the difference between Barro et al. (labeled stimates (SFRx), and a vertical dashed line that marks t at −0.03 dex. mbination of ∆and χ2 SOM metrics is an efficient issues related to observational (statistical) errors failures of the SOM when the weight is not close despite the precision of the latter. Besides the h cell, we can also inspect the reduced χ2 (i.e., suming Ndim degrees of freedom) of individual which ones are misrepresented by their weight. t edges of the grid, for instance, where the SOM ck together sparse objects from the outskirts of ution. A bad match between certain galaxies and ed to them can also concern cells in the interior e the cells marked in yellow in Fig. B.1) since a ifold such as our 0 < z < 1.8 galaxy sample is e with a relatively limited network of weights. Appendix B: Additional tests for SOM implementation A more detailed description of how galaxies with different stellar masses are distributed across the SOM. Upper panel: Aver- age log(MLePh/M⊙) of COSMOS2020 galaxies within the same cell, coded according to the color bar above the SOM grid (white cells are empty). Upper panel: Standard deviation of the same log(MLePh/M⊙) distribution per individual cell, coded according to the color bar above the SOM grid (also in this case, empty cells are colored in white). A34, page 21 of 22 A&A 665, A34 (2022) sured as in Ilbert et al. (2015), any difference between that and Barro et al. (2019) will propagate in the comparison between SFRSOM and SFRUVIR from Barro et al. (2019). Figure B.2 shows our SFRUVIR versus Barro et al. values (after homogeniza- tion, see Appendix A). The latter are obtained by averaging four sets of FIR templates, including Dale & Helou (2002) which are the ones we used. Another important distinction between our approach and Barro et al. concerns objects with only the 24 µm detection: they use an analytical prescription (Wuyts et al. 2011) to convert the 24 µm flux into LIR, while we fit templates also in that case. It is also possible that the input SEDs differ, for example in Barro et al. (2019) the prior for the FIR photometric extraction and deblending are the CANDELS sources. Neverthe- less, the SFR estimates are in good agreement (Fig. B.2) with a contained scatter comparable to the typical (0.3 dex) uncertainty of this kind of measurement. Our SFRUVIR values are slightly larger, by about 8% (0.03 dex in the logarithmic comparison in the figure). Another test we made is summarized in Fig. B.3. The goal is to verify the “clustering” of low-mass galaxies in the SOM grid, which alleviate the scatter in the rescaling process of Eq. (3). Even though we did not use individual fluxes as fea- tures, the clustering is a consequence of two combined proper- ties: the relationship between colors and M-to-light ratio (e.g., Bell & de Jong 2000, 2001; Zibetti et al. 2009) and the dis- tinctive M-to-light ratios that low-mass galaxies used as com- parisons to those at M > 1010 M⊙(e.g. Bundy et al. 2006; Moffett et al. 2016; Cui et al. 2021). A34, page 22 of 22

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https://zenodo.org/records/2525150/files/article.pdf

de

Das Chlormetakresol in der Desinfectionspraxis und die Schnelldesinfection

Archiv für Gynäkologie

public-domain

(Aus dem bakteriologischen Institut der K(Snigl. Universit~it in Budapest. Director: Prof. Dr. H. Preisz.) Das Chlormetakresol in der Desinfectionspraxis und die Schnelldesinfection. Yon Dr. E u g e n Konr~d. Seitdem R e i n i c k e 0 zuerst die Aufmerksamkeit darauf zu lenken versuehte, dass dio zdtraubende und auch die H~inde ziem]ieh angreifende P i i r b r i n g e r ' s e h e 5Iethode dureh einfaehe Alkoholwasehung ganz gut ersetzt wird und kliniseh ebenso gute Ergebnisse aufweist, sind mehrere Jahre verflossen, bis man auf die ~Sehnelldesinfeetion a der Hgnde wieder z u r i i e k k a m . - Es ist nieht zum mindesten die GewShnung an eine alte, gu~ befundene, wenn aueh unbectueme und zeitraubende 3Iethode, die die Ursaehe war, dass R e i n i e k e keinen Anklang fand. Das Sublimat hatte einen solehen Ruf als Desinfeetionsmit~el sieh erworben, dass man die unangenehmen Nebenwirkungen , wie auch die Giftigkeit desselben gerne in Kauf nahm. Die frSheren tI~indedesinfeetionsmethoden batten ein so gutes Zeugniss fiir das Sublimat ausgestellt, dass man es nicht missen wollte, trotz A h l f e l d ' s Bem/ihungen, tier naehzuweisen suehte~ dass das Sublimat in der tt/indedesinfeetion ganz gut entbehrlieh ist, und dass man mit Alkohol, naeh vorhergegangenen HeisswasserSeifendesinfeetion, die denkbar besten Erfolge haben kann~ ohne die H'ande dureh alas Sublimat zu sehs -Die in den ersten Versuehon A h l f e l d ' s offenbar vorhandenen kleineren Fehlerquellen waren die Ursache, dass die Kritik hier 1) Centralbl. f. Geburtsh. u. Gyn. 1894. No. 47. Bd. 9l. H. 2. Archiv ffir Gyn~ikologio. 17 24:4= Konrgd~ Chlormetakreso[ in der Desinfeetionspraxis u. s. w. scharf eingriff, und wenn aueh A h l f e l d seine Versuehe spgter meines Erachtens - - auf einwandfreier Basis wiederholte, konnte er keine vollkommene Anerkennung linden, wenn auch eine ganze Reihe yon Forschern seine Befunde besttitigen konnte. - - Dieser Kampf ist zu genau bekannt, als dass ieh mit der Wiederholung der Literatur die Zeit verschwenden miisste. - - Wie jeder Kampf, so hat auch dieser ~Kampf sich niitzlich erwiesen, indem wit zur Erkenntniss gelangten, dass keine Desinfectionsmethode fghig ist, die tt/inde keimfrei zu maehen. Wit verdanken diese Erkenntniss den scharfsinnigen Untersuchungsmethoden yon KrSnig und Paul, S a r w e y und P a u l , S a r w e y , dann auch P a u l und Prall. Aueh gelangten wir zur Erkenntniss, dass das Sublimat eben in Bezug auf die, den Operateur am meisten interessirenden Staphylokokken, Streptokokken, B. cell in seiner keimtSdeenden Wirkung zweifellos iibersehtttzt worden war. Schon Ahlfeld hat darauf hingewiesen, dass alas Sublimat auf die sporogenen Mikroorganismen eine bedeutend st/trkere keimtSdtende Kraft ausiibt, als auf die asporogenen Mikroorganismen, die der Alkohol viel intensiver und schneller verniehtet, als das Sublimat. Deswegen verlangte er auch, dass die H/indedesinfectionsversuche mit vegetativen Formen, und zwar mit dem resistenten Staphylococcus angestellt werden sollten. Wie wir sehen kSnnen, ist in den neuesten Arbeiten diesem berechtigten Verlangen Rechnung getragen worden. Wie wenig intensiv das Sublimat auf Staphylokokken einzuwirken im Stande ist, zeigen z. B. die Untersuehungen yon O t t o lenghil), der naehwies, dass in Wasser suspendirte Staphylokokken in einer 2~712 prec. SublimatlSsung erst nach 3 Stunden abgetSdtet werden, und in einer 0,542 prec. LSsung erst nach 7 Stunden. Ausserdem darf man night ausser Acht lassen, dass selbst mit den K r 6 n i g - P a u l - S a r w e y ' s e h e n Methoden der Desinfectionspriifung der Httnde alas Sublimat nieht giinzlich neutralisirt war, denn Opitz ~) hat nachgewiesen, dass dies nut mit intensiver Behandlung der Hgnde mit Ammoniumsulfat mSglich ist: was aber dig I-I/tnde h/isslicherweise schwarz ttirbt. Opitz hat in diesen Untersuchungen den Nachweis gefiihrt, dass mit Natronlauge abgespiilte Hautstiickchen, die stundenlanger Sublimateinwirkung aus- - 1) Desinf. 1909. H. 2. 2) Berliner klin. Wochensehr. 1898. No. 39. Konrs Chlormetakresol in der Desinfectionspraxis u. s.w. 245 gesetzt waren und sich bakteriologiseh erst s~eril erwiesen, naeh der Ammoniumsulfatbehandlung sich keimhaltig (Staphylokokken) zeigten. Sehon friiher batten einige Forscher sich dureh /~hnliehe Ergebnisse na.eh anderen, weniger giftigen und mSgliehst reizlosen Desinficientien umgesehen. Haupts/iehlich die Kresole und die ihnen verwandten ehemisehen Produete waren as, weiehe die Aufmerksamkeit auf sieh lenkten. Es ist ja seitdem eine Unmasse yon mehr oder minder werthvollen Desinfieientien bekannt geworden, ":on denen ieh z.B. nut das Lysol, das Lysoform und Solveol nennen mSehte. W/ihrend dieser Untersuehungen hat C. Fr/tnkel sehon im Jahre 1890 gefunden, class unter den Kresolen eines, das Metakresol, eine ganz besonders energisehe keimtSdtende Kraft besitzt. Das 4proe. Metakresol tSdtete naeh C. F r / i n k e l 1) Anthraxsporen in 8 Stunden, wogegen Parakresol erst in 10, das Orthokresol erst in 20 Stunden sieh keimtOdtend erwies. Diese Thatsaehe blieb aueh 1/ingere Zeit unbeachtet, und as war erst L a u b e n heimer2), tier sieh wieder, und zwar ganz eingehend mit den Kresolen und aueh dem Metakresol befasste. Er land, dass das Metakresol, und zwar das Chlormetakresol, das in rieinolsaurem Kali in 50 pC~. 15slieh, in dieser LSsung sowohl in Wasser, wie in Alkohol leieht 15slieh ist, eine ganz hervorragende Wirkung auf .die pathogenen vegetativen Formen der Mikroorganismen besitzt. Nach seinen Untersuehungen tSdtet das Chlormetakresol die an Grana~en angetroekneten Staphylokokken in 30 See. sieher ab und seine Desinfeetionskraft nimmt his 0,25 pCt. aueh nur unbedeutend ab. Seine Dosis letalis ist 21/2 real hSher als die des Lysols und as eignet sieh besonders in alkoholiseher LSsung ganz vorziiglieh zur tt/indedesin feetion. Laubenheimer's orientirende Htindedesinfeetionsversuehe :haben in der That reeh~ gut e Ergebnisse gehabt. Ieh habe dutch L a u b e n h e i m e r ' s Versuehe es fiir angezeigt gefunden, mieh mit dem Chlormetakresol als Desinfieiens zu beseh//ftigen. Da mir besonders viel daran lag, die Sehnelligkeit der Tiefenwirkung des Chlormetakresols zu beobaehten, habe ieh bei den Desinfeetionsversuehen doeh die heute so versehrieene Seidenfaden1) Zeitschr. f. Hyg. Bd. VI. S. 521. 2) Habilitationsschr. Giessen 1909. 17" 246 Konr~;d, Chlormetakresolin der Desinfoetionspraxisu. s. w. methode und daneben die E h r l i c h - B e e h o l d ' s c h e Uebersahiehtungsmethode verwendet. Die Anwendung der Seidenfadenmethode war folgende: Die sterilen, 2--3 em langen Seidenf~den wurden auf 1 Minute in eine physiologische Kochsalzsuspension yon vollvirulenten Staphylokokken und von B. coli gebraeht. Die Suspensionen werden durch Abschwemmen einer 24 Std. alten Agarcultur yon Staphylokokken mit 10 ecru einer physiologischen Kochsalzl6sung hergestellt und diese dann filtrirt; das Gleiche gesehah mit der~ Colisuspensionen. Nach 1 Minute wurden die FSden aus den Suspensionen mit steritem Platinspatel entfernt und 24 Stunden lang im Brutsehrank bei 370 C. getroeknet. Diese F/iden wurden zu den Desinfectionsversuehen verwandt und ihren Keimgehalt controlirte ich bei jedem Versuche; aueh wurde jeden 5. Tag ein friseher Vorrath yon Staphylokokkenf~tden und Colifiiden zubereitet. Zu den Uebersehichtungsversuehen beniitzte ieh 18 Stunden alte Schr~tgagareulturen des Staphylococcus aureus und albus und frisehe Coliculturen. Zu den Desinfeetionsversuehen verwendete ich drei versehiedene L/3sungsmittel und zwar: 1. reines Leitungswasser, 2. 98proe. Alkohol, 3. das v. Herff'sche Gemiseh (2 Theile Alkoho~ und 1 Theil AeetOn)o Jedes dieser L~Ssungsmittel enthielt 1 pCt. des yon der Firma G. R i c h t e r in Budapest hergestellten, unter dem Namen L y s o e h l o r in den Handel gebraehten Chlormetakresols. Die aus den desinfieirenden L~3sungen entnommenen Staphylokokkenseidenf~iden wurden in sterile, mit 5 eem steriler physiologiseher Koehs~lzlSsung gefiillte RiShrehen gebraeht und dort durc~t~ ttin- und Hersehwenken mit der PlatinSse das Desinficiens m~iglichst ausgespiilt. Dann wurden mit den Seidenf~den Agarplatten gegossen, ebenso mit einigen Tropfen der Spiilfliissigkeit. Bei den Ueberschiehtungsversuehen wurde nach Abgiessen des Desinficiens 3 mal mit physiologiseher steriler Koehsalzl6suug naehgespiilt, und dann yon dem Sehrggagar in fliissigen t30 @. warmen Agar geimpft und dieser zu Platten ausgegossen. Die Agarplatten wurden eine Woche lang beobaehtet und dann das. Ergebniss registrirt. Die kurz mitgetheilten, jetzt folgenden Ergebnisse stehen im Einklang mit denen L a u b e n h e i m e r ' s , nur ist bei mir die Zeitdauer, innerhalb der alas Chlormetakresol abt/3dtend wirkte, etwas l~inger, was aber leicht verst~indlich ist, da ich mit Seidenf~der~ gearbeitet hat,e, zu deren Durehdringung es doch einer kleinen. Spanne Zeit bedarf. Konr~d, Chlormetakreso[ in der Desinfectionspraxis u. s.w. 2,) 1. V e r s u e h e 247 mit w/~sseriger 1 prec. Lysoehlorl6sung a u f S t a p h . alb. nach 15 See. Einwirkung 70 Colonien 30 , , 8 , 75 , , 0 7~ 2. W i r k u n g der l p r o e , w~sserigen LysoehlorlSsung B. eoli. auf nach 15 See. Einwirkung cx~ Colonien 30 7~ , ~,D ~, (mehr als 100) 11/2 3Iin. , 9 ~, , 3 See. ,, 0 7~ B) W i r k u n g d e r l p r o e , alkoholisehen Lysoehlor]Ssung. 1. Auf Staphylokokken: Vollkommene AbtSdtung naeh 30 See. 2. Auf B. eoli naeh 11/2 Min. 83 Colonien ,, 2 See. 31 ~ , 3 , 0 , G) W i r k u n g der l p r o e . A l k o h o l - A e e t o n l S s u n g d e s Lysoehlor. 1. Auf Staphylokokken naeh 15 See. 17 Colonien, ,, 30 , vollk. AbtSdtung. 2. Auf B. eoli , 15 See. o<~ Colonien, , 30 ~ 2 ~ , 11/2 Min. vollk. AbtSdtung. Wie aus diesen Angaben leieht ersichtlich ist~ hat das Chlormetakresol sozusagen eine elektive Wirkung auf die pathogenen Kokken, da es z . B . das B. eoli langsamer abtSdtet, als die Staphylokokken. Ausserdem ist es augenf/illig, dass das Lysoehlor in dem v. t I e r f f ' s e h e n Gemiseh gelSst eine ganz besonders starke bakterieide Kraft entwiekelt, indem es die Golibacillen schneller abzut6dten vermag, als die rein alkoholische oder rein w/~sserige LSsung. Dass aber das v. I-]erff'sehe Gemiseh allein die Abt6dtung in so kurzer Zeit zu erzielen vermag, haben wir durch Versuehe dargethan, indem im v. H e r f f ' s e h e n Gemiseh, ohne Lysoehlorzusatz, die Colibaeillen erst naeh 5 Minuten abstarben. Diese intensive keimtSdtende Kraft braehte mieh auf den Gedanken, die Alkohol-Aeeton-LysochlorlSsung zur Sehnelldesinfeetion der Gummihandsehuhe und der Hgnde auf ihre Wirksamkeit zu erproben. ~248 Konrgd~ Chlormetakresol in tier Desinfectionspraxis u. s. w. Mi{ der F r o m m e - G a w r o n s k y ' s c b e n 1) Methode kann man die Handsehuhoberfl/iche, wie bekannt ist, in 4: Minuten keimfl'ei machen. Nun hat in der ]etzten Zeit B e c k e t 2) erw/ihnt, dass auf Veit's Veranlassung und Fingerzeig die Hallenser Klinik ein festes 8 prec. Formoipr@arat, Formogen genannt, in Anwendung bringen will, mit dem in einer halben Minute die Oberfl'ache der Gummihandschuhe vollkommen keimfrei gemachte wiirde, ohne dass man grSssere Soheidenschleimhautreizungen bekgme. Besonders das Letztere ersohien mir nur sehwer mSglieh, da ja alle Formalinprgparate yon etwas hSherer Concentration eine sehr starke Reizwirkung besitzen. Datum lag es sehr nahe, das Lysoehlor zu versuehen, dem eine derartige Reizwirkung fehlt, das dabei stark bakterieid wirkt und kein besonderes Herstellungsveffahren erfordert. Da nun das Lysochlor in Alkohol-Acetonl6sung sowohl Staphylokokken, 8treptokokken in 30 Sec. wie aueh den B. cell in 11/2 Minute vernichtet, so versuchte ieh ohne vorhergehende Wasser8eifenwaschung die Gummihandsehuhe in der LysochlorlSsung zu sterilisiren. Die angezogenen @ummihandsehuhe wurden erst mit Staphy]okokkenbouillon inficirt, das Eintrocknen der 8taphylokokkenbouilion abgewartet und dann die Vcrsuehe begonnen. Die Ergebnisse sind bei 30 Versuchen: naeh einer Waschung yon 30 See. 20 Colonien ~ ,~ :~ ,~ ,, 11/2 , 28 mal 0 und 2 mal 1 Colonie , ~ ,, 2 , jedesma] sterile Platten. Dasselbe Ergebniss kann verzeiehnet werden, wenn man nach 30 See. Wasser-Seifenwaschung eine 11/2 Min. lange Lysoehlorwasehung vornimmt. ~{an verwendet am besten einen Flanellappen, oder eine sehr weiehe Biirste, oder aueh einen nieht zu kleinen Wattebauseh, mit dem sieh die Itandsehuhe besonders gut behandeln lassen. Dass bei diesen Versuehen auf die Aussehaltung der naehtr~igliehen Desinfeetionswirkung des Lysoehlors vollste Aufmerksamkeit verwendet wurde, versteht sioh yon selbst. Wie die mitgetheilten Versuchsergebnisse zeigen, gelingt es mit einer 2 ~Iin. langen Lysoehlorwasehung, oder 11/2 Min. Lysoehlorbehandlung naeh einer 11/2 Min. langen Seifenwaschung, yell1) Miinchener med. Woehenschr. 1904. No. 40. 2) Deutsche reed. Wochenschr. 1909. Konrgd, Chlormetakresol in der Desinfectionspraxis u. s.w. 249 kommene Keimfreiheit der Handschuhoberflgche zu erzielen. Dabei hat man keine Reizwirkung, keine Vergiftungsgefahr zu befiirchten, aueh schadet das Verfahren den Handschuhen nicht im Geringten. Da sich Chlormetakresol im v. H e r f f ' s c h e n Gemisch so wirksam erwies, hielt ich es fiir angezeigt, damit Hgndedesinfectionsversuche vorzunehmen. Vor Allem interessirte es mich~ wie sieh diese L/Ssung zur Schnelldesinfection der Hgnde eignen wiirde. Die F S b r i n g e r ' s c h e Methode leistet ja Vorziigliches, aber sie greifr die ttgnde an und ist zeitraubend. Und wenn wit dieselbe Keimarmuth der H/inde in viel kiirzerer Zeit und unter besserer Schonung unserer tt/inde erreichen k~Snnen, warum sollten wir diesen neuen Weg nicht beschreiten? Hgtten wit nur noch vor einigen Jahren gedacht, dass es zur Vorbereitung des Operationsfeldes geniigend ist, dasselbe zwei Mal mit Jod einzupinseln, ohne Anwendung von Seife, Biirste, Alkohol und Sublimat? Und doch hat sich diese O-rossich'sche 1) Methode, die noch einfacher und kiirzer ist als die D o e d e r l e i n ' s c h e Gaudaninmethode, vollkommen bew~ihrt. Was die Schnelldesinfeetion der H'ande anbetrifft, so hat die ersten Versuche darin t l e i n i c k e angestellt, der als erster, unter Ausschaltung der Seifenwasserwaschung und Weglassen des Sublimats, eine 5 Minuten ]ange Alkoholwaschung tier Hgnde empfahl und damit auch gute Resultate hatte. Verdienstvoll haben in dieser Ilichtung H e u s n e r , S e h u m burg, v. B r u n n und v. H e r f f mit seinen Sc.hiilern, Oeri und P fi s t e r e r weitergearbeitet. Ueber H e u s n e r ' s 3odbenzindesinfection sind d i e Ansiehten nieht so ganz einig2), da versehiedene Autoren unangenehme Reizwirkungen erlebt haben. Dasselbe kann man auoh yon W e d e r h a k e ' s a) Dermagummit sagen. S e h u m b u r g 4) hat bei seinem ersten Gemisch Aether angewendet und dabei nieht ggnzlich zufriedenste[lende Er~ebnlsse gehabt, his auch er anstatt des Aethers Aceton nahm. v. B r u n n s) hat a ueh mit dem 96 proe. klkohol (5 Min. Wasehung) gute kliniseho Resultate gefunden. V. H e r f f 6) hat an seiner Klinik, nun sehon fiber ein Jahr, 1) CentrMbl. f. Chirurg. 1909. No. 441 2) Mfinehener med. Woshensobr. 1007. S. 1872. 3) CentrMbl. f. Geburtsh. u. Oyn. 1008. 4) Archiv f. Min. Chirnrg. Bd. 79. H. 1. 5) Beitr. z. klin. Chirurg. Bd. 58. 6) Mtinehener med. Wochenschr. 1910. 250 Konrgd~ ChlormetakresoI in der Desinfectionspraxis u. s. w. eine Misohung zur Desinfection verwendet, die crsf aus Alkohol nnd Aceton 1 : 1 , dann 2 : 1 bestand. Die H-ando werden nur 1/2 Min. ]ang ein wenig in warmem Wasser mit Seife, ohne Btirste gewasohen, haup~s//ehlich zur Erleiehterung der Nageltoilet~e. ])ann folgt eine 5 Min. lange Waschung in dem Gemisoh; mit Flanelllapper~ werden die Hgnde sorgf/iltig abgerieben, naoh Vollendung dieser einfaehen Procedur sind die ttgnde operationsfertig. Sohon die wissensehaftlie, h bakteriologisehe Controle des Verfahrens berechtigte zur ttoffnung, eine bran&bare Sehnelidesinfectionsmothode zu bekommen. Oeri's 1) VerSffentliehung hat sich dann ouch bewahrheitet, denn die vor kurzer Zeit yon v. H e r f f gomachte Mittheilung tiber die klinisch-praktisehen Ergebnisse seiner Desinfeetionsmethode sind wirklioh vorziiglioh zu nennen. Da sioh dos Chlormetakresol ganz besonders in dor v. tterff'sehen Mischung gelSst so bakterieid erwies~ und ganz besonders gegen die Eiterkokken nnd pathogenen vegetativen Spaltpilzformen, konnte ich a priori annehmen, dass die Desinfeotion mit dieser LSsung vielleicht noeh schneller, als mit der reinen v. Herff'schon Misohung bewerkstelligt werden kSnnte. Jedenfalls konnte man denken, in der yon v. H e r f f angegebenen Zeit eine nooh sicherere und lgnger dauernde Reimarmuth erreiehen zu kSnnen. Die H~indedesinfeotionsversuche habe ieh r.ungohst an meinen eigenen tt/inden angestellt~ dann habe ich Versuohe an den H~inden yon 3 Collegen, die als Volontb;rassistenten des bakteriologisohen Instituts th~itig sind, vorgenommen. Die Ergebnisse dieser Versuehe halte ich ftir besonders wichtig, da die betreffenden Collegen st'andig mit Bakterien, zwei yon ihnen sogar speeiell mit den verschiedensten Eitererregern gearbeitet haben. Ausserdem hatten die betreffenden Herren nicht dieselbe Desinfeotionsteohnik, ouch nioht so sorgfgltig gepflegte Hgnde, wie ein geiibter Chirurg odor Geburtshelfer sie hat. Die Versuche mit meinen eigenen H'anden habe ieh in Intervallen yon 3 - - 4 Tagen gomaeht. Jedesmal impfe ich ouch yon der undesinfioirten Hautoberflgche und Unternagelranm, um reich yon der Besehaffenheit mciner It~indebakterienflora zu unterriohten und diese Ergebnisse mit donen der sich ansehliessenden Desinfectionsversuche zu vergleichen. Diese Untersuchungen orgaben, dass unter den 12mal vorgenommenen Impfungen kein einziges Mal 1) Zeitschr. f. Geburtsh. u. Gyn. Bd. 63. H. 3. Konrg~d 7 Chlormetakresolin der Desinfeotionspraxis u. s.w. 251 Streptokokken zu finden waren. Aueh Staphylokokken (weisse) wurden nur in 3 Fallen gefunden. Dagegen enthielt der Nagelsehmutz fast jedesmal B. eoli, und aueh 5fters ein kleines, sehr resistentes Saprophytenst/ib~hen. Auf Agar ist es den Staphylokokkeneolonien b;hnlieh, doch ist der Rand der runden Colonien nieht so soharf wie bei den Staphylokokken, aueh haben diese Oolonien einen dunkleren Kern, sind also nieht gleiehm~issig gefgrbt. Die jetzt allgemein anerkannte hohe Wiehtigkei~ der rationellen Hitndepflege fiir den Chirurgen und Gyn/ikologen wird dutch diese Versuehe yon Neuem gestiitzt; denn wenn man die yon meinen Hgnden erhobenen Befunde mit denen von den H/inden der Bakteriologen vergleicht, so is~ der gntersehied seharf ins kuge springend. Die Impfungen yon den undesinfieirten H/inden der 3 Herren des bakteriologisehen Institutes ergaben folgendes Durehsehnittsresultat: 1. Herr Dr. H. (nach 3 maliger kbimpfung) 62 Colonien, darunter 9 Colonien Streptokokken, 3 Colonien der Saroina lutea und 2 Colonien des Staphylococcus aureus, sonst lauter weisse Staphylokokkeneolonien (merkwiirdigerweise keine Colibaeillen). 2. Herr Dr. B. (3 malige Abimpfung) 71 Colonien, vorwiegend Staphylokokken und Colibaoillen, darunten 7 Colonien yon Staphylococcus aureus. 3. Herr Dr. G. ~ (iiber 100) Staphylococcus albus- und Colibaeilleneolonien, daneben einige Colonien mit Sareina lutea. Es ist angenfiillig, dass unter 12 Untersuehungen meiner Itgnde nieht annghernd so viel Staphylokokkeneolonien gefunden wurden, wie bei einem einzigen Versuoh bei den 3 Herren. Und ieh glaube, dass dies aueh auf den grossen Nutzen der Gummihandsehuhe hindeutet. Es ist ganz riehtig, wenn man aueh die kleinsten Manipulationen in der gynS;kologisehen Behandlung unter Gummihandsehuhsehutz vornimmt, manehe, z.B. L e o p o l d , fassen sogar bei tier Wundenbehandlung Alles mit Pineetten an, um ihre H/inde soweit wie m6glieh zu sehiitzen, leh glaube aueh entsehieden, class ein derart vorsiehtiger Operateur, wenner einmal eine Nothoperation zu maehen, wenig Zeit far Desinfeetion seiner H/tnde und keine Gummihandsehuhe zur Hand hat, selbst bei einer ungeniigenden H/indedesinfeetion kein grosses Unheil stiftet. Die Hgndedesinfeetionsteehnik und ihre Oontrolle wurde folgendermaasssen ausgefiihrt. Naeh eine halbe Minute langer 252 Konr~d~ Chlormetakresol in der DesinfeGtionspraxis u. s. w. Seifenwasehung (ohns Biirsts) Nagelrsinigung. Naeh der Nageltoilette 31/2 Min. lang Wasehen der H~inde in siner 1 prec. L~sung des Chlormetakresols in d e r v . Herff'sehen Misehung. Naeh der vollendeten Desinfeetion wird das Troekuen der H~inde abgewartet. Dies tritt bald sin, gew6hnlieh in 60--75 See. Darnaeh Abspiilen der H/inde mit geniigender Menge yon stsriler KoehsalzliSsung und hernaeh Aussehaben der Untsrnagelrg.ume mit sterilen H61zehsn, die in t2 0 warmem Agar zu Platten. ausgegossen werden. Beobaehtungszeit sine Woehe. Um zu sshsn i o b dis troeknende, gerbende Wirkung disser Dssinfeetionsmsthode aueh l~nger anhg~lt, habe ieh noeh fo]gendes einfaehe Verfahren in A~wendung gebraeht. Ueber die desinfieirten H~nde wurden sterile Gummihandsehuhe gezogen. Naehdem die Handsehuhe sehon 45 Min. lang auf den H~ndsn sassen, wurden dis behandsehuhten H~nde noeh 10--15 Min. lang in 40 o C. hsissem Wasser gebadst, darnaeh die ttandsehuhoberfl/iehs wieder mit Chlormetakresol desinfieirt und vorsiehtig abgezogen. Naeh Entfsrnung der Handschuhe dasselbe Verfahrsn wie sehon angegeben. Steriler Kasten~ wie ihn S a r w s y nnd P a u l gebraueht haben~ stand mir nicht zur Verfiigung, und ieh meine, dass es nut interessant sein kann, was ffir Keime aus der Luft wShrend und naeh der Desinfeetion auf die Hautoberfl/~ehs niederfallen. Der Operatsur kana ja seine H~nde im stsrilen Kasten desinfieiren; abet am Ends muss er doeh herausriieksn, wsnn er dis Operation beginnen will. Uebrigens haben versehiedene Untersueher, so z. B. S e h m i d l e e h n e r l ) , die Luft der Operationsr~ume auf Luftkeime untersueht und gefunden, dass dieselbe besondsrs bei Gebraueh einer l~sgenwasehvorriehtung, wie ihn dis Tauffer'sehe Klinik besitzt, sozusagen als ksimfrei angesehen werden kann. Ish habe die Vsrsuehe in einem natiirlieh nieht gerade so geeigneten Raume ausgeffihrt, denn die Luft des bakteriologisehen Institutes ist ganz entsehieden etwas ksimreichsr. Bei versehiedenen~ zu diesem Zweeks angestellten Plattenversuehen habe ieh gefunden, dass die Laboratoriumsluft des 5fteren B. subtilis~ Sareina lutea und versehiedene Saprophytenst~behsn enthielt, ja ein-zweimal habe ieh aueh sinige Staph. alb.-Colonisn angehen sshsn. 1) Verhandlungen der deutschen Gosellschaft fiir Geburtshfilfe und Gyn~kologie. 1907. Konr~d, Chlormetakresol in der Desinfactionspraxis u. s.w. Die Ergehnisse der H~inden sind folgende. Versuch , , , , ,~ , , H~ndedesinfectionsversuche an 253 meinen 1 = 2 = 3 = I = 5 = 6 = 2 Colonien (B. subtilis), Platten steril, 4 Colonien (1 B. subtilis, 3 Streptothryx), Plat~en steril, Platten steril, 2 Colonien (kleine St~behen in Ketten, triiben die Bouillon), 7 ~--- 1 Colonie (B. sub~ilis), 8 - - 1 2 = Platten steril. Da die Laboratoriumsluft, wie ieh fand, 5fters B. subtilis enthielt, so kann ieh annehmen, dass die auf den Platten in den Versuehen 1, 4, 7 gewachsenen Subtiliseolonien aus der Luft stammen, dasselbe kann ieh vom Streptothryx vermuthen. Nur die 2 Colonien des Versuches No. 6 kann ieh nicht als Luftinfeetion ansehen, da ieh diese Saprophytenst~bchen bei mir und aueh bei Anderen oft aus dem Nagelsehmutz geziiehtet habe. Demnaeh kann ieh sagen, dass ieh in der Desinfeetion meiner H~nde vom praktiseh-chirurgisehen Standpunkt aus, in jedem Falle, streng bakteriologiseh betrachtet, yon 12 Versuehen 11 ma| geniigende Keimarmuth erzielt habe. Die Versuche an den H/inden der Herren Dr. H., B. und G. ergaben : Herr Dr. H. Versueh ,, ,, Herr Dr. B. , Herr Dr. G. :~ 1 = 2 Colonien (Sareina lutea), 2 = Platten steril, 3 =- Platten steriI, 1 - - 3 z Platten steril, 1 - - 3 = Platten steril. Diese Ergebnisse sind, wie ich glaube und sehon betont habe, ganz besonders werthvoll, denn sie sind an den HSnden yon Fachbakteriologen erreieht worden. ]ch babe noeh naehtr/iglieh 2 - - 3 Versuehe an meinen H';inden angestellt und jedesmal sterile Platten bekommen. Meine Versuehe haben aueh gezeigt, dass die mit Chlormetakresol-Alkohol-AeetonlSsung desinficirten l:I//nde, wenn man die Handsehuhe nahezu 1 Stunde lang anhat, und die H~nde dabei 1 0 - - 1 5 ~lin. lang in warmem Wasser badet, diese noeh sehSn 254 Konrs Chlormetakresol in der Desinfectionspraxis u. s. w. trocken bleiben and nicht schwitzen. Die Haut bleibt, selbsg wenn man nach Entfernung der Handschuhe den Talg mit warmem Wasser abspiil% merkwiirdig trocken und es ist ordentlich angenehm, wenn man die Ilgnde etwas einfetten kann. Es giebt viele Pr~parate, aber um die Haut weich und geschmeidig zu machen und auch jeder noch so geringen Reizwirkung vorzubeugen ist es am besten die H~nde mi~ Glycerin zu behandeln, in dem man 5 pot. Extraetum fluidum Hamamelis Virginicae gelSst hat. Der Gerueh dieser LSsung ist, wenn auch nieht unangenehm, etwas eigenartig, weshalb man, je naeh Geschmaek, Geruchseorrigentien zusetzen kann. Bei nut einigermaassen sachgem~;sser H'/tndepflege wird man bei dot Chlormetakresol-Alkohol-keetondesinfeotion gar keine Reizerseheinung erleben, wogegen bei Verwendung der PSrbringer'sehen Methodik selbst der wetterfesteste Praktiker hier und da sein Sublimatekzem zu behandeln hat. Nach Beendigung der Versuche habe ich es fiir berechtigt gefunden, die Methode praktisoh zu erproben. Ieh habe mit Anwendung der Chlormetakresol-Alkohol-Aeetondesinfection absichtlich ohne Handsohuhsehutz folgende Eingriffe ausgef(ihrt: 4 Gebgrmutterausrgumungen bei Fehlgeburten (mit dem Finger), 1 Colporrhaphie, 2 Dammnghte, 4 Curettagen bei Endometritis. GrSssere Eingriffe auszufiihren ist mir in der kurzen Zeit, die seit der Beendigung verstrichen ist, nieht m6glich gewesen, kber die oben genannten Eingriffe batten kein Fieber, keine Infection zur Polge. Besonders tier fieberfreie Verlauf der Geb/irmntterausr~umungen zeigt, dass diese Desinfectionsmethode lebensf~ihig ist. Gegenw~irtig wird sic an einem gr6sseren chirurgiseh-operariven Material erprobt, iiber deren abgesehlossene Ergebnisse ich noch berichten werde. Diese Verbesserung und auch eine Besehleunigung derv. Herffsehen Desinfectionsmethodik, die auch eine wirkliche Schnelldesinfeetionsmethode ist, seheint mir besonders fiir die Geburtshelfer und Kriegsehirurgen geeignet zu sein. Im Falle dringlicher Operationen wird sie Vorziigliches leisten. Abet aueh im klinisehen Operationsbetrieb~ wie aueh im Kreisssaa[ wird sic, da man mit ihr viel Zeit sparen nnd aueh die H~nde schonen kann, hoffentlich sich langsam aber sicher Eingang verschaffen. Konrs Chlormetakresolin der Desinfoc~ionsioraxisu. s.w. 255 Zusammenfassung. I. Das C h l o r m e t a k r e s o l (Lysochlor) ist ein mSchtiges D e s i n f e c t i o n s m i t t e l . I n s b e s o n d e r e schne]] vern i e h t e t es vegetative Spaltpilze, d a r u n t e r die Eiterkokken. 2. Da seine Wirkung, wie die der /ibrigen Kresole, in eiweisshal~ig'en oder s e i f e n h a l t i g e n Medien kaum geringer ist, eignet sich in Anbe~raeht seiner s t a r k e n bakt e r i c i d e n Kraft ganz besonders zur H a u t d e s i n f e e t i o n . 3. Das C h l o r m e t a k r e s o l wirkt am st~;rksten in Alkohol, noeh besser in A l k o h o l - A c e t o n g e m i s e h (naeh v. Herff). 4. Zur Desinfeetion der ttaut eignet sich yon den S c h n e l l d e s i n f e e t i o n s m e t h o d e n diev. Herff'sehe am besten. Diese wird dutch d i e Z u g a b e yon 1 proe. C h l o r m e t a k r e s o l noeh v e r b e s s e r t und besehleunigt, so dass man in 31/2 his 4 Minuten geniigende t ( e i m a r m u t h der Itaut erzielen kann. Die F r o m m e - G a w r o n s k y ' s c h e G u m m i h a n d s e h u h m e t h o d e kann dutch d a s C h l o r m e t a k r e s o l - A l k o h o ] - A e e t o n g e m i s e h um die H/ilfte der dazu nSthigen Zeit (4Min.) verk[irzt werden. 5. Wegen der s t a r k b a k t e r i e i d e n Kraft der wgsserigen LSsungen und wegen g e r i n g e r e r G i f t i g k e i t (s. Laubenheimer) wird sich das C h l o r m e t a k r e s o l zu desinfieirenden Spiilungen sehr gut g e b r a u e h e n lassen. Wegen dieser Vorziige wgre es keine verlorene Miihe, das Chlormetakresol in anderen therapeutischen Beziehungen zn erproben. 

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Evolutionary study of duplications of the miRNA machinery in aphids associated with striking rate acceleration and changes in expression profiles

BMC evolutionary biology

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Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Open Access Evolutionary study of duplications of the miRNA machinery in aphids associated with striking rate acceleration and changes in expression profiles Benjamín Ortiz-Rivas1, Stéphanie Jaubert-Possamai1,2, Sylvie Tanguy1, Jean-Pierre Gauthier1, Denis Tagu1 and Rispe Claude1* Abstract Background: The sequencing of the genome of the pea aphid Acyrthosiphon pisum revealed an unusual expansion of the miRNA machinery, with two argonaute-1, two dicer-1 and four pasha gene copies. In this report, we have undertaken a deeper evolutionary analysis of the phylogenetic timing of these gene duplications and of the associated selective pressures by sequencing the two copies of ago-1 and dcr-1 in different aphid species of the subfamily Aphidinae. We have also carried out an analysis of the expression of both copies of ago-1 and dcr-1 by semi-quantitative PCR in different morphs of the pea aphid life cycle. Results: The analysis has shown that the duplication of ago-1 occurred in an ancestor of the subfamily Aphidinae while the duplication of dcr-1 appears to be more recent. Besides, it has confirmed a pattern of one conserved copy and one accelerated copy for both genes, and has revealed the action of positive selection on several regions of the fast-evolving ago-1b. On the other hand, the semi-quantitative PCR experiments have revealed a differential expression of these genes between the morphs of the parthenogenetic and the sexual phases of Acyrthosiphon pisum. Conclusions: The discovery of these gene duplications in the miRNA machinery of aphids opens new perspectives of research about the regulation of gene expression in these insects. Accelerated evolution, positive selection and differential expression affecting some of the copies of these genes suggests the possibility of a neofunctionalization of these duplicates, which might play a role in the display of the striking phenotypic plasticity of aphids. Keywords: Aphids, miRNAs, Gene duplication, Neofunctionalization © 2012 Ortiz-Rivas et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH ARTICLE Open Access © 2012 Ortiz-Rivas et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: [email protected] 1INRA, UMR 1349 IGEPP, Le Rheu 35653, France Full list of author information is available at the end of the article Background Further- more, the ratio of non-synonymous to synonymous rates (dN/dS or ω) was well above 1 for ago-1b, which suggested the existence of positive selection driving the evolution of this gene, possibly towards a new function [17]. However, most of these preliminary analyses included only one aphid species, A. pisum, which might have given very rough estimates of the dates of duplication events and of the selective pressures acting on the different copies. the nucleus by the RNase III enzyme Drosha with the help of the double stranded RNA binding protein Pasha (DGCR8 in mammals) [6]. The resulting pre- miRNAs are then translocated to the cytoplasm by Exportin-5, where they are subsequently cleaved by a second RNase III enzyme called Dicer in association with the double stranded RNA binding protein Loqua- cious (TRBP in mammals), yielding the mature miRNA in the form of an RNA duplex. Commonly, one of the strands of the duplex is degraded while the other is loaded into an effector complex named RISC (RNA-induced silencing complex) which has a protein of the Argonaute family as key component. The miRNA guides the RISC to a target messenger RNA (mRNA) by recognition of a complementary or nearly complementary sequence usually located in the 3’UTR of the target mRNA. MicroRNA-mRNA recognition leads to the degradation or the inhibition of the translation of the targeted mRNA [7-9]. Several instances of duplications have been reported for the machinery of small non-coding RNAs, in par- ticular for the miRNA pathway in animals. Some of these duplications have been also linked to the neo- functionalization or subfunctionalization of one or both duplicate pairs. For example, only one Dicer is present in vertebrates and in the nematode Caenor- habditis elegans, which cleaves both miRNAs and small interfering RNAs (siRNAs), but two copies of this protein have been described in Drosophila mela- nogaster and other insects: Dicer-1, involved in the miRNA pathway, and Dicer-2, associated with the production of siRNAs [10]. Expansions of the Argonaute family have been more common, the most impressive example being the 27 members found in C. elegans [11]. The duplications in this nematode have involved subfunctionalization and neofunctionalization of the copies, with only two of the 27 copies associated with miRNAs. Background These ~20-30 nucleotides long molecules have been described (e.g. endo- and exo-small interfering RNAs, micro RNAs, piwi associated RNAs. . .) and classified depending on their endogenous or exogenous origin, their mechanisms of biogenesis and function and their biological role. Regulation of gene expression by small non-coding RNAs is always carried out in association with a protein of the Argonaute family and is executed at multiple levels, ranging from chromatin modification to post-transcriptional control [4,5]. Evolutionary novelty may brought by different processes, their different weights having been debated in the last years [1]. These processes include changes in protein se- quence, regulatory changes and duplication [2], or com- binations of all these factors [3]. The present study illustrates a possible new example of a combination of duplication, modulation of gene expression and changes in evolutionary rates, which may be of relevance to adaptation in a group of insects. Small non-coding RNAs have been shown to play a fundamental role in the regulation of gene expression. MicroRNAs (miRNAs) are small non-coding RNAs of ~20-24 nucleotides which derive from the transcription of endogenous genes by RNA pol II. In animals, they are first produced as long stem-loop hairpin primary miRNA precursors (pri-miRNA) which are cleaved in * Correspondence: [email protected] 1INRA, UMR 1349 IGEPP, Le Rheu 35653, France Full list of author information is available at the end of the article * Correspondence: [email protected] 1INRA, UMR 1349 IGEPP, Le Rheu 35653, France Full list of author information is available at the end of the article Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 2 of 13 Apidcr-1b), and two copies of Argonaute-1 (Apiago-1a and Apiago-1b) [17]. This is the first such general ex- pansion of the miRNA machinery observed for a coel- omate animal. In all other insects studied to date, only one copy of each of these genes specific to the miRNA pathway exists. Preliminary molecular evolutionary analyses suggested that the duplications of ago-1 and dcr-1 in aphids occurred roughly simultaneously, and that for each gene one of the duplicated copies was characterized by a higher evolutionary rate and relaxation of selection (ago-1b and dcr-1b), while the other was conserved and subject to strong purifying selection (ago-1a and dcr-1a). Background Other expansions of this family have also been observed in insects like the mosquitoes Culex pipiens and Aedes aegypti [12], or in the crustacean Daphnia pulex, with 9 copies described [13]. In D. melanogaster five Argonaute proteins exist, of which AGO-1 (mainly associated with miRNAs) and AGO-2 (mainly with siRNAs) are related to the RISC. On the contrary, all of the 4 Argonaute proteins described in vertebrates are able to load miRNAs [4,14]. To further evaluate the biological implications of the expansion of the miRNA machinery in aphids, we have carried out an evolutionary and expression analysis of dcr-1 and ago-1 duplicated genes. For this, the two copies of dcr-1 and ago-1 were sequenced in several aphid spe- cies representing a gradient of evolutionary distance from A. pisum in order to better evaluate the phylogenetic timing of the duplications as well as the evolutionary pattern of nucleotide evolution and selective pressures affecting these genes. The existence of different copies of the miRNA ma- chinery raises the question of whether these duplicates could be recruited differently along the complex bio- logical life-cycle of aphids, and if the different copies could play a role in the modulation of gene expression along this cycle. Aphids typically alternate between several generations of parthenogenetic females, which take place during spring and summer, and one annual generation of sexual reproduction at the end of sum- mer, which produces overwintering eggs. They display a remarkable degree of polyphenism, with several morphs resulting from a same genotype during the parthenogenetic phase of their life cycle (e.g. winged/ wingless individuals, viviparous/oviparous females . . .). As a first attempt to evaluate the functional implication of the miRNA machinery duplications, semi-quantitative RT-PCRs were carried out in the different reproductive morphs of the pea aphid and revealed different levels of gene expression in parthenogenetic and sexual aphids, suggesting a role for ago-1b and dcr-1b regulation in the sexual polyphenism of A. pisum. We discuss the data in the perspective that the duplication of ago-1 and dcr-1 might be related to the neofunctionalization of one of the copies of each of these genes. The sequencing of the genome of the pea aphid Acyrthosiphon pisum [15] allowed the identification of homologues of the small non-coding RNAs machinery for the first time in a hemimetabolous insect. Phylogenetic distribution of the duplications of the miRNA machinery in aphids We have investigated the duplication of dcr-1 and ago-1 in several aphid species from the subfamily Aphidinae. Two copies of ago-1 were found in all of these species, belonging to two tribes, Aphidini and Macrosiphini (see Table 1). However, some regions of ago-1b could not be obtained in some species, due to unsuccessful PCR amplifications. The separation of ago-1a and ago-1b copies was clear when amino acid sequences were used for the phylogen- etic inference, for the three regions of the gene as well as for the concatenated alignment, and independently of the method used (Figure 1), while with nucleotide sequences the groups differed depending on the phylo- genetic method. All amino acid sequences of ago-1a in the Aphidini+Macrosiphini were identical except for one amino acid substitution in Region 3 in A. svalbardicum. By contrast, ago-1b copies were much more variable, as revealed by long branches for all species. None of the seven hypotheses were rejected by the SH test, while the ELW test only rejected hypotheses 6 and 7 (Table 2). The hypothesis 1 (Figure 2) suggests one gene duplication of dcr-1 that would have happened in the ancestor of the tribe Macrosiphini, after the split between Aphidini and Macrosiphini; in addition, the dcr1-b copy would have been lost secondarily in M. per- sicae or just could not be amplified in that species. The hypotheses 2 and 3 (not rejected in the tests) imply a complex scenario for the evolution of this gene, involv- ing several duplications during the evolution of the sub- family Aphidinae. Both hypotheses 2 and 3 would also The sequencing of dcr-1 yielded two different copies only in the three species of the genus Acyrthosiphon. Despite the use of up to 4 combinations of PCR primers, no partial dcr-1b sequence was obtained in the other species. The name of the gene was kept as dcr-1 for those species where only one copy was sequenced, while the two copies found in the Acyrthosiphon species were named dcr-1a or dcr-1b. Identical trees were obtained with different methods and whether analyzing nucleotide or amino acid data. Background Their pre- liminary characterization revealed an unusual expansion of the machinery specific to piRNAs [16] and miRNAs, with the existence of four copies of Pasha (Apipasha-1 to Apipasha-4), two copies of Dicer-1 (Apidcr-1a and Page 3 of 13 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 obtained for dcr-1 were not supported by high bootstrap values, thus preventing an unambiguous determination of the phylogenetic timing of the duplication of this gene in aphids. We conducted SH and ELW [18,19] tests to compare among seven different phylogenetic hypotheses concerning the time of duplication of dcr-1. The seven hypotheses were constructed by independent permuta- tion of each of the three oldest nodes in the dcr-1 ML tree (Figure 2; nodes supported by 57%, 74% and 83% bootstrap values). The ML tree was named as hypothesis 1. The seven hypotheses can be seen in Additional file 1: Figure S3. Hypotheses 2 and 3 placed either R. padi dcr- 1 or A. gossypii dcr-1 sequences in a basal position, leav- ing the other one as sister of the rest of aphid sequences. Hypotheses 4 and 5 included R. padi and A. gossypii dcr- 1 sequences in the dcr-1/1a group or the dcr-1b group, respectively. Hypotheses 6 and 7 altered the phylogen- etic position of M. persicae dcr-1 sequences, either pla- cing them as sister to the rest of Macrosiphini or included in the dcr-1b group. Phylogenetic distribution of the duplications of the miRNA machinery in aphids By contrast with results obtained for ago-1, the oldest nodes in the maximum likelihood tree Table 1 EMBL Accession numbers of genes from aphid species analysed in this studya Speciesb ago-1a ago-1b dcr-1/dcr-1ac dcr-1b Region 1 Region 2 Region 3 Region 1 Region 2 Region 3 Macrosiphini Acyrthosiphon pisum HE585884 HE585889 HE585923 HE585940 HE585898 HE585904 HE585918 HE585919 HE585950 HE585952 HE585973 HE585961 HE585966 Acyrthosiphon kondoi HE585892 HE585893 HE585926 HE585935 HE585905 HE585912 HE585953 HE585979 HE585980 HE585963 Acyrthosiphon svalbardicum HE585896 HE585927 HE585934 HE585906e HE585913 HE585947 HE585968 HE585962 Sitobion avenae HE585902 HE585929 HE585930 HE585942 HE585943 HE585883 HE585914 HE585945 HE585978 Aulacorthum solani HE585894 HE585895 HE585928 HE585941 HE585907e HE585920 HE585944 HE585948 HE585976 HE585977 Myzus persicae HE585886 HE585931 HE585946 HE585897 HE585921 HE585969 HE585970 Aphidini Aphis gossypii HE585882 HE585910 HE585911 GW549708d HE585981 HE585982e HE585955 HE585959 Rhopalosiphum padi HE585880 HE585881 HE585908 HE585909 HE585932 HE585933 HE585956 HE585957 HE585958 aEMBL accession numbers are given for each copy and region of ago-1 and for each copy of dcr-1. Two numbers are given for those species where two alleles were obtained. A blank in a cell means that the sequence for that gene/region in that particular species was not obtained. bAphid species were classified following Remaudière and Remaudière (1997). cThe gene was named dcr-1a in the Acyrthosiphon species, where a dcr-1b sequence was found, and dcr-1 in the rest of species, where a second copy was not found. dSequence from an Aphis gossypii cDNA library (Webb, B.A. and Shelby, K.S., unpublished data). eSequences for which the complete targeted region could not be obtained. Table 1 EMBL Accession numbers of genes from aphid species analysed in this studya aEMBL accession numbers are given for each copy and region of ago-1 and for each copy of dcr-1. Two numbers are given for those species where two alleles were obtained. A blank in a cell means that the sequence for that gene/region in that particular species was not obtained. bAphid species were classified following Remaudière and Remaudière (1997). cThe gene was named dcr-1a in the Acyrthosiphon species, where a dcr-1b sequence was found, and dcr-1 in the rest of species, where a second copy was not found. dSequence from an Aphis gossypii cDNA library (Webb, B.A. and Shelby, K.S., unpublished data). eSequences for which the complete targeted region could not be obtained. aEMBL accession numbers are given for each copy and region of ago-1 and for each copy of dcr-1. Phylogenetic distribution of the duplications of the miRNA machinery in aphids The value was calculated as the weighted mean among the ω values of the three coding Figure 1 Maximum likelihood tree inferred from the amino acid concatenated alignment of ago-1 sequences (m um likelihood tree inferred from the amino acid concatenated alignment of ago-1 sequences (model JTT+G+F). The as found using maximum parsimony and Bayesian inference with only slight differences in the relationships inside the ago Figure 1 Maximum likelihood tree inferred from the amino acid concatenated alignment of ago-1 sequences (model JTT+G+F). The same topology was found using maximum parsimony and Bayesian inference, with only slight differences in the relationships inside the ago-1b group. For those species for which two alleles were obtained only one consensus sequence was included, since no correlation could be made between different alleles of the three different regions. An asterisk (*) marks the suggested moment of the duplication of ago-1. Bootstrap values are shown only when higher that 50%. For each copy of the gene, the value of the ratio of non-synonymous to synonymous rates (ω) resulting from the “two-ratio” model is shown (see Results). The value was calculated as the weighted mean among the ω values of the three coding nucleotide regions analyzed. A strong purifying selection characterizes the ago-1a sequences, contrasting with relaxed selection of ago-1b. ated alignment of ago-1 sequences (model JTT+G+F). The Figure 2 Maximum likelihood tree inferred from the amino acid alignment of dcr-1 sequences. The same topology was found with Bayesian inference and maximum parsimony except for the lack of monophyly of the Aphidini in the latter method. Bootstrap values are shown only when higher that 50%. a1: allele 1; a2: allele 2. For each copy of the gene, the value of the ratio of non-synonymous to synonymous rates (ω) resulting from the “two-ratio” model is shown (see Results), reflecting the difference in evolutionary pressures acting on the different copies, though less marked than for ago-1. Figure 2 Maximum likelihood tree inferred from the amino acid alignment of dcr-1 sequences. The same topology was found with Bayesian inference and maximum parsimony except for the lack of monophyly of the Aphidini in the latter method. Bootstrap values are shown only when higher that 50%. a1: allele 1; a2: allele 2. Phylogenetic distribution of the duplications of the miRNA machinery in aphids Two numbers are given for those species where two alleles were obtained. A blank in a cell means that the sequence for that gene/region in that particular species was not obtained. bAphid species were classified following Remaudière and Remaudière (1997). cThe gene was named dcr-1a in the Acyrthosiphon species, where a dcr-1b sequence was found, and dcr-1 in the rest of species, where a second copy was not found. dSequence from an Aphis gossypii cDNA library (Webb, B.A. and Shelby, K.S., unpublished data). eSequences for which the complete targeted region could not be obtained. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 4 of 13 Figure 1 Maximum likelihood tree inferred from the amino acid concatenated alignment of ago-1 sequences (model JTT+G+F). The same topology was found using maximum parsimony and Bayesian inference, with only slight differences in the relationships inside the ago-1b group. For those species for which two alleles were obtained only one consensus sequence was included, since no correlation could be made between different alleles of the three different regions. An asterisk (*) marks the suggested moment of the duplication of ago-1. Bootstrap values are shown only when higher that 50%. For each copy of the gene, the value of the ratio of non-synonymous to synonymous rates (ω) resulting from the “two-ratio” model is shown (see Results). The value was calculated as the weighted mean among the ω values of the three coding nucleotide regions analyzed. A strong purifying selection characterizes the ago-1a sequences, contrasting with relaxed selection of ago-1b. Figure 1 Maximum likelihood tree inferred from the amino acid concatenated alignment of ago-1 sequences (model JTT+G+F). The same topology was found using maximum parsimony and Bayesian inference, with only slight differences in the relationships inside the ago-1b group. For those species for which two alleles were obtained only one consensus sequence was included, since no correlation could be made between different alleles of the three different regions. An asterisk (*) marks the suggested moment of the duplication of ago-1. Bootstrap values are shown only when higher that 50%. For each copy of the gene, the value of the ratio of non-synonymous to synonymous rates (ω) resulting from the “two-ratio” model is shown (see Results). Phylogenetic distribution of the duplications of the miRNA machinery in aphids For each copy of the gene, the value of the ratio of non-synonymous to synonymous rates (ω) resulting from the “two-ratio” model is shown (see Results), reflecting the difference in evolutionary pressures acting on the different copies, though less marked than for ago-1. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 Page 5 of 13 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 5 of 13 Table 2 Results from ELW and SH tests performed on the set of seven different hypotheses concerning the phylogenetic timing of dcr-1 duplication in aphids Hypothesis Brief description of topology ELW SH δ c p-value 1 Amino acid ML tree (Figure 2) 0.87 0.1665 0.762 2 R. padi dcr-1 basal 0.87 0.1673 0.761 3 A. gossypii dcr-1 basal Best 0.3947 1.000 4 Aphidini dcr-1 in dcr-1/1a group 1.73 0.1122 0.546 5 Aphidini dcr-1 in dcr-1b group 1.73 0.1123 0.546 6 M. persicae dcr-1 basal to Macrosiphini 10.03 0.0292* 0.111 7 M. persicae dcr-1 in dcr-1b group 10.36 0.0177* 0.091 The complete set of topologies can be found in Additional file 1: Figure S3. δ: difference in -lnL from best topology as calculated by TREE-PUZZLE. c: confidence value (expected likelihood weigth). A * denotes topologies significantly out of the confidence set in the ELW test and topologies significantly worse than the ML tree in the SH test. d SH tests performed on the set of seven different hypotheses concerning the duplication in aphids es can be found in Additional file 1: Figure S3. δ: difference in -lnL from best topology as calculated by TREE-PUZZLE. c: confidence igth). A * denotes topologies significantly out of the confidence set in the ELW test and topologies significantly worse than the ML The complete set of topologies can be found in Additional file 1: Figure S3. δ: difference in -lnL from best topology as calculated by TREE-PUZZLE. c: confidence value (expected likelihood weigth). A * denotes topologies significantly out of the confidence set in the ELW test and topologies significantly worse than the ML tree in the SH test. The complete set of topologies can be found in Additional file 1: Figure S3. δ: difference in -lnL from best topology as calculated by TREE-PUZZLE. c: confidence value (expected likelihood weigth). A * denotes topologies significantly out of the confidence set in the ELW test and topologies significantly worse than the ML tree in the SH test. Phylogenetic distribution of the duplications of the miRNA machinery in aphids involve that one of the copies would have been lost in the Macrosiphini or simply not amplified in the PCR experiments. The hypotheses 4 and 5 (not rejected in the tests) imply a simpler scenario that would imply that the duplication of dcr-1 would have preceded the split between Aphidini and Macrosiphini, and that the sequences obtained for R. padi and A. gossypii would be- long either to the dcr-1a (hypothesis 4) or the dcr-1b (hypothesis 5) copies –but this again implies a loss of or failure to amplify the other copy in several species. Finally, the hypotheses 6 and 7, which involve a change in the branching of the M. persicae dcr-1 sequences, were rejected by the ELW test, which gives strong support to the belonging of these sequences to the dcr-1/1a group. Taken together, these results do not allow making a clear statement about the phylogenetic timing of the duplica- tion of dcr-1 in aphids, although scenarios of a duplica- tion after the split between Aphidini and Macrosiphini are more parsimonious. Further duplication and pseudogenization of dcr-1 in the genus Acyrthosiphon The sequencing of dcr-1a and dcr-1b in the species A. kondoi and A. svalbardicum revealed a third copy of this gene, which was called dcr-1c. Furthermore, a deeper search on the A. pisum LSR1 genome led us to find also a third copy of dcr-1 in this species, although partial and fragmented into two different positions of the currently available scaffolds of the genome (version 2 Assembly). Besides, the region of dcr-1 analyzed in this study was not found in this third copy of A. pisum. The inclusion of these sequences in different phylogenetic trees showed that they correspond most likely to two inde- pendent further duplications of dcr-1b (see Additional file 2: Figures S1 and 3: Figure S2), one for A. pisum and another one for A. kondoi and A. svalbardicum. On the other hand, several features of these sequences, like the presence of frameshifts, stop codons and mutations in Consistent with this pattern, site models and branch- site models in which a class of positively selected codon positions is allowed (M8 and MA with omega estimated) were significantly better than neutral models (M7 and MA with omega fixed to 1) for Regions 1 and 2 of ago-1, but not for Region 3 of ago-1 nor for dcr-1. Bayesian em- pirical Bayes estimates in model M8 showed 4 and 5 codon positions with a probability P>0.95 of ω being higher than 1 for Regions 1 and 2 respectively. The esti- mates from the branch-site model MA showed 52 and 74 codon positions with P>0.95 of ω>1 for the same regions. For both models the positively selected sites detected for Regions 1 and 2 did not aggregate in any particular domain. Figure 3 Gene structure of ago-1 and dcr-1 in Acyrthosiphon pisum. For each gene, both copies have a similar exon/intron structure. Apiago-1a and Apiago-1b have 18 exons, most of them packed in three different regions. The gene encodes a protein with an N-terminal domain of unknown function (DUF1785), a Piwi/Argonaute/Zwille domain (PAZ) and a C-terminal PIWI domain. Apidcr-1a and Apidcr-1b have 20 exons and encode a protein with an N-terminal DexD/H-box helicase domain, a dsRNA binding domain, a PAZ domain and two tandem C-terminal RNase III domains (RNase IIIa and IIIb). The RNase IIIa domain is truncated in Apidcr-1b by a deletion of 47 amino acids. The regions analyzed in this study are marked under the genes. Gene conversion GENECONV was used to search fragments in the alignments of dcr-1 and ago-1 that were unusually similar between pairs of sequences, a possible result from gene conversion. Some pairwise fragments were detected for two regions of ago-1 and for dcr-1 but always concerned two sequences of the same gene duplicate (two -1a copies or two -1b copies). On the contrary, no global fragments were significant for any of the three regions of ago-1 or for dcr-1, thus showing no evidence for gene conversion occurring between the -1a and -1b copies of each of these genes. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 6 of 13 regions and genes. Regions 1 and 2 of ago-1 showed the minimal value of ω=0.0001 for ago-1a copies, due to lack of non-synonymous substitutions, so reflecting strong purifying selection acting on this copy. On the contrary, we found values of ω=0.8621 for Region 1 and ω=0.6236 for Region 2 for the ago-1b copies, suggesting highly relaxed selection. These two regions analyzed for ago-1 encode an N-terminal domain of unknown function (DUF1785), a PAZ domain and the N-terminal portion of the PIWI domain of the protein (see Figure 3 for more details). The C-terminal portion of the PIWI domain is encoded in Region 3, where a similar pattern was found, but with a low value of ω=0.0039 for ago-1a, and a not so relaxed ω=0.2476 for the ago-1b copies. The difference be- tween the slow- and fast-evolving copies of dcr-1 was less pronounced, with ω=0.1247 for dcr-1/1a and ω=0.3577 for dcr-1b. Analyses of evolutionary pressures on sequences y y p q The best fit branch model for each region of ago-1 and for dcr-1 was the two-ratio model, with one ratio of non-synonymous to synonymous rates (ω=dN/dS) for the fast evolving copies (−1b copies) and a different ratio for the slow evolving copies (ago-1a or dcr-1/1a) copies (Table 3). This result is in agreement with the difference in branch lengths observed in the phylogenetic recon- structions (see above and Figures 1 and 2). However, se- lective pressures on each of the copies varied among Table 3 Summary of the analyses of selective pressures on ago-1 and dcr-1 with PAML Branch models Site models Branch-site models ago-1 Region 1 (726 nt) one-ratio:two-ratio P<0.0001 one-ratio:free-ratio P<0.0001 two-ratio:free-ratio P=0.6706 M7:M8 P<0.0001 MAof:MAoe P=0.0285 ωago1a=0.0001 ωago1b=0.8621 4 sites with P>0.95 52 sites with P>0.95 ago-1 Region 2 (909 nt) one-ratio:two-ratio P<0.0001 one-ratio:free-ratio P<0.0001 two-ratio:free-ratio P=0.0506 M7:M8 P<0.0001 MAof:MAoe P=0.0218 ωago1a =0.0001 ωago1b=0.6236 5 sites with P>0.95 74 sites with P>0.95 ago-1 Region 3 (276 nt) one-ratio:two-ratio P<0.0001 one-ratio:free-ratio P<0.0001 two-ratio:free-ratio P=0.4675 M7:M8 P=0.9802 MAof:MAoe P=1 ωago1a=0.0039 ωago1b=0.2476 No positively selected sites No positively selected sites dcr-1 (957 nt) one-ratio:two-ratio P=0.0002 one-ratio:free-ratio P=0.0515 two-ratio:free-ratio P=0.6424 M7:M8 P=0.4677 MAof:MAoe P=1 ωdcr-1/1a =0.1247 ωdcr-1b=0.3577 No positively selected sites No positively selected sites For branch models, the best overall model is highlighted (two-ratio model for all the alignments) with its corresponding ω values. For site and branch-site models, the best model is highlighted as well as the number of positively selected sites when relevant. MAof: model MA with omega fixed to 1; MAoe: model MA with omega estimated. See text for further explanations about the models. Table 3 Summary of the analyses of selective pressures on ago-1 and dcr-1 with PAML Branch models Site models For branch models, the best overall model is highlighted (two-ratio model for all the alignments) with its corresponding ω values. For site and branch-site models, the best model is highlighted as well as the number of positively selected sites when relevant. MAof: model MA with omega fixed to 1; MAoe: model MA with omega estimated. See text for further explanations about the models. Further duplication and pseudogenization of dcr-1 in the genus Acyrthosiphon Functional domains of the proteins are shown with approximate indication of the corresponding span in nucleotide sequence. Figure 3 Gene structure of ago-1 and dcr-1 in Acyrthosiphon pisum. For each gene, both copies have a similar exon/intron structure. Apiago-1a and Apiago-1b have 18 exons, most of them packed in three different regions. The gene encodes a protein with an N-terminal domain of unknown function (DUF1785), a Piwi/Argonaute/Zwille domain (PAZ) and a C-terminal PIWI domain. Apidcr-1a and Apidcr-1b have 20 exons and encode a protein with an N-terminal DexD/H-box helicase domain, a dsRNA binding domain, a PAZ domain and two tandem C-terminal RNase III domains (RNase IIIa and IIIb). The RNase IIIa domain is truncated in Apidcr-1b by a deletion of 47 amino acids. The regions analyzed in this study are marked under the genes. Functional domains of the proteins are shown with approximate indication of the corresponding span in nucleotide sequence. Figure 3 Gene structure of ago-1 and dcr-1 in Acyrthosiphon pisum. For each gene, both copies have a similar exon/intron structure. Apiago-1a and Apiago-1b have 18 exons, most of them packed in three different regions. The gene encodes a protein with an N-terminal domain of unknown function (DUF1785), a Piwi/Argonaute/Zwille domain (PAZ) and a C-terminal PIWI domain. Apidcr-1a and Apidcr-1b have 20 exons and encode a protein with an N-terminal DexD/H-box helicase domain, a dsRNA binding domain, a PAZ domain and two tandem C-terminal RNase III domains (RNase IIIa and IIIb). The RNase IIIa domain is truncated in Apidcr-1b by a deletion of 47 amino acids. The regions analyzed in this study are marked under the genes. Functional domains of the proteins are shown with approximate indication of the corresponding span in nucleotide sequence. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 7 of 13 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 splice sites, strongly suggest that they have undergone a process of pseudogenization. 15 in A. pisum would yield a separation of 19 aa between the active/metal binding sites and the polypeptide binding sites of these set. In A. gossypii and R. padi, this retention would entail the loss of these polypeptide binding sites and of the whole RNase IIIb domain of DCR-1 (see Figure 4). Alternative transcription of dcr-1 in aphids The cloning of cDNA sequences from A. pisum, A. gos- sypii and R. padi revealed the existence of alternative transcription in dcr-1/1a (Figure 4). Two alternative transcripts were found for each of these three species, differing by a pattern of intron retention: in each species, intron 15 was retained in one of the transcripts but spliced in the other. In A. pisum, this intron is 57 nucleotides long, thus yielding a 19 amino acids longer protein, but in A. gossypii and R. padi this intron is 59 and 56 nucleotides long respectively, thus a not multiple of three. In these two latter species, intron retention introduces a frameshift and the appearance of premature stop codons. Intron 15 of Apidcr-1 is located in the region of the gene that encodes the second set of functional sites of the RNase IIIa domain of the pro- tein. The translation of the transcript retaining intron Expression profiles in the reproductive morphs Expression profiles in the reproductive morphs Aphids typically alternate between several generations of parthenogenetic reproduction and one annual gener- ation of sexual reproduction taking place at the end of summer in response to shortening of photoperiod [20]. Transcripts levels of dcr-1a, dcr-1b, ago-1a and ago-1b genes were measured by semi-quantitative RT-PCR in different reproductive morphs of the life cycle of the pea aphid Acyrthosiphon pisum: adult parthenogenetic virgi- noparae females (that contain parthenogenetic embryos in their ovaries), adult parthenogenetic sexuparae females (that contain sexual embryos in their ovaries), sexual oviparae females (that contain eggs in their Figure 4 Alternative transcription in dcr-1/1a in A.pisum, A. gossypii and R.padi. a) Representation of the C-terminal region of DCR-1, the 3’ region of dcr-1 and the transcripts sequenced for the three species. For each of them, two alternative transcripts were found for dcr-1/1a, one retaining intron 15 and another one splicing it. Only one transcript was found for A. pisum dcr-1b, with intron 15 spliced. Apidcr-1b lacks a portion of exon 15 as compared to Apidcr-1a which corresponds to a deletion of 47 aa in the middle of the RNase IIIa domain. b) Fragment of the alignment of the translated amino acid sequence of the transcripts, showing the region around intron 15. The retention of intron 15 results in an insertion of 19 aa in A. pisum DCR-1a. In A. gossypii and R.padi this retention results in premature stop codons (marked by an *) and the loss of the entire RNase IIIb domain of DCR-1. Figure 4 Alternative transcription in dcr-1/1a in A.pisum, A. gossypii and R.padi. a) Representation of the C-terminal region of DCR-1, the 3’ region of dcr-1 and the transcripts sequenced for the three species. For each of them, two alternative transcripts were found for dcr-1/1a, one retaining intron 15 and another one splicing it. Only one transcript was found for A. pisum dcr-1b, with intron 15 spliced. Apidcr-1b lacks a portion of exon 15 as compared to Apidcr-1a which corresponds to a deletion of 47 aa in the middle of the RNase IIIa domain. b) Fragment of the alignment of the translated amino acid sequence of the transcripts, showing the region around intron 15. The retention of intron 15 results in an insertion of 19 aa in A. pisum DCR-1a. In A. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 8 of 13 in this study to determine the age, evolutionary rates, and expression-specificities of the gene duplicates of dicer-1 and argonaute-1 described for A. pisum. The preliminary characterization of the expansion of the miRNA machinery in the pea aphid pointed out a strong divergence of ago-1b in A. pisum, a feature that suggested a change in function for AGO-1b protein in aphids [17]. ovaries), and males. The two ago-1 gene copies and dcr- 1a were expressed in all morphs (Figure 5). For dcr-1a, expression levels showed no statistically significant dif- ferences among morphs. By contrast, ago-1a, ago-1b and dcr-1b showed significant differences of expression according to the reproductive morph: ago-1a, was over- expressed in sexuparae and under-expressed in oviparae, and ago-1b was under-expressed in virginoparae while dcr-1b was over-expressed in sexuparae and not detected in males. These results suggest that the duplication of ago-1 and dcr-1 in the pea aphid is associated with specialization of transcription according to the repro- ductive morph. The sequencing of ago-1a and ago-1b in different aphid species in the present study has revealed the pres- ence of both copies of this gene in all the species ana- lyzed, including the two tribes of the aphid subfamily Aphidinae: Aphidini and Macrosiphini. This result indi- cates that ago-1 was duplicated in an ancestor of this subfamily (Figure 1). By contrast, our analysis provided a different estimation of the timing of duplication of dcr-1. We found a dcr-1b copy only in the three species of the genus Acyrthosiphon, which suggests a more recent du- plication event. However due to the moderate bootstrap support values, we cannot presently propose a solid con- clusion on the timing of this duplication. However, sce- narios of a more recent duplication than found for ago-1 (scenarios in which the duplication of dcr-1 would only concern Macrosiphini) seem more parsimonious, as they imply less events of gene loss (or failure to amplify one copy in several species). Future genome sequencing pro- jects for different aphids species will give the opportun- ity of estimating whether the duplication of dcr-1 may have occurred earlier during aphid evolution. Discussion The sequencing of the genome of the pea aphid Acyrtho- siphon pisum indicated an unusual expansion of the small non coding RNA machinery specific to miRNAs [15,17]. The discovery of these gene duplications opens new perspectives of research about the regulation of gene expression by miRNAs in aphids, which might play a crucial role in the striking polyphenism displayed by these insects. Several scenarios may occur after gene duplications: non-functionalization, subfunctionalization and, more rarely, neofunctionalization [2]. Given that the miRNA machinery appears to be a fundamental mechanism in metazoa, with genes very conserved and as unique copies in most animal genomes known to date, it was particularly interesting to evaluate the differ- ent fates of these duplications. More precisely, we tried Aphid genomes seem to have been affected by recur- rent gene duplications through their evolution, with the Figure 5 Expression profiles of Apiago-1a, Apiago-1b, Apidcr-1a and Apidcr-1b in the four reproductive morphs of A. pisum LSR1. Expression levels of the four genes were estimated by semi-quantitative RT-PCR in the four reproductive morphs: parthenogenetic virginoparae females (Vp), parthenogenetic sexuparae females (Sxp), sexual oviparae females (O) and sexual males (M). Expression was normalized with the gene encoding the ribosomal protein 7 (Apirpl7) as a reference gene [45]. The ratio between the expression of the amplified gene and of the Apirpl7 reference gene was calculated to normalize for variations in experimental conditions, with three replicates. For each gene and morph, gene expression was measured from three batches of 10 adult aphids resulting from three independent biological experiments. The histograms show the averages of normalized expression levels, while letters indicate statistically significant differences among morphs (Duncan grouping in ANOVA). of Apiago-1a, Apiago-1b, Apidcr-1a and Apidcr-1b in the four reproductive morphs of A. pisum LSR1. Figure 5 Expression profiles of Apiago-1a, Apiago-1b, Apidcr-1a and Apidcr-1b in the four reproductive morphs of A. pisum LSR1. Expression levels of the four genes were estimated by semi-quantitative RT-PCR in the four reproductive morphs: parthenogenetic virginoparae females (Vp), parthenogenetic sexuparae females (Sxp), sexual oviparae females (O) and sexual males (M). Expression was normalized with the gene encoding the ribosomal protein 7 (Apirpl7) as a reference gene [45]. The ratio between the expression of the amplified gene and of the Apirpl7 reference gene was calculated to normalize for variations in experimental conditions, with three replicates. Expression profiles in the reproductive morphs gossypii and R.padi this retention results in premature stop codons (marked by an *) and the loss of the entire RNase IIIb domain of DCR-1. Figure 4 Alternative transcription in dcr-1/1a in A.pisum, A. gossypii and R.padi. a) Representation of the C-terminal region of DCR-1, the 3’ region of dcr-1 and the transcripts sequenced for the three species. For each of them, two alternative transcripts were found for dcr-1/1a, one retaining intron 15 and another one splicing it. Only one transcript was found for A. pisum dcr-1b, with intron 15 spliced. Apidcr-1b lacks a portion of exon 15 as compared to Apidcr-1a which corresponds to a deletion of 47 aa in the middle of the RNase IIIa domain. b) Fragment of the alignment of the translated amino acid sequence of the transcripts, showing the region around intron 15. The retention of intron 15 results in an insertion of 19 aa in A. pisum DCR-1a. In A. gossypii and R.padi this retention results in premature stop codons (marked by an *) and the loss of the entire RNase IIIb domain of DCR-1. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 9 of 13 Acyrthosiphon spp and the unique dcr-1 copy in species where the duplication was not found, (ω=0.1247), although the sequence was less conserved than the slow-evolving ago-1a. By contrast, the selective pressures on dcr-1b appeared to be more relaxed (ω=0.3577) and similar to the value found for Region 3 in ago-1b. As for Region 3 of ago-1b, no sign of positive selection was detected in dcr-1b. pea aphid genome showing the highest number of gene families among the insect genomes sequenced to date [15,21]. In a previous study, we found similar pairwise synonymous distances between duplicates of genes of the miRNA machinery in the pea aphid, which suggested that the duplications of ago-1 and dcr-1 had occurred roughly simultaneously [17]. In contrast, in this study, with more phylogenetic evidence, we found that the du- plication of ago-1 probably preceded that of dcr-1. In- deed, we have also found that dcr-1 has been further duplicated two independent times after the dcr-1a/b duplication in the genus Acyrthosiphon, although these new copies have most likely become pseudogenes. We finally evaluated differences of expression of the duplicated copies among different reproductive morphs characteristic of the aphid life-cycle, which alternates sexual and asexual reproduction. The display of such polyphenism must involve a finely tuned mechanism of regulation of gene expression from their gene families- rich genomes, in which miRNAs could play a key role, along with other phenomenon like alternative transcrip- tion. In this report, we have obtained a first insight on the functional characterization of the gene duplicates of ago-1 and dcr-1 by means of semiquantitative PCRs car- ried out on four morphs of the life cycle of the pea aphid. Our results have shown a differential regulation of the expression of ago-1a, ago-1b and dcr-1b among the morphs. The most striking pattern is that of dcr-1b, which seems to be only expressed in the sexupara, the parthenogenetic female that gives birth to the sexual morphs. Although no signature of positive selection could be detected for dcr-1b, the structure of its se- quence gives an interesting clue supporting a possible change in function. The three species of Acyrthosiphon where dcr-1b was sequenced share a deletion of 47 amino acids in the protein sequence with respect to dcr- 1a, which is located inside the first RNase III domain. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 This deletion does not affect any of the active sites, but has probably changed the distance between them inside the RNAse IIIa domain, as well as the distance between the RNAse IIIa and RNase IIIb domains. Interestingly, the two RNase III domains in Drosha and Dicer seem to interact with each other to make an intramolecular dimer with the two catalytic sites located close to each other, and the distance between them seems to deter- mine the length of the small RNA produced after cleav- age [14,23]. It is tempting to hypothesize that the deletion in DCR-1b, which has been evolutionarily con- served in the genus Acyrthosiphon, may have determined a change in the distance between catalytic centers and consequently in its function, leading to the production of small non coding RNAs of different length. All active sites in the two RNase III domains of DCR-1 have remained conserved in the two copies of the protein found in aphids and also in the three outgroup species except for two changes. By contrast, the region of the RNase IIIa domain that contains the deletion in DCR-1b is highly variable and shows other deletions in the out- group species, which could affect the regulation of gene The analysis of the patterns of non-synonymous and synonymous substitutions in the duplicates of ago-1 and dcr-1 in aphids has also provided, for each of the genes, a similar picture of a slow evolving copy subject to puri- fying selection (ago-1a and dcr-1/1a) and a fast evolving copy characterized by relaxed selection (ago-1b and dcr-1b). The difference among copies was much more pronounced for ago-1 than for dcr-1. The evolution of ago-1a has clearly been driven by strong purifying se- lection, as detected by branch models in PAML, with extremely low values of ratios of non-synonymous to synonymous substitution rates (dN/dS or ω) and near- absence of replacements for the aphids of the subfam- ily Aphidinae. Such strong purifying selection implies sequence stability for this protein over a period of ap- proximately 50–70 millions of years, the estimated age of the split between Aphidini and Macrosiphini [22]. Contrastingly, the ago-1b copies have been character- ized by a strong acceleration of evolutionary rates, with different intensity in the three regions analyzed (Region 1: ω=0.8621; Region 2: ω=0.6236; Region 3: ω=0.2476). Discussion For each gene and morph, gene expression was measured from three batches of 10 adult aphids resulting from three independent biological experiments. The histograms show the averages of normalized expression levels, while letters indicate statistically significant differences among morphs (Duncan grouping in ANOVA). Figure 5 Expression profiles of Apiago-1a, Apiago-1b, Apidcr-1a and Apidcr-1b in the four reproductive morphs of A. pisum LSR1. Expression levels of the four genes were estimated by semi-quantitative RT-PCR in the four reproductive morphs: parthenogenetic virginoparae females (Vp), parthenogenetic sexuparae females (Sxp), sexual oviparae females (O) and sexual males (M). Expression was normalized with the gene encoding the ribosomal protein 7 (Apirpl7) as a reference gene [45]. The ratio between the expression of the amplified gene and of the Apirpl7 reference gene was calculated to normalize for variations in experimental conditions, with three replicates. For each gene and morph, gene expression was measured from three batches of 10 adult aphids resulting from three independent biological experiments. The histograms show the averages of normalized expression levels, while letters indicate statistically significant differences among morphs (Duncan grouping in ANOVA). Aphid species analyzed The aphid species used in this study (Table 1) belong to the subfamily Aphidinae (Hemiptera: Aphididae) and were chosen to cover a gradient of evolutionary distance from the pea aphid A. pisum. These include four species from the Macrosiphini tribe (including two other species of the genus Acyrthosiphon) and two species from the tribe Aphidini [24]. Sequences for outgroup species were retrieved from public databases, including two other Hemiptera species (Bemisia tabaci and Rhodnius pro- lixus) and a representative of the order Phthiraptera (Pediculus humanus). Future experiments will allow to study whether the gene duplications of the miRNA machinery play a primary role in the sexual/asexual polyphenism in aphids. It is import- ant to note that cyclical parthenogenesis is an ancient and ubiquitous trait in these insects, and that most of its sub- families display this same reproductive polyphenism with only one copy of DCR-1. This means that the duplication of the miRNA machinery genes was not essential for set- tling the sexual/asexual alternation of reproductive modes that define the aphid life-cycle. But the different copies might have evolved differently and specific roles at differ- ent steps of the life-cycle. The research on the implications of the miRNA machinery in the aphid polyphenism will benefit not only from comparative genomics analysis that will allow knowing precisely the phylogenetic distribution of its gene duplications but also comparative functional studies between aphids having and not having the gene duplicates. The strong purifying selection acting on ago-1a and dcr-1a suggests that these proteins probably have retained their function in the miRNA machinery. On the other side, both relaxed and positive selection acting on DCR-1b and AGO-1b might have led these copies to evolve a new function. Further studies will be needed to explore their potential new role in the miRNA system and at the different steps of the reproductive cycle of these organisms. PCR amplification of ago-1 and dcr-1 gene duplicates For ago-1, three different regions were chosen for the analyses, covering overall ~72% of the coding sequence. These regions were chosen to maximize the total coding length of the sequences obtained. Each region comprised a group of exons separated by short introns in both cop- ies of ago-1 in A. pisum (Figure 3), and we expected this pattern to be conserved in the different species. Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Branch models for the analysis of selective pressures estimate a dN/dS value that is averaged over the entire sequence analyzed. However, the gene sequence may contain both positions with very low or no variability due to purifying selection and positions in which change is promoted by positive selection. Site-models allow the detection of positive selection acting on specific positions of the sequence that might be unseen by branch models. Indeed, site and branch-site models led us to detect the signature of positive selection acting on Regions 1 and 2 of ago-1b. Branch-site models in particular showed that positive selection has played a role in the evolution of the ago-1b copies, with many codon positions with dN/dS ratios significantly higher than 1 (54 and 74 in Regions 1 and 2 respectively, which represent 22% and 24% of the codons of each region). The difference of rates between the two copies of dcr 1 The difference of rates between the two copies of dcr-1 was less pronounced than for the two copies of ago-1. The branch model of best fit estimated that purifying selection has characterized the dcr-1a copy of the Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 10 of 13 Page 10 of 13 expression by small non coding RNAs. Consistent with this hypothesis, the alternative transcription described for dcr-1a in A. pisum would yield two translated DCR- 1a proteins differing by the absence or presence of 19 aa located inside the second set of functional sites of the first RNase III domain of the protein, which might also alter the length between the catalytic centers and allow the production of small non coding RNAs of different length. The same alternative transcription observed in A. gossypii and R. padi entails the loss of the RNase IIIb domain in one of the alternative transcripts, which probably leads to an non-functional protein, given that the interaction of both RNase III domains seem to be needed for cleavage. evolutionary rates in ago-1b and dcr-1b and the existence of positive selection acting on ago-1b suggests the possi- bility of a neofunctionalization for these copies which might have interesting implications on the regulation of gene expression by miRNAs in aphids. Aphid species analyzed The regions were named Region 1 (exons 3 to 6), Region 2 (exons 8 to 14) and Region 3 (exons 15 to 17) and included several predicted functional domains of the protein. For dcr-1, a single sequence spanning exons 15 and 16 was analyzed. Genomic DNA was extracted from a single individual or from several individuals of a same clone following a salting-out protocol [25]. In order to obtain transcript data to support predicted exon/intron boundaries, and also to amplify some regions not obtained from genomic DNA, RNA was extracted from whole body samples of A. pisum LSR1, A. gossypii and R. padi, with subsequent RT protocol carried out with Superscript III (Invitrogen). Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Besides, semi- quantitative PCRs have revealed a differential expression of ago-1b and dcr-1b among the different morphs of the parthenogenetic and sexual phases of the pea aphid life cycle, thus suggesting their implication in the striking polyphenism displayed by this group of insects. Conclusions The sequencing of the two gene duplicates of argonaute- 1 and dicer-1 in several aphid species has allowed us to better estimate the phylogenetic timing and the evolu- tionary pressures that have affected the duplication of the miRNA machinery in these insects. This study has revealed that the duplication of argonaute-1 occurred in the ancestor of the aphid subfamily Aphidinae, while the duplication of dicer-1 seems to have occurred in the ancestor of the aphid tribe Macrosiphini. A common pattern for both genes has been shown with one fast- evolving copy (ago-1b and dcr-1b) and one slow-evolving copy (ago-1a and dcr-1/1a). The acceleration of Two alternative PCR approaches were carried out for the amplification of ago-1 and dcr-1 sequences: one using degenerate primers intended to amplify simultan- eously both copies of the genes in a same reaction and another one using specific primers of copy -1a or -1b. PCR primers were based on the available sequence of A. pisum (primer sequences in Additional file 4: Table S1). In some instances, the initial primers did not allow the Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 11 of 13 the oldest nodes in the maximum likelihood tree obtained from the amino acid alignment of dcr-1. amplification of the targeted region so a smaller fragment was obtained by designing internal primers. A typical PCR reaction consisted in 94°C 3’, 10x(94°C 30”, 60°C de- creasing to 50°C 60”, 72°C 60-120”), 30x(94°C 30”, 50°C 60”, 72°C 60-120”), 72°C 7’, 4°C hold, and was carried out with GoTaq system (Promega). Each amplified fragment was cloned using the Strataclone Package (Stratagene) following manufacturer’s instructions. For each sequence to be used in further analyses three or more clones were sequenced in order to detect Taq errors, deducing the final sequence as the consensus of the clones. Analyses of evolutionary pressures on sequences The nature and distribution of the selective pressures acting on the gene duplicates of ago-1 and dcr-1 was evaluated using PAML 4.4 [38] on nucleotide coding regions. The analyses were carried out separately for each region of ago-1 and for dcr-1, and outgroup sequences were excluded. Branch models were imple- mented to test the hypothesis of different ratios of non- synonymous to synonymous substitution rates (ω=dN/ dS) acting on the -1a and -1b copies of these genes. Phylogenetic analyses Ch Chromatograms were analyzed and assembled using the Staden Package 2.0b [26]. Nucleotide sequences were aligned with Clustal X 2.0.9 [27] and corresponding amino acid alignments were obtained with MEGA 4.0.2 [28]. g Phylogenetic trees were inferred from nucleotide and amino acid alignments by maximum likelihood, max- imum parsimony and Bayesian inference. The analyses of ago-1 were done separately for each of the three regions as well as for concatenated alignments. The phylogenetic analyses on nucleotide sequences were only carried out on coding regions. The model of se- quence evolution for maximum likelihood analyses was chosen using jModeltest [29] for nucleotides and Prot- Test [30,31] for amino acids. Maximum likelihood reconstructions were obtained with PhyML [32] using the NNi algorithm. PAUP* 4.0b10 [33] was chosen for maximum parsimony analyses with TBR branch swap- ping and 5000 repetitions of random sequence addition. Statistical support to nodes was evaluated for maximum likelihood and maximum parsimony by the bootstrap method [34] with 200 and 1000 pseudorreplicates re- spectively. The Bayesian inference of phylogeny was car- ried out as implemented in MrBayes 3.1 [35,36]. Two parallel runs, each one consisting of three cold and one heated chains were set. 106 and 5 × 105 generations for nucleotides and amino acids respectively were enough for reaching convergence between the runs, which was checked using Tracer v1.5 [37]. A burn-in fraction of the initial 25% generations was eliminated and posterior prob- abilities of trees were obtained by sampling every 100th gen- eration afterwards. Conclusions Likelihood ratio tests (LRTs) were used to compare among: i) a “one-ratio” model that assumed no differ- ence in the ratio across the phylogeny, ii) a “two-ratio” model that fitted one ratio for the fast evolving copies (−1b copies) and a different ratio for the slow evolving copies, and iii) a “free-ratio” model, that assumed the ex- istence of a different ratio for each branch of the tree [39,40]. In the “two-ratio” model for ago-1, the ago-1b sequences were set as fast evolving copies and the ago- 1a sequences as slow evolving copies. For dcr-1, the dcr- 1b sequences were set as fast evolving copies and the rest of aphid dcr-1 sequences as slow evolving copies (including the dcr-1a copies of Acyrthosiphon). Site- models were also used for the search of positively selected codon positions in the alignments. Two models were compared by an LRT test: model M7, assuming no positively selected sites in the alignment, and model M8, which allows for their existence [40,41]. The search for positively selected sites with these site models was made only for the -1b copies of the alignments. Finally, branch-site models were also implemented, comparing by an LRT the null model MA with ω fixed to 1 and the alternative model MA with ω estimated [42,43]. For branch-site models, both copies were included, labeling the clade of -1b copies as foreground branches, where positively selected sites are analyzed. All models were implemented allowing the four nucleotide frequencies to vary among codon positions (model F3X4), which gave significantly better likelihood values than not allowing variation (model F1X4). Comparison of alternative hypotheses for dcr-1 duplication The existence of gene conversion, particularly between the -1a and -1b copies of each of the genes, was evalu- ated by GENECONV [44]. The alignments used included all aphid sequences, with only one allele per species and no partial sequences. Each region of ago-1 was analyzed separately. The program was run allowing the existence of mismatches in the fragments susceptible of having experienced gene conversion. SH tests [1] and ELW tests [2] were implemented in TREE-PUZZLE to compare among alternative scenarios concerning the phylogenetic timing of the duplication of dcr-1 in aphids. Seven different hypotheses were simul- taneously tested in each test. The seven hypotheses were constructed by permutation of the groups branched to Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Page 12 of 13 Ortiz-Rivas et al. BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 4. Ghildiyal M, Zamore PD: Small silencing RNAs: an expanding universe. Nat Rev Genet 2009, 10(2):94–108. 5. Ketting RF: The many faces of RNAi. Dev Cell 2011, 20(2):148–161. 6. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ: Processing of primary microRNAs by the Microprocessor complex. Nature 2004, 432(7014):231–235. 7. Hutvagner G, Simard MJ: Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol 2008, 9(1):22–32. 8. Liu N, Okamura K, Tyler DM, Phillips MD, Chung WJ, Lai EC: The evolution and functional diversification of animal microRNA genes. Cell Res 2008, 18(10):985–996. 8. 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Yigit E, Batista PJ, Bei Y, Pang KM, Chen CC, Tolia NH, Joshua-Tor L, Mitani S, Simard MJ, Mello CC: Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell 2006, 127(4):747–757. 12. Campbell CL, Black WC, Hess AM, Foy BD: Comparative genomics of small RNA regulatory pathway components in vector mosquitoes. BMC Genomics 2008, 9:425. 12. Campbell CL, Black WC, Hess AM, Foy BD: Comparative genomics of small RNA regulatory pathway components in vector mosquitoes. BMC Genomics 2008, 9:425. Acknowledgments The authors wish to thank Dr. Flavie Vanlerberghe, Dr. Owain Edwards, Dr. Maurice Hullé, Joël Bonhomme and Jean-François Le Gallic for kindly providing aphid material. Benjamín Ortiz Rivas was funded by an INRA-SPE postdoctoral contract. This work was funded by INRA-SPE projects and INRA- INRIA AIP. Authors’ contributions BOR, SJP, DT and CR were in charge of the conception and design of the study. BOR gathered the sequence data for the phylogenetic and evolutionary analyses, which were carried out by BOR and CR. ST made the semi-quantitative PCR experiments, which were analyzed by SJP and JPG. The writing of the manuscript was done by BOR, SJP and CR, with revision by DT. All authors read and approved the final manuscript. Received: 11 June 2012 Accepted: 4 November 2012 Published: 12 November 2012 Received: 11 June 2012 Accepted: 4 November 2012 Published: 12 November 2012 Gene expression profiling in different reproductive morphs Additional file 5: Table S2. PCR primers used for semiquantitative RT- PCRs. p The transcription of dcr-1a, dcr-1b, ago-1a, and ago-1b genes was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in four reproductive morphs (all adult) of Acyrthosiphon pisum clone LSR1: parthenogenetic females (virginoparae), mothers of oviparae (sexuparae), sexual females (oviparae) and males. The gene encoding ribosomal protein 7 (rpl7) was used as a reference gene [45] to normalize dcr-1 and ago-1 expression. For each morph, three experimental replicates were done, consisting each of batches of 10 individuals which were frozen in liquid nitrogen 48 h after adult moult and used for RNA extraction. The concentra- tion and quality of the extracted RNA was estimated with a NanoDrop (Thermo Scientific). First strand cDNAs were produced from 500 ng total RNA using the SuperscriptIII reverse transcriptase (Invitrogen) and random nonamers (Promega) following the supplier’s instructions. DNA con- tamination was removed by treating RNA extraction pro- ducts with RNase-free DNAse (Promega). As a negative control, RT-PCR experiments were performed on total RNA without SuperscriptIII reverse transcriptase. The PCR program consisted of an initial step of 4 minutes (min) at 94°C, then multiple cycles composed of 2 min at 94°C, 30 seconds (sec) at the annealing temperature and 1 min 30 sec at 72°C, and a final elongation step of 5 min at 72°C. The appropriate number of cycles corresponding to the exponential range was defined for every gene in order to quantify the amplification products. Additional file 5: Table S2 lists the sequences of PCR primers, the corre- sponding annealing temperatures and the appropriate number of cycles used to measure expression level of the different dcr-1 and ago-1 genes. Images of the RT-PCR Sybr-safe (Invitrogen) stained agarose gels were acquired with a G:BOX Imager (Syngene) and quantification of the bands was performed using Image-J (http://rsbweb.nih. gov/ij/). For each batch of each reproductive morph, the ratio between the band intensity of the amplified gene and rpl7 reference gene was calculated to normalize for initial variations in sample concentration. The significance of variation of expression level (p-value >0.05) was tested by analysis of variance (ANOVA) with un-transformed data. Competing interests The authors declare that they have no competing interests. Additional file 1: Figure S3. Test of alternative hypotheses for the phylogeny of dcr-1 in aphids. Additional file 2: Figure S1. Further duplication of dcr-1 in Acyrthosiphon kondoi and Acyrthosiphon svalbardicum. Additional file 3: Figure S2. Further duplication of dcr-1 in Acyrthosiphon pisumLSR1. Additional file 4: Table S1. PCR primers used in this study, ordered in relative positions from 5’ to 3’ of the corresponding gene/region. 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BMC Evolutionary Biology 2012, 12:216 http://www.biomedcentral.com/1471-2148/12/216 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 39. 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Analysis of nanopore detector measurements using Machine-Learning methods, with application to single-molecule kinetic analysis

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BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-2105/8/S7/S12 This article is available from: http://www.biomedcentral.com/1471-2105/8/S7/S12 © 2007 Landry and Winters-Hilt; licensee BioMed Central Ltd. © 2007 Landry and Winters-Hilt; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2007 Landry and Winters Hilt; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit Abstract Background: A nanopore detector has a nanometer-scale trans-membrane channel across which a potential difference is established, resulting in an ionic current through the channel in the pA-nA range. A distinctive channel current blockade signal is created as individually "captured" DNA molecules interact with the channel and modulate the channel's ionic current. The nanopore detector is sensitive enough that nearly identical DNA molecules can be classified with very high accuracy using machine learning techniques such as Hidden Markov Models (HMMs) and Support Vector Machines (SVMs). Results: A non-standard implementation of an HMM, emission inversion, is used for improved classification. Additional features are considered for the feature vector employed by the SVM for classification as well: The addition of a single feature representing spike density is shown to notably improve classification results. Another, much larger, feature set expansion was studied (2500 additional features instead of 1), deriving from including all the HMM's transition probabilities. The expanded features can introduce redundant, noisy information (as well as diagnostic information) into the current feature set, and thus degrade classification performance. A hybrid Adaptive Boosting approach was used for feature selection to alleviate this problem. Conclusion: The methods shown here, for more informed feature extraction, improve both classification and provide biologists and chemists with tools for obtaining a better understanding of the kinetic properties of molecules of interest. channel current-based signal analysis platform. Emission inversion and the addition of a spike density feature are shown to noticeably improve performance and are folded channel current-based signal analysis platform. Emission inversion and the addition of a spike density feature are shown to noticeably improve performance and are folded y Matthew Landry1 and Stephen Winters-Hilt*1,2 ddress: 1Department of Computer Science, University of New Orleans, New Orleans, LA, 70148, USA and 2The Rese 00 Henry Clay Ave., New Orleans, LA 70118, USA Address: 1Department of Computer Science, University of New Orleans, New Orleans, LA, 70148, USA and 2The Research Institute for Children, 200 Henry Clay Ave., New Orleans, LA 70118, USA Email: Matthew Landry - [email protected]; Stephen Winters-Hilt* - [email protected] Email: Matthew Landry - [email protected]; Stephen Winters-Hilt* - [email protected] * Corresponding author from Fourth Annual MCBIOS Conference. Computational Frontiers in Biomedicine New Orleans, LA, USA. 1–3 February 2007 BMC Bioinformatics 2007, 8(Suppl 7):S12 doi:10.1186/1471-2105-8-S7-S12 Open A Proceedings Analysis of nanopore detector measurements using Machine-Learning methods, with application to single-molecule kinetic analysis Matthew Landry1 and Stephen Winters-Hilt*1,2 Open Access y Matthew Landry1 and Stephen Winters-Hilt*1,2 Background g Classification results are the ultimate judge of the success of whether a given feature or feature set is useful in the Page 1 of 16 (page number not for citation purposes) Page 1 of 16 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/S7/S12 BMC Bioinformatics 2007, 8(Suppl 7):S12 test of pattern recognition informed (PRI) sampling con- trol. As the nanopore detector generates data, a simplified time-domain Finite State Automaton (τFSA), shown in the figure in Additional File 1, is used for signal acquisi- tion (see [4,11] for full model). The Bottom part of the fig- ure in Additional File 1 describes the FSA that is used to find captures in a channel current data file. Only transi- tions between states are shown. Staying in the current state does not require any updating of the state of the FSA. Transitioning to another state requires only the recording of that sample index if the capture state is entered or exit. Note that only the current reading of the current observa- tion and the current level count are needed to determine the state of the current observation. The current reading is used to determine the level and the current level count is used to ensure an actual level and not noise in the chan- nel. The Top part of the figure shows a sample channel current blockade signal colored to correspond with the FSA. Once the signal is acquired, it is passed on to a generic HMM that is used to characterize current block- ades and extract features [1-3,6]. During this step, the parameters of a generic-HMM are estimated using Expec- tation Maximization (EM) to effectively de-noise the sig- nal [12]. After this stage, the extracted feature vector is passed on to an off-line-trained SVM. The classification result yielded by the SVM is then used to close the sam- pling control loop, i.e., undesirable molecules, or undesir- able orientations of "capture", can promptly be ejected (by potential reversal). Further details on recent results on pattern-recognition-informed sampling control are pre- sented in [3]. In this paper machine learning techniques and results, primarily in feature extraction and feature selection, are presented. into a previously presented architecture [1]. Background It is also shown that Emission Variance Amplification (EVA) greatly reduces computation complexity and makes anal- ysis of levels that are not well defined possible, while over- zealous use of tuning parameters can destroy kinetic infor- mation and thus render a channel current blockade signal useless. A new, efficient HMM-with-Duration is proposed as a solution [2-4]. Finally, although AdaBoost was not able to reproduce the best classification results obtained from a carefully selected feature set, AdaBoost is shown to be useful in several situations, including ab initio feature selection, and post feature selection pruning that offers similar results (not shown) to PCA-based feature selection on the same data (see [5], and references cited there, for a more comprehensive discussion of Adaboosting-based selection). Moreover, AdaBoost serves to validate the cur- rent, manually designed feature set. HMM feature extraction An HMM is used to de-noise and extract features from the acquired channel current signal. The HMM is imple- mented with fifty states. The only parameters necessary for determining a state is the current reading (which is given in picoamps) at a given point in the signal. This current reading is normalized to the baseline – taken to be the average current reading just before the capture event occurred. An average baseline reading of 120pA and a cur- rent reading of 70pA, for example, corresponds to a nor- malized value of 58.33% baseline. Then, using a bin size of one, the value of 58 is used as the current state. (Other bin sizes have been considered, but 1% granularity was sufficient for discerning structure, without burdening the HMM processing with too many states, and is used in what follows.) For most of the data studied in these exper- iments, almost all capture events take place between 20- and 70% blockade. Thus, only fifty states are used in an effort to help ease computational complexity – as input scales linearly, computation time scales quadratically. In the implementation of the HMM, the states are chosen Nanopore detector The nanopore detector generates the data used in later stages of the channel current cheminformatics signal anal- ysis architecture. A lipid bilayer supports the biologically- based channel. The channel used in what follows consists of a protein heptamer formed by protein monomers secreted by Staphylococcus aureus. Alpha-Hemolysin is used as the channel in the nanopore device due to its sta- ble conformation (minimal gating) and its overall geom- etry (see Figure 1). The data consists of current reading through this channel. DNA and RNA interaction with the channel during translocation is non-negligible, but not strong enough for the molecule to get "stuck." Although dsDNA is too large to translocate, about ten base-pairs at one end can still be drawn into the large cis-side vestibule. This permits very sensitive experiments since the ends of "captured" dsDNA molecules can be observed for exten- sive periods of time to resolve features, allowing highly accurate classification of the captured end of dsDNA mol- ecules [1,6-10]. In previous experiments, single molecules such as DNA have been examined in solution with nanometer-scale precision using nanopore blockade detection [1,6-8]. In early studies [8], it was found that complete base-pair dissociations of double stranded DNA to single stranded DNA could be observed for sufficiently short DNA hairpins. In later work [1,6], the nanopore detector was used to read the ends of double stranded DNA molecules and was operated as a chemical biosen- sor. In [6,9,10], the nanopore detector is used to observe the conformational kinetics of the end regions of individ- ual DNA hairpins. Page 2 of 16 (page number not for citation purposes) Cheminformatics overview The prototype channel current cheminformatics signal processing architecture "closes the loop" on the architec- ture previously presented in [1] (see Figure 2). The signal processing architecture is used to perform a preliminary Page 2 of 16 (page number not for citation purposes) Page 2 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 A l d b l h l h h l h h h l d ll h d Left Panel: A lipid bilayer supports the alpha-hemolysin heptamer that creates a pore, or channel used to collect the data, as shown left Figure 1 Left Panel: A lipid bilayer supports the alpha-hemolysin heptamer that creates a pore, or channel used to collect the data, as shown left. The channel is supported by an aperture, which allows the flow of ions between cis (here, left) and trans (here, right) wells. Right Panel: The assembled α-hemolysin pore shown to scale, with a captured dsDNA molecule. As shown, the double stranded form is too wide to pass through the pore, while a single strand may pass through. Bottom Panel: First 100 ms blockade patterns of four DNA hairpins, part of a test set of nine base-pair hairpins, with 4dT hairpin loops, that have been studied extensively, and an eight base-pair control. The nine base-pair molecules only differ in their terminal base-pairs, yet their channel current blockade signals, "signatures", are easily resolved [1]. Left Panel: A lipid bilayer supports the alpha-hemolysin heptamer that creates a pore, or channel used to collect the data, as shown left Figure 1 Left Panel: A lipid bilayer supports the alpha-hemolysin heptamer that creates a pore, or channel used to collect the data, as shown left. The channel is supported by an aperture, which allows the flow of ions between cis (here, left) and trans (here, right) wells. Right Panel: The assembled α-hemolysin pore shown to scale, with a captured dsDNA molecule. As shown, the double stranded form is too wide to pass through the pore, while a single strand may pass through. Bottom Panel: First 100 ms blockade patterns of four DNA hairpins, part of a test set of nine base-pair hairpins, with 4dT hairpin loops, that have been studied extensively, and an eight base-pair control. The nine base-pair molecules only differ in their terminal base-pairs, yet their channel current blockade signals, "signatures", are easily resolved [1]. Page 3 of 16 (page number not for citation purposes) Cheminformatics overview Page 3 of 16 (page number not for citation purposes) Page 3 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 The channel current cheminformatics signal processing architecture Figure 2 The channel current cheminformatics signal processing architecture Figure 2 The channel current cheminformatics signal processing architecture. The channel current cheminformatics signal processing architecture Figure 2 The channel current cheminformatics signal processing architecture. with this observation in mind. In the previous example, our state of 58 would correspond to state 38. This process of scaling raw data to actual states is referred to as "quan- tization". After the Viterbi algorithm is run, a 150-component fea- ture vector is created for the given signal. Each feature vec- tor consists of three distinct sets of information. The first 50 components come directly from the 50 previously described states of the HMM. These components are level occupation probabilities (a histogram view) for each state calculated after the Viterbit trace back algorithm yields the most likely path. The second set of 50 components is com- posed of the variances of the emission probabilities. The third and final set of 50 components is composed of a weighted sum of transition probabilities from the domi- nant levels of a given signal. After the data is quantized, five rounds of Expectation Maximization are run to obtain accurate estimates of emission and transition probabilities. Initially, emissions for each state 'L', corresponding to a blockade at level 'L', is set to a gaussian with mean at L and unit variance. In addition, all transitions are equally likely. Expectation Maximization serves to obtain a more accurate measure of emissions and transitions based on the observed signal. A standard Viterbi algorithm is then run in order to de-noise the signal – that is, obtain the most likely path of states that created the observed signal. The process of finding the most likely path of states obtained by the Viterbi algo- rithm typically reduces the noise in the channel current signal. One refinement to the standard implementation of an HMM, presented here, involves the initial manipulation of the emission probabilities as they are entered in to the HMM. SVM classification bility of emitting a hidden or true state given an actual or observed state. By exchanging the roles of the true and actual states, an additional contribution arises that is approximately a locally distributed entropy that is intro- duced at the cellular level in the standard Viterbi dynamic programming table (see Methods). While the exact theo- retical underpinnings of this method are still being researched, it is clear that this "emission inversion" improves classification performance. bility of emitting a hidden or true state given an actual or observed state. By exchanging the roles of the true and actual states, an additional contribution arises that is approximately a locally distributed entropy that is intro- duced at the cellular level in the standard Viterbi dynamic programming table (see Methods). While the exact theo- retical underpinnings of this method are still being researched, it is clear that this "emission inversion" improves classification performance. Support Vector Machines (SVMs) are variational-calculus based methods that are constrained to have structural risk minimization (SRM) such that they provide noise tolerant solutions for pattern recognition [13,14]. Simply put, an SVM determines a hyperplane that optimally separates one class from another (see Figure 4). Once learned, the hyperplane allows data to be classified according to the region in which it resides. In addition to the 150-component feature vectors and the emission inversion technique already described, addi- tional information can also be extracted. The effects of the addition of a spike density feature are explored, where a spike is defined as an anomalous, deep blockade of chan- nel current from the lower level of a given signal. The SVM approach encapsulates a significant amount of model-fitting information in its choice of kernel. In some sense, the SVM kernel provides a notion of distance to the decision hyperplane. Novel, information-theoretic, ker- nels were successfully employed for notably better per- formance over standard kernels in prior work[1,15]. Thus, SVMs are fast, easily trained, discriminators [13,14], for which strong discrimination is possible without the over-fitting complications common to neural net discrim- inators [13]. In these experiments, SVM classification per- formance is used as the benchmark for testing the validity of the various feature extraction permutations that are explored. This idea is a natural fit since one of the over- arching goals of the nanopore detector is to be able to clas- sify molecules based on their behavior in the channel. Cheminformatics overview The emission probabilities are the main place where the observed data is brought into the HMM-EM algorithm and can be viewed conceptually as the proba- Page 4 of 16 (page number not for citation purposes) Page 4 of 16 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/S7/S12 BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 Results h In what follows, results are described for the proposed extensions and improvements to existing methods in the feature extraction architecture. Improvements in feature extraction and types of features are discussed. Specifically, emission inversion, the addition of a spike density fea- ture, and HMM with EVA are discussed. In addition, a new method of feature selection is shown. The effects of using AdaBoost on a full set of transition probabilities versus a scheme for manually compressing transition probabilities are shown. AdaBoost Adaptive Boosting (AdaBoosting) is typically used for classification purposes. In general, AdaBoost is an iterative process that uses a collection of weak learners to create a strong classifier. Training data is given a weight, and at each iteration, the weak learners are trained on this weighted data. Weights for these data points are then updated based on the error rate of the weak learner and whether a given data point was classified correctly or not. The consensus vote at each iteration is treated as a hypoth- esis, and weights are given to a hypothesis based on its accuracy. At the end of the iterative process, final classifi- cation is done using all hypotheses and their correspond- ing weights (see Figure 3). In this way, AdaBoost is able to use a set of weak learners to generate a strong classifier. SVM classification Furthermore, SVMs provide a natural confidence factor that can be leveraged when closing the sampling control loop. Another variation on a standard HMM, Emission Variance Amplification is discussed. Here, the goal is to obtain dwell time information for the levels of a given molecule. From this information, the half-life, and thus, the stability of a given level can be determined. However, channel cur- rent data is noisy and building a Finite State Automaton to accurately model this noisy data can be difficult. More- over, this model would not be easily re-usable for other channel current analysis without significant restructuring and re-tuning. Here, an HMM with EVA is used to reduce the gaussian noise bands around a given level while still strictly retaining transitions between levels. This method was first introduced in [2] and is used here to obtain the new results. The tradit Figure 3 Th d The traditional AdaBoost algorithm (graphic taken from [5]) Figure 3 The traditional AdaBoost algorithm (graphic taken from [5]). problems considered, three different feature sets were chosen to analyze the effect of data inversion on SVM clas- sification performance. The three sets selected for compar- ison were the manually designed 150-component feature vectors described in Background, the first set of 50 level occupation features from that 150-component set, and the second set of 50 variances on the emission probabili- ties from that 150-component set. The 9AT vs. 9TA, 9CG vs. 9TA, and 9GC vs. 9TA binary classification cases were selected to be shown here as they provide typical examples of the entire result set. increase in accuracy. This result is stable over a range of kernel parameter. For the case where the first 50 compo- nents were studied, a slight increase in classification per- formance as well as an increase in stability is observed. In Fig. 5c, a slight boost in classification performance is observed while a significant increase in stability is observed. In nearly all cases studied, inverting the emissions pro- vides a performance increase in accuracy, stability, or both accuracy and stability. For some molecules, this perform- ance increase was more significant than others and in one case, out of the ten permutations studied, performance was marginally better using a standard HMM without emission inversion (but suffered from being less stable in its kernel parameter, so wouldn't be preferred anyway). Experimentally, this emission inversion works well with channel current data as shown in Figure 5. These figures show SVM classification performance for the various fea- ture sets just described using both a standard HMM imple- mentation and a HMM implemented with data inversion as described here. The y-axis measures classification accu- racy (sensitivity plus specificity) and the x-axis shows a tuning over the kernel parameter. The symmetric entropic kernel was used in this study as it has been shown to work well with channel current data in previous experiments [1]. The performance benefit is shown most notably in Figure 5a. In the case where the 150-component feature set was used, inverting the emissions yields a 5% peak Emission inversion b d d Observed data is brought into the HMM/EM process chiefly through the emission probabilities. Through run- ning the HMM in debug mode and observing the interac- tions of various components, an interesting twist on traditional emission probabilities was found – when the observed states and emitted states share the same alphabet the roles of observed states and emitted states can be reversed for possible improvement to classification per- formance. As a classification method, one of the main disadvantages of AdaBoost is that it is prone to over training. However, AdaBoost is a natural fit for feature selection. Here, over training is not a problem, as AdaBoost finds diagnostic features and those features are passed on to a classifier that does not suffer from over training such as a SVM. For this function, a modified form of AdaBoost is introduced. Data used from these experiments were the 9bphp data shown in Figure 1. For each of the binary classification Page 5 of 16 (page number not for citation purposes) Page 5 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 The hyper classifier fo a thickness mization c Figure 4 yp p p y y g , yp p ( ) g The hyperplane separability heuristic underlying the SVM classifier formulation, where the hyperplane is endowed with a thickness that is maximized (the SVM's structural risk mini- mization criterion). Dwell time analysis using emission variance amplification Another important feature of a channel current blockade signal is the duration of blockade levels. However, acquir- ing level duration information is a non-trivial task due to a significant gaussian noise band around blockade levels. The goal here is to use Emission Variance Amplification in the HMM with EM to drastically reduce noise in the signal while still precisely retaining level transitions. By retaining the level transitions, the integrity of the kinetic informa- tion – level dwell times in this case – remain in tact. sity can yield information about the stability of the final few base pairings. For this analysis, data obtained from collaborators at NASA/AMES was used (see [16] for details). Here, the analysis is centered on two very similar 9GC molecules. On one of the molecules, the terminal guanine base was modified in an effort to simulate radia- tion damage. A blockade level histogram of the two sig- nals (figure in Additional File 2) shows that there is high similarity between the blockades produced by the two molecules. Data used for this analysis was gathered from a simple study of DNA-DNA annealing using the nanopore detec- tor and a Y-aptamer transduction platform. Results on blockade states observed for Y-aptamer overhang+com- plement binding study are shown in the figures in Addi- tional Files 5 and 6. Additional File 5 shows the 150- component feature vector profiles for the Y-aptamer that binds a 6A ssDNA, for signals before and after introduc- tion of that six adenosine ssDNA (from [18]). Additional File 6 shows the dwell time distributions for the three dominant levels of the Y-aptamer (without 6A target). For further details and Results, see the work presented in [18] in this same Journal. The spike detection method presented in [16] was used to identify spikes and extrapolate true spike counts as shown in the figures in Additional files 3 and 4. In those figures the blue curve represents actual spike counts observed ver- sus a given cutoff. The red curve is drawn tangent to the observed curve. Thus, the true spike count is the reading as the tangent line crosses the x-axis. Spike analysis In addition to the level occupation probability, emission probability, and transition probability, the spike density from the lower level of a given molecule has been identi- fied as a possibly significant feature. A spike event – an anomalous, deep blockade of channel current – from the lower level is conceptually seen as a fraying of the last few termini of a given molecule. Thus, a measure of spike den- Page 6 of 16 (page number not for citation purposes) Page 6 of 16 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/S7/S12 BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 The hyperplane separability heuristic underlying the SVM classifier formulation, where the hyperplane is endowed with a thickness that is maximized (the SVM's structural risk mini- mization criterion) Figure 4 The hyperplane separability heuristic underlying the SVM classifier formulation, where the hyperplane is endowed with a thickness that is maximized (the SVM's structural risk mini- mization criterion). ing in the captured DNA hairpin (as shown in [7]). The radiated form of the molecule frayed 17.6 times on aver- age (while in the LL state), and is shown in Additional File 3, while the non-radiated molecule only frayed 3.58 times a second, on average, while in its lower-level state (Addi- tional File 4). Building on the efforts in [16], this spike density feature was used as a single feature and concatenated to the end of the 150-component feature vector (described in Back- ground). The results of this analysis are shown in Figure 6 (similar to the description of the emission inversion results in the previous section). Incorporation of this spike feature for this data set leads to classification with approximately 5% greater accuracy over a wide range of tuning parameters. It is noteworthy that the addition of only one extra feature, the spike density feature, yields a significant performance increase. Page 7 of 16 (page number not for citation purposes) The hyper classifier fo a thickness mization c Figure 4 9AT9TA 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 Sigma SN + SP Inverted - all 150 Inverted - 1st 50 Inverted - 2nd 50 Standard - all 150 Standard - 1st 50 Standard - 2nd 50 9CG9GC 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 Sigma SN + SP Inverted - all 150 Inverted - 1st 50 Inverted - 2nd 50 Standard - all 150 Standard - 1st 50 Standard - 2nd 50 9GC9TA 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 Sigma SN + SP Inverted - all 150 Inverted - 1st 50 Inverted - 2nd 50 Standard - all 150 Standard - 1st 50 Standard - 2nd 50 (c) (b) (a) 9AT9TA 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 Sigma SN + SP Inverted - all 150 Inverted - 1st 50 Inverted - 2nd 50 Standard - all 150 Standard - 1st 50 Standard - 2nd 50 9CG9GC 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 SN + SP Inverted - all 150 Inverted - 1st 50 Inverted - 2nd 50 Standard - all 150 Standard - 1st 50 Standard - 2nd 50 (b) (a) 9AT9TA 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 SN + SP Inverted - all 150 Inverted - 1st 50 Inverted - 2nd 50 Standard - all 150 Standard - 1st 50 Standard - 2nd 50 (a) SN + SP (b) SN + SP Sigma (a) SVM performance with different feature sets, for different binary classification data sets: 9AT vs 9TA Figure 5 (a) SVM performance with different feature sets, for different binary classification data sets: 9AT vs 9TA. The hyper classifier fo a thickness mization c Figure 4 The molecule studied is a 9 base-pair hairpin that is the radiation damaged DNA model (a terminal guanine is oxolated) (see [16] for details), with terminal guanine unaltered in the "non- radiated" molecule. The spike count plots show increasing counts as spike cut-off thresholds are relaxed (to where eventually any downward deflection will be counted as a spike). The linear phases of spike count increase, with threshold relaxation, is associated with instances of anomalous "spike noise" and forms the basis for a heuris- tic for defining the spike feature. Plots are automatically generated using gnuplot and automatically fit with extrap- olations of their linear phases at the group's tools website. The extrapolations provide an estimate of "true" anoma- lous spike counts – counts associated with terminus fray- Visually, the results of EVA can be seen in Figure 7. Note that as the variance is amplified from the original setting of 1, the noise band around a given level is reduced signif- icantly. Moreover, even though many spike events are destroyed, transitions between dominant levels – and thus level dwell times – are strongly retained. After EVA pre-processing, a trivial Finite State Automaton can now extract dwell time information. This FSA only needs a cur- rent reading and a duration (in sample counts) to charac- terize any given level. Without EVA, a wide range of current cutoffs or even some more complex model would Page 7 of 16 (page number not for citation purposes) Page 7 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 SVM performance with different feature sets, for different binary classification data sets: 9AT vs 9TA ure 5 SVM performance with different feature sets, for different binary classification data sets: 9AT vs 9TA. The Y-axis, "SN shows the sum of the Sensitivity and the Specificity. The X-axis is the kernel parameter σ. Note: standard ROC curve used here, and in what follows, for two reasons: (i) a comparison with common sigma parameterization was being ored, and (ii) SN and SP are not evaluated individually, whereas (SN + SP) is the measure employed for overall fitnes on of a given feature set, kernel, and kernel sigma. (b) SVM performance with 9CG vs 9TA. (c) SVM performance w C vs 9TA. Throughout, the SVM shows that the feature set produced using the inverted emissions performs consiste er than the standard implementation of a HMM. Page 8 of 16 (page number not for citation purposes) Example c Figure 6 Example classification results with and without spike analysis Figure 6 Example classification results with and without spike analysis. Note that adding a spike feature significantly improves classifica- tion accuracy over a wide range of kernel parameters. The Y-axis, "Accuracy", is taken to be (SN+SP)/2 expressed as a per- centage, to be consistent with the prior SN+SP based measure, so is not the conventional accuracy = (TP+TN)/ (TP+TN+FP+FN). The X-axis is the kernel parameter σ. be needed to characterize a given level. But, using this sim- plified FSA, dwell time distributions for the studied data were easily obtained (see the figure in Additional File 6). From these dwell time distributions, the half-life – and thus a measure of level stability – can be gathered. This half-life is an important kinetic characteristic for a biolo- gist or chemist studying the properties of a molecule. Future work will evaluate whether half-lives of levels or even entire dwell time distributions can be useful in improving classification performance. desired to solve the issue of feature selection. Here, a hybrid AdaBoost approach is used as an automated, objective means of feature selection. The data studied for feature selection include the 9CG vs 9GC and 9GC vs 9TA binary classification problems from the 9bphp data used in the data inversion analysis (Figure 1). The 9GC vs 9TA set was studied first. Since the 9GC vs 9TA case is one of the easier classification problems with this dataset, the 9CG vs 9GC case was also analyzed. This case is among the hardest binary classification problems in this dataset. The hyper classifier fo a thickness mization c Figure 4 The Y-axis, "SN + SP", shows the sum of the Sensitivity and the Specificity. The X-axis is the kernel parameter σ. Note: standard ROC curves are not used here, and in what follows, for two reasons: (i) a comparison with common sigma parameterization was being explored, and (ii) SN and SP are not evaluated individually, whereas (SN + SP) is the measure employed for overall fitness eval- uation of a given feature set, kernel, and kernel sigma. (b) SVM performance with 9CG vs 9TA. (c) SVM performance with 9GC vs 9TA. Throughout, the SVM shows that the feature set produced using the inverted emissions performs consistently better than the standard implementation of a HMM. Page 8 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 Example classification results with and without spike analysis Figure 6 Example classification results with and without spike analysis. Note that adding a spike feature significantly improves classifica- tion accuracy over a wide range of kernel parameters. The Y-axis, "Accuracy", is taken to be (SN+SP)/2 expressed as a per- centage, to be consistent with the prior SN+SP based measure, so is not the conventional accuracy = (TP+TN)/ (TP+TN+FP+FN). The X-axis is the kernel parameter σ. Example classification results with and without spike analysis Figure 6 Example classification results with and without spike analysis. Note that adding a spike feature significantly improves classifica- tion accuracy over a wide range of kernel parameters. The Y-axis, "Accuracy", is taken to be (SN+SP)/2 expressed as a per- centage, to be consistent with the prior SN+SP based measure, so is not the conventional accuracy = (TP+TN)/ (TP+TN+FP+FN). The X-axis is the kernel parameter σ. Page 9 of 16 (page number not for citation purposes) Spike analysis h l d The results described above clearly show that spike den- sity from the lower level is an important feature. Obtain- ing the spike density feature (described in Methods) is straightforward. However, adding this feature to the exist- ing 150- or 2600-component feature sets currently requires tuning. Simply adding the spike density feature to an existing feature vector already containing 150 features will obscure the effect of the spike density feature almost completely. Thus, a weight must be added to this new fea- ture. Should the weight be too heavy, though, the effect of the other features will be obscured. Currently, the weight- ing factor is tuned over in order to arrive at a weighting Feature selection with AdaBoost As the EVA factor increases, the gaussian noise surrounding the levels is reduced significantly, yet level transitions are strictly retained Figure 7 As the EVA factor increases, the gaussian noise surrounding the levels is reduced significantly, yet level transitions are strictly retained There are currently a couple of caveats. Emission inversion only works when the emitted and observed states share the same alphabet – with the channel current blockade analysis platform this restriction holds. Another caveat is that this method may be strongly data dependent. Only channel current data has been studied using this method for feature extraction, and it is entirely possible that emis- sion inversion does not improve classification on other datasets. In this particular application, the AdaBoost fea- ture selection provides a simple fix to the choice of what features to use. Simply create datasets that include extracted features from both a standard HMM implemen- tation and a HMM implementation with emission inver- sion and let AdaBoost select the most diagnostic features in an automated way. features and passed on to the SVM for classification. In this case, classification outperforms both the full 2600- component set and the manually designed 150-compo- nent set. The curve denoted by "First 50" represents the first 50 blockade level probabilities. This set is the best performing manually designed set, and outperforms the AdaBoost selected feature set in both performance and stability. Figure 10 shows the results of AdaBoosting off of the manually designed 150-component feature set in the case of the 9GC vs 9TA binary classification problem. There is a notable performance increase in classification accuracy and stability. features and passed on to the SVM for classification. In this case, classification outperforms both the full 2600- component set and the manually designed 150-compo- nent set. The curve denoted by "First 50" represents the first 50 blockade level probabilities. This set is the best performing manually designed set, and outperforms the AdaBoost selected feature set in both performance and stability. Figure 10 shows the results of AdaBoosting off of the manually designed 150-component feature set in the case of the 9GC vs 9TA binary classification problem. There is a notable performance increase in classification accuracy and stability. Discussion In what follows, the pros and cons of each proposed method presented in the Background and Results sections are discussed. In addition, proposed fixes and future work is discussed. Feature selection with AdaBoost As has been shown in the spike analysis, careful selection of features plays a significant role in classification per- formance. However, adding non-characteristic or noisy features will hurt classification performance. In addition, recall from the discussion in Background that the last set of 50 components from the baseline 150-component fea- ture vector are compressed transition probabilities. With a 50 state HMM, there would be 50*50 or 2500 possible transitions. However, a means of compression is neces- sary because many of these transitions are very unlikely and contribute noise to the feature vector. Without com- pression, classification performance suffers as a result, yet it is uncertain as to whether diagnostic information has been inadvertently discarded in the manual compression of the transition probabilities. An automated approach is Figures 8, 9, 10 show the results of this automated feature selection analysis (these figures have a similar description to the figures described in the Emission Inversion results section). Figure 9 shows the effects of AdaBoosting off of the full, uncompressed feature vectors. These feature vec- tors are comprised of the 50 blockade level components (same as from the 150-component set), the 50 variances on the emission probabilities (same as from the 150-com- ponent set), and the full 2500 transition probabilities. Using a SVM to classify all 2600 features shows a notable decrease in classification accuracy and a significant decrease in the stability of classification results. AdaBoost is used to select the top 100 diagnostic features. These 100 features are extracted from the full 2600-component set of Page 9 of 16 (page number not for citation purposes) Page 9 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 As the EVA factor increases, the gaussian noise surrounding the levels is reduced significantly, yet level transitions are strictly retained Figure 7 As the EVA factor increases, the gaussian noise surrounding the levels is reduced significantly, yet level transitions are strictly retained. As the EVA factor increases, the gaussian noise surrounding the levels is reduced significantly, yet level transitions are strictly retained Figure 7 As the EVA factor increases, the gaussian noise surrounding the levels is reduced significantly, yet level transitions are strictly retained. Page 10 of 16 (page number not for citation purposes) If Adaboos improvem Figure 8 If Adaboos improvem Figure 8 If Adaboost operates from the 150 component manual set, a reduced feature set of 30 is found to work best, and with notable improvement in kernel parameter stability in the region of interest Figure 8 If Adaboost operates from the 150-component manual set, a reduced feature set of 30 is found to work best, and with notable improvement in kernel parameter stability in the region of interest. (The Y-axis, "SN + SP", shows the sum of the Sensitivity and the Specificity. The X-axis is the kernel parameter σ.) such that the spike density feature improves classification without obscuring the contribution of other features. can be destroyed and the channel current signal will be mangled beyond use. Although this problem is not signif- icant for a wide range of EVA factor, a HMM with Dura- tion [2-4] will retain transitions and can eliminate this problem altogether. A few automated solutions are suggested for future work. One proposed solution is to simply add the un-weighted spike density feature to the existing feature vector and use AdaBoost to select the most diagnostic features. This approach will essentially create a weight for the spike den- sity feature. That is, by removing many components that only add noise to a given feature vector, the remaining fea- tures are given more weight. Another solution that is cur- rently being worked on is to fold the definition of a spike into the HMM. This solution requires a non-trivial amount of work as the entire definition of a state has to be entirely reworked. Moreover, the definition of a state must be considered carefully such that a state explosion (as seen in higher order HMMs) does not occur. Another aspect of the dwell time analysis that will be explored in future work is the effect of dwell time infor- mation on classification. Dwell time distributions for dominant levels should be characteristic for a given signal and thus improve classification performance. However, a significant amount of data is generally necessary to gener- ate accurate dwell time distributions. In the current archi- tecture, 100 ms of channel current blockade are analyzed to create one feature vector. It is unclear as to whether 100 ms will be enough data to overcome this limitation on sparseness of data. A longer signal trace could be ana- lyzed, but computational complexity grows quadratically as signal input increases linearly. If Adaboos improvem Figure 8 Here, the use of a distrib- uted HMM has been developed to allow for the processing of enough data to provide accurate dwell time statistics while still meeting reasonable time constraints (paper in preparation), using a simple distribution analogous to the chunk processing that is employed for the SVM training [17]. Emission inversion Emission inversion involves exchanging of the roles of emitted states and observed states. The exact theoretical underpinning of exchanging these roles is not yet com- pletely understood (see Methods for details). In some sense, however, classification performance is the ultimate judge of the validity of a given method. As described in the Results section, the SVM classification performance is strongest when using emission inversion. Page 10 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 If Adaboost operates from the 150-component manual set, a reduced feature set of 30 is found to work best, and with notable improvement in kernel parameter stability in the region of interest Figure 8 If Adaboost operates from the 150-component manual set, a reduced feature set of 30 is found to work best, and with notable improvement in kernel parameter stability in the region of interest. (The Y-axis, "SN + SP", shows the sum of the Sensitivity and the Specificity. The X-axis is the kernel parameter σ.) AdaBoosting from Original 150 Features - 8GC9AT 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 Sigma SN + 30 All AdaBoosting from Original 150 Features - 8GC9AT AdaBoosting from Original 150 Features - 8GC9AT AdaBoosti the SVM Figure 9 AdaBoosting to select 100 from the full set of 2600 features improves classification over just passing all 2600 components to the SVM Figure 9 AdaBoosting to select 100 from the full set of 2600 features improves classification over just passing all 2600 components to the SVM. However, the best performance is still obtained when working with the Adaboosting from the manual set. (The Y- axis, "SN + SP", shows the sum of the Sensitivity and the Specificity. The X-axis is the kernel parameter σ.) Feature selection the choice of D does not present a great problem as SVMs are robust and can learn well in the presence of noise and non-diagnostic features. Experimentally it has been observed that it is more important that D not be chosen too small as opposed to too large. Typically AdaBoost is used as a classification method. But due to the limitations discussed in the Background sec- tion, SVMs provide a much more robust means of classifi- cation for channel current data. However, AdaBoost is still useful in feature selection, and that is the main use we have for AdaBoost in the work presented here. The weight- ing schemes in the AdaBoost algorithm are a natural fit for feature selection as the weights indicate which features are most diagnostic for a given classification problem. It is also important to note that automated feature selec- tion using AdaBoosting was not able to reproduce results obtained from the "best-case" manually designed feature set (see Figure 9). Nonetheless, feature selection using AdaBoost is an important technique. It allows for the automated exploration of the effect of many different fea- tures and feature sets. In addition, AdaBoosted feature selection would be useful in problems where the defini- tion of states do not lead to an easily designed manual set of features. AdaBoost does require some subtle tuning. As can be seen in the algorithm shown in Figure 3, AdaBoost does not have a natural end point. Unlike an SVM, AdaBoost does not converge on a solution. The number of iterations in the AdaBoosting algorithm must be tuned over in order to ensure accurate results. Another tuning parameter is the number of diagnostic features "D" to select from the orig- inal feature set "O". Should D be chosen too small, diag- nostic features existing in O will be excluded and classification performance will suffer. Should D be chosen too large, noisy features existing in O will exist in D and classification performance will suffer. In general, though, Dwell time analysis Preprocessing channel current blockade data using a HMM with EVA significantly reduces the complexity of dwell time analysis. Within a reasonable range of values for EVA factor, the noise bands around levels are signifi- cantly reduced while level transitions are retained. How- ever, if too large of an EVA factor is used then transitions Page 11 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 AdaBoosting to select 100 from the full set of 2600 features improves classification over just passing all 2600 components to the SVM Figure 9 AdaBoosting to select 100 from the full set of 2600 features improves classification over just passing all 2600 components to the SVM. However, the best performance is still obtained when working with the Adaboosting from the manual set. (The Y- axis, "SN + SP", shows the sum of the Sensitivity and the Specificity. The X-axis is the kernel parameter σ.) AdaBoosting off of 2600 Features - 9CG9GC 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 1.00E-05 0.001 0.003 0.005 0.007 0.009 0.02 0.04 0.06 0.08 0.1 0.3 0.5 0.7 0.9 2 4 10 1000 Sigma SN + First 50 Original 150 100 All 2600 AdaBoosting off of 2600 Features - 9CG9GC AdaBoosting off of 2600 Features - 9CG9GC Sigma Methods Emission inversion The data inversion implementation simply exchanges the roles of the actual state and the observed state as follows: Classificatio Figure 10 Classification improvement with Adaboost taking the best 50 from the Inverted-emission 150 feature set Figure 10 Classification improvement with Adaboost taking the best 50 from the Inverted-emission 150 feature set. 95% accuracy is pos- sible for discriminating 9GC from 9TA hairpins with no data dropped with use of Adaboost, without Adaboosting, the accuracy is approx. 91%. This demonstrates a significant robustness to what the SVM can "learn" in the presence of noise (some of the 2600 component have richer information, but even more are noise contributors). This also validates the effectiveness with which the 150-parameter compression was able to describe the two-state dominant blockade data found for the nine base-pair hairpin and other types of "toggler" blockades, as well as the utility of the inverted features. Classification improvement with Adaboost taking the best 50 from the Inverted emission 150 feature set Figure 10 Classification improvement with Adaboost taking the best 50 from the Inverted-emission 150 feature set. 95% accuracy is pos- sible for discriminating 9GC from 9TA hairpins with no data dropped with use of Adaboost, without Adaboosting, the accuracy is approx. 91%. This demonstrates a significant robustness to what the SVM can "learn" in the presence of noise (some of the 2600 component have richer information, but even more are noise contributors). This also validates the effectiveness with which the 150-parameter compression was able to describe the two-state dominant blockade data found for the nine base-pair hairpin and other types of "toggler" blockades, as well as the utility of the inverted features. where b = observed_value and k = state. A standard imple- mentation of a HMM would be implemented in the fol- lowing manner: data and final classification performance. Previous meth- ods for spike feature extraction were folded into the cur- rent architecture. In addition, a new method for analyzing dwell times, Emission Variance Amplification was applied to the HMM. Finally, a hybrid AdaBoost approach was introduced in an effort to improve the feature selection process. Not only are these techniques useful improve- ments for the current signal process architecture, but sev- eral techniques introduced here also provide means to move forward with future research as detailed in the Dis- cussion section. Emission inversion As previously discussed in the Background and Discussion sections, the main place where data is introduced into the HMM/EM algorithm is through the emission probabili- ties. In the HMM, emissions are defined as a multidimen- sional array and can be viewed conceptually as the probability of a hidden state emitting an observed state: Conclusion Several new techniques and improvements on existing techniques in the channel current signal analysis platform have been introduced. Data inversion was introduced and was shown to be an improvement over the standard implementation of a HMM in regards to channel current Page 12 of 16 (page number not for citation purposes) Page 12 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 Classification improvement with Adaboost taking the best 50 from the Inverted-emission 150 feature set Figure 10 Classification improvement with Adaboost taking the best 50 from the Inverted-emission 150 feature set. 95% accuracy is pos- sible for discriminating 9GC from 9TA hairpins with no data dropped with use of Adaboost, without Adaboosting, the accuracy is approx. 91%. This demonstrates a significant robustness to what the SVM can "learn" in the presence of noise (some of the 2600 component have richer information, but even more are noise contributors). This also validates the effectiveness with which the 150-parameter compression was able to describe the two-state dominant blockade data found for the nine base-pair hairpin and other types of "toggler" blockades, as well as the utility of the inverted features. 9GC vs 9TA Classification Results for AdaBoost vs Inv150 1.5 1.6 1.7 1.8 1.9 2 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 0.200 Kernel Parameter Sensitivity + Specificity Inverted 150 AdaBoost Inv150 9GC vs 9TA Classification Results for AdaBoost vs Inv150 1.5 1.6 1.7 1.8 1.9 2 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 0.200 Kernel Parameter Sensitivity + Specificity Inverted 150 AdaBoost Inv150 9GC vs 9TA Classification Results for AdaBoost vs Inv150 9GC vs 9TA Classification Results for AdaBoost vs Inv150 Inverted 150 AdaBoost Inv150 Forward [0] [I] = emission_probabilities [I] [observed_data[0]] * Forward [0] [I] = emission_probabilities [I] [observed_data[0]] * [I] exp(-(k-i)*(k-i)/(2*variance*eva_factor)). Break if the overall composite learner's error rate is 0% or 50% Essentially EVA boosts the variance of the distribution and yields the following effect: for states near a dominant level in the blockade signal, the transitions are highly favored to points nearer that dominant level. This is a simple sta- tistical effect having to do with the fact that far more points of departure are seen in the direction of the nearby dominant level than in the opposite direction. When in the local gaussian tail of sample distribution around the dominant level, the effect of transitions towards the dom- inant level over those away from the dominant level can be very strong. In short, a given point is much more likely to transition towards the dominant level than away from it. In an example where there is a set of 150-component fea- ture vectors, 150 weak learners would be created. As pre- viously mentioned, each weak learner corresponds to a single component and classifies a given feature vector based solely on that one component. Then, weights for these weak learners are introduced. In each iteration of this modified AdaBoost process, weights for both the input data and the weak learners are updated. The weights for the input data are updated as in the standard AdaBoost implementation while weights on the individual weak learners are updated as if each were a complete hypothesis in the standard AdaBoost implementation (see figure in Additional File 7). At the end of the iterative process, the weak learners with the highest weights, that is, the weak learners that represent the most diagnostic features, are selected and those features are passed on to a SVM for clas- sification. Thus, the benefits of both AdaBoost and SVMs are obtained. For (I = 0; I < NUM_STATES; I++) { Forward [0] [I] = emission_probabilities [observed_data[0]] [I] * Prior_probability [I]; Prior_probability [I]; emission_probabilities [state] [observed_value] ≡ P(X = b|S = k), emission_probabilities [state] [observed_value] ≡ P(X = b|S = k), } Page 13 of 16 (page number not for citation purposes) Page 13 of 16 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/S7/S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 BMC Bioinformatics 2007, 8(Suppl 7):S12 this idea can be seen in the figure shown in Additional File 7. Training data can be viewed as a two dimensional array of feature components. F1 - Fj are individual feature vec- tors representing a single capture event. E1 - Ei are the experts or weak learners assigned to an individual compo- nent in feature space. In the implementation described in this paper, naïve bayes classifiers were used as weak learn- ers. this idea can be seen in the figure shown in Additional File 7. Training data can be viewed as a two dimensional array of feature components. F1 - Fj are individual feature vec- tors representing a single capture event. E1 - Ei are the experts or weak learners assigned to an individual compo- nent in feature space. In the implementation described in this paper, naïve bayes classifiers were used as weak learn- ers. This simple inversion introduces another information fac- tor into the Viterbi algorithm and improves performance as discussed in the Results section. So, with inversion, instead of P(X = b|S = k) we now have P(X = k|S = b). In our analysis we have P(X = k|S = b) ≈ P(S = k|X = b), so the change with inversion is approximately a factor of [P(S = k)/P(X = b)] introduced at each column position. For the Viterbi calculation, with sums on log contributions from each column, i.e., log [P(S = k)/P(X = b)], the new term sums to the length-weighted relative entropy between the state prior probability and emission posterior probability: - L D(X||S), where L is the length of data parsed and 'D(* || *)' is the Kullback-Leibler Divergence (or relative entropy). For a given number of iterations T, the process is as fol- lows: Initialize weights on weak learners Initialize weights on training data Normalize the two weights. exp(-(k-i)*(k-i)/(2*variance*eva_factor)). Emission variance amplification As mentioned in the Discussion section, a HMM with EVA is used to significantly reduce the gaussian noise band around levels. In a non-EVA approach, emission probabil- ities are initialized with a gaussian profile. The initializa- tion is as follows: Train weak learners Update the weights for each weak learner – just like the hypothesis emission_probabilities [i] [k] = exp(-(k-i)*(k-i)/(2*vari- ance)) emission_probabilities [i] [k] = exp(-(k-i)*(k-i)/(2*vari- ance)) update in the standard AdaBoosting method Update the weights for each training point – just like the original where "i" and "k" are each a state with 0 <= {i, k} <= 49 in a 50 state system. To perform EVA, the variance is sim- ply multiplied by a factor that essentially widens the gaus- sian distribution imposed on possible emissions, and the equation simply becomes where "i" and "k" are each a state with 0 <= {i, k} <= 49 in a 50 state system. To perform EVA, the variance is sim- ply multiplied by a factor that essentially widens the gaus- sian distribution imposed on possible emissions, and the equation simply becomes AdaBoosting method Page 14 of 16 (page number not for citation purposes) p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors' contributions The initial submission was written by ML and SWH, with revisions by SWH. The core feature extraction and pattern recognition software was developed by SWH. The Ada- Boost refinement and test dataruns were done by ML. Feature selection As introduced in the Backgrounds and Discussion sec- tions, AdaBoost is used in feature selection. In this hybrid implementation, weights are given to the weak learners as well as the training data. The key modifications here are to give each column of features in a training set a weak learner and to update each weak learner every iteration, not just updates the weights on the data. Conceptually, Page 14 of 16 (page number not for citation purposes) Page 14 of 16 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/S7/S12 BMC Bioinformatics 2007, 8(Suppl 7):S12 Additional file 6 The dwell time distributions for the three dominant levels of the Y- aptamer (without 6A target) The dwell time distributions for the three dominant levels of the Y-aptamer (without 6A target). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S6.doc] Additional file 7 The key Adaboost modifications are to give each column of features in a training set a weak learner, and to update each weak learner every iteration, not just update the weights on the data The key Adaboost modifications are to give each column of features in a training set a weak learner, and to update each weak learner every itera- tion, not just update the weights on the data. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S7.doc] Additional file 3 This article has been published as part of BMC Bioinformatics Volume 8 Sup- plement 7, 2007: Proceedings of the Fourth Annual MCBIOS Conference. Computational Frontiers in Biomedicine. The full contents of the supple- ment are available online at http://www.biomedcentral.com/1471-2105/ 8?issue=S7. Additional material The key Adaboost modifications are to give each column of features in a training set a weak learner, and to update each weak learner every iteration, not just update the weights on the data The key Adaboost modifications are to give each column of features in a training set a weak learner, and to update each weak learner every itera- tion, not just update the weights on the data. Click here for file Additional file 1 A simplified time-domain Finite State Automaton (τFSA) is used for signal acquisition (see [4,11] for full model) A simplified time-domain Finite State Automaton (τFSA) is used for sig- nal acquisition (see [4,11] for full model). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S1.doc] Additional file 2 A blockade level histogram of two DNA hairpin channel blockade signals A blockade level histogram of two DNA hairpin channel blockade signals. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S2.doc] Additional file 3 The extrapolated true spike counts for the radiated DNA hairpin blockade The extrapolated true spike counts for the radiated DNA hairpin blockade. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S3.doc] Additional file 4 The extrapolated true spike counts for the non-radiated DNA hair- pin blockade The extrapolated true spike counts for the non-radiated DNA hairpin blockade. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S4.doc] Additional file 5 The 150-component feature vector profiles for the Y-aptamer that binds 6A ssDNA, for signals before and after introduction of that six-adenosine ssDNA The 150-component feature vector profiles for the Y-aptamer that binds 6A ssDNA, for signals before and after introduction of that six-adenosine ssDNA. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S5.doc] [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S7.doc] Acknowledgements Federal funding was provided by NIH K-22 (PI, 5K22LM008794), NIH NNBM R-21 (co-PI), and NIH R-01 (sub-award). State funding was provided from a LaBOR Enhancement (PI), a LaBOR Research Competitiveness Sub- contract (PI), and a LaBOR/NASA LaSPACE Grant (PI). Thanks to Eric Morales and Iftekhar Amin for gathering data. Funding also provided by New Orleans Children's Hospital and the University of New Orleans Com- puter Science Department. References 1. Winters-Hilt S, Vercoutere W, DeGuzman VS, Deamer DW, Akeson M, Haussler D: Highly Accurate Classification of Watson- Crick Basepairs on Termini of Single DNA Molecules. Biophys J 2003, 84:967-976. J 2. Winters-Hilt S: Hidden Markov Model Variants and their Application. BMC Bioinformatics 2006, 7(Suppl 2):S14. 3. Baribault C, Winters-Hilt S: A novel, fast, HMM-with-Duration implementation – for application with a new, pattern recog- nition informed, nanopore detector. BMC Bioinformatics 2007, 8(Suppl 7):S19. Competing interests Additional file 6 The dwell time distributions for the three dominant levels of the Y- aptamer (without 6A target) The dwell time distributions for the three dominant levels of the Y-aptamer (without 6A target). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-8-S7-S12-S6.doc] p g The authors declare that they have no competing interests. Additional file 5 ( pp ) 4. Churbanov A, Baribault C, Winters-Hilt S: Duration learning for nanopore ionic flow blockade analysis. BMC Bioinformatics 2007. 4. Churbanov A, Baribault C, Winters Hilt S: Duration learning for nanopore ionic flow blockade analysis. BMC Bioinformatics 2007. 5. Iqbal R, Landry M, Winters-Hilt S: DNA Molecule Classification Using Feature Primitives. BMC Bioinformatics 2006, 7(Suppl 2):S15. p y 5. Iqbal R, Landry M, Winters-Hilt S: DNA Molecule Classification Using Feature Primitives. BMC Bioinformatics 2006, 7(Suppl 2):S15. 6. Winters-Hilt S, Akeson M: Nanopore cheminformatics. DNA and Cell Biology 2004. gy 7. Vercoutere W, Winters-Hilt S, DeGuzman VS, Deamer D, Ridino S, Rogers JT, Olsen HE, Marziali A, Akeson M: Discrimination Among Individual Watson-Crick Base-Pairs at the Termini of Single DNA Hairpin Molecules. Nucl Acids Res 2003, 31:1311-1318. 8. Vercoutere W, Winters-Hilt S, Olsen H, Deamer DW, Haussler D, Akeson M: Rapid discrimination among individual DNA hair- pin molecules at single-nucleotide resolution using an ion channel. Nat Biotechnol 2001, 19(3):248-252. ( ) 9. Winters-Hilt S, Landry M, Akeson M, Tanase M, Amin I, Coombs A, Morales E, Millet J, Baribault C, Sendamangalam S: Cheminformat- ( ) 9. Winters-Hilt S, Landry M, Akeson M, Tanase M, Amin I, Coombs A, Morales E, Millet J, Baribault C, Sendamangalam S: Cheminformat- Page 15 of 16 (page number not for citation purposes) Page 15 of 16 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 7):S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 http://www.biomedcentral.com/1471-2105/8/S7/S12 ics Methods for Novel Nanopore analysis of HIV DNA ter- mini. BMC Bioinformatics 2006, 7(Suppl 2):S22. 10. ( pp ) 10. Winters-Hilt S, Davis A, Amin I, Morales E: Nanopore current transduction analysis of protein binding to non-terminal and terminal DNA regions: analysis of transcription factor bind- ing, retroviral DNA terminus dynamics, and retroviral inte- grase-DNA binding. BMC Bioinformatics 2007, 8(Suppl 7):S10. g g ( pp ) 11. Cormen TH, Leiserson CE, Rivest RL: Introduction to Algo- rithms. MIT-Press, Cambridge, USA; 1989. rithms. MIT-Press, Cambridge, USA; 1989. 12. Durbin R: Biological sequence analysis : probalistic models of proteins and nucleic acids. Volume xi. Cambridge, UK New York: Cambridge University Press; 1998:356. g y 13. Vapnik VN: The Nature of Statistical Learning Theory. 2nd edition. Springer-Verlag, New York; 1998. p g g 14. Burges CJC: A tutorial on support vector machines for pattern recognition. Data Min Knowl Discov 1998, 2:121-67. 15. Additional file 5 Winters-Hilt S, Yelundur A, McChesney C, Landry M: Support Vec- tor Machine Implementations for Classification & Cluster- ing. BMC Bioinformatics 2006, 7(Suppl 2):S4. g ( pp ) 16. Prabhakaran A: Power Signal analysis of Channel Current Sig- nal using HMM-EM and Time-domain FSA. University of New Orleans Masters Thesis in Computer Science; 2005. 17. Osuna E, Freund R, Girosi F: An improved training algorithm for support vector machines. In Neural Networks for Signal Processing VII Edited by: Principe J, Gile L, Morgan N, Wilson E. IEEE, New York; 1997:276-85. 18. Thomson K, Amin I, Morales E, Winters-Hilt S: Preliminary Nano- pore Cheminformatics Analysis of Aptamer-Target Binding Strength. BMC Bioinformatics 2007, 8(Suppl 7):S11. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 16 of 16 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Page 16 of 16 (page number not for citation purposes)

https://openalex.org/W2166824973

https://www.ajol.info/index.php/wsa/article/download/76758/67197

English

Framework for assessing the viability of implementing dual water reticulation systems in South Africa

Water S.A./Water SA

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The South Africa water resources situation: background and motivation off arising from 20% of the land area. In some places the runoff can be highly variable and below normal for up to 10 consecu­ tive years (DWAF, 2004a). Water is increasingly becoming a limiting resource in South Africa and the scarcity of this resource affects national, pro­ vincial and local development in critical areas (Eberhard and Robinson, 2003). South Africa is an arid to semi-arid country with high water stress due to the low mean annual precipitation, MAP (which is approximately 500 mm/a – significantly below the world average of about 860 mm/a) and high mean annual evaporation, MAE (approximately 350% of MAP) (Eberhard and Robinson, Ibid). Of interest is the highly seasonal occur­ rence of rainfall over virtually all of the country resulting in a wide range of climates, from winter rainfall and warm windy summers in the south-western Cape, to erratic, non-seasonal rainfall and extreme temperatures in the Karoo. The variation in annual rainfall from the long-term mean is especially pro­ nounced in the more arid areas where unpredictable droughts of extended durations often contribute to the harshness of existing water scarcity. Also, as a consequence of topographical and cli­ matic conditions, the natural availability of surface water across the country is unevenly distributed with more than 60% of run­ Many of the metropolitan and industrial centres of South Africa (e.g. Johannesburg, Kimberley, Rustenburg, Mokopane, Durban and Cape Town) have developed around mineral depos­ its and harbour sites, and are located a significant distance away from major freshwater sources. Some irrigation developments in the country are also located in sub-optimal regions with respect to water use efficiency, having been established in times of rela­ tive water abundance and lower demand for water in upstream reaches. Thus, the location of several South African metropoles, industrial and agricultural areas has added to the challenges of freshwater availability. To manage existing water resources, the country’s hydrolog­ ical basins have been divided into 19 water management areas with total available water resources of approximately 49 x 109 m3/a. This includes water inflows of about 4.8 x 109 m3/a and 0.7 x 109 m3/a originating from Lesotho and Swaziland respectively (DWAF, 2004a and Mukheibir, 2005). Of the total available water resources, only about 25% (13.23 x 109 m3/a) is harnessed as usable yield – this figure includes usable return flows, surface water and groundwater. Abstract In many settlements across the world (e.g. Pimpama Coomera and Mawson Lakes – Australia, Hong Kong – China, Majuro – Marshall Islands, Tarawa – Kiribati, and Windhoek – Namibia), dual water reticulation systems have been implemented in response to increasing water demands and decreasing freshwater availability. A dual water reticulation system comprises separate pipes that supply different water qualities to the end consumer. A set of pipes supply potable water while another set of pipes supply non-potable water. The non-potable water is targeted at meeting water requirements traditionally met using potable water (e.g. toilet and urinal flushing, landscaping irrigation, and industrial cooling). This therefore frees potable water to be used for previously unmet or increasing potable water requirements. For several reasons including the dearth of relevant national regulatory and guideline documents, consumer and decision-maker perceptions, ignorance, and appropri­ ate decision-making tools, the use of dual water reticulation systems in South Africa has been limited. The aim of this study was therefore to develop a decision-making framework, using robust criteria, for assessing the viability of implementing dual systems in South Africa. This aim was achieved through undertaking literature reviews on the subject, an investigation of non-potable water consumers’ and decision-makers’ perceptions using questionnaires, and the actual development of a framework using data obtained from the literature review and questionnaires. The questionnaires were developed using seven key issues i.e. public health and safety, economics, technical feasibility, legislation/regulations and guidelines, organisational capacity, social acceptance, and public education. The various aspects of the Triple Bottom Line of sustainability (i.e. eco­ nomic, environmental and social) provided structure to the framework while the Triple Bottom Line approach was utilised in the assessment of the different criteria. Keywords: dual water reticulation systems, non-potable water recycling Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Framework for assessing the viability of implementing dual water reticulation systems in South Africa AA Ilemobade1*, JR Adewumi1 and JE van Zyl2 1School of Civil and Environmental Engineering, University of the Witwatersrand, South Africa 2Department of Civil Engineering Science, University of Johannesburg, South Africa This paper was originally presented at the 2008 Water Institute of Southern Africa (WISA) Biennial Conference, Sun City, South Africa, 18-22 May 2008. * To whom all correspondence should be addressed.  +2711 717 7153 ; fax: +2711 717 7045; e-mail: [email protected] The South Africa water resources situation: background and motivation Internationally, different categories of dual water reticu­ lation systems with diverse design specifications, conveying diverse non-potable water qualities for different water require­ ments, have been implemented. Many of these systems can be found in the United Kingdom, United States of America, Singa­ pore and Australia (Dimitriadis, 2005; Po et al., 2005; Po et al., 2003), Namibia (van der Merwe, 2006), Japan, China (Tang et al., 2007), the Caribbean nations of Trinidad and Tobago (Busi­ ness and Economy, 2003), Netherlands (Health Stream, 2003), and Republics of Kiribati and the Marshall Islands (Parr et al., 1997). In South Africa, the use of dual systems was investigated in the past (Botha and Pretorius, 1998). The report concluded that dual systems offer new possibilities for maintaining ade­ quate water supply and appropriate use of the available water resources in South Africa. Dual systems were reported to be especially beneficial in the following areas: Potable water refers to water that is safe for human consump­ tion while non-potable water refers to non-consumable water. Non-potable water, after some level of treatment, may be suit­ able for some water requirements e.g. toilet and urinal flushing, car washing, fire-fighting, landscape irrigation, dust suppression and a variety of industrial and commercial water requirements – this process is called water recycling/reuse. Potable water, which is of a better quality and higher cost, is however, commonly used for these non-potable water requirements. This practice is unsustainable if South Africa is to effectively manage increas­ ingly scarce freshwater resources. • Where sea or brackish water (with high total dissolved solids (TDS) concentrations) is the closest available water source; • Where intensive indirect reuse of water may cause high Total Dissolved Solids concentrations in the source waters (as with the Vaal River barrage). • Where the incremental cost of developing new freshwater sources may be high and therefore less attractive in com­ parison to recycling sewage effluent. The mass balances and cost comparisons conducted in Botha and Pretorius (1998) study indicated that using dual systems would result in smaller desalination streams, less salts to be removed from sewage, better water utilisation indices and probably, better economics than the reclamation of treated effluent for direct potable reuse. Non-potable water conveyed through dual water reticulation systems (henceforth, dual systems) therefore presents a viable option to supplementing existing water supplies. The South Africa water resources situation: background and motivation Groundwater accounts for only about 2.2% of the total available water resources as the country is mainly underlain by hard rock formations which, although rich in minerals, do not contain major groundwater aquifers that can be used on a large scale for water supply (Mukheibir, 2005). The incidence of groundwater salinity in especially the coastal areas of the country also adds to the unavailability of fresh ground­ water. Nevertheless, groundwater has played a pivotal role in the 216 al., 2006), Spain (March et al., 2004), Australia (Po et al., 2003; Po et al., 2005), Namibia (Van der Merwe, 2006), and some parts of South Africa (Sustainability Institute, 2006; CoCT, 2007b). Also, due to the advantages of implementing reuse to supple­ ment potable water supplies, reuse has been embraced in some water rich countries such as China (Junying et al., 2004), Japan (Dixon et al., 1999), Germany (Nolde, 1999), United Kingdom (Jimenez and Asano, 2008) and the United States of America (Okun, 1996). settlement and initial development of the country, and continues to do so, especially in rural areas of the country (Basson et al., 1997). Total water use in the year 2000 grouped into six catego­ ries, amounted to about 12.87 x 109 m3/a (a figure almost match­ ing the exploitable supply) with the agricultural sector consum­ ing the largest proportion of supply (62%), while urban, mining and industry, rural, afforestation, and power generation sectors consuming 23%, 6%, 4%, 3% and 2% respectively (DWAF, 2004c). ) The scenarios painted above have therefore resulted in dire water scarcity problems in several areas of South Africa, with the result that in several river catchments, the water require­ ments already far exceed the natural availability of water. It was projected in 1996 that the water resources supply for the country may be unable to cater for anticipated overall demands by 2030 if demands did not go unchecked (Basson et al., 1997). Supply and demand have thus had to be balanced by large water resources development projects (fresh and saline water) and extensive inter-basin transfers from areas of surplus to areas of deficit. Some water demand management initiatives (e.g. leak­ age management, meter management, use of efficient plumbing fittings and non-potable water use) have also been implemented. Non-potable water use has in particular, become an area of inter­ est in recent times. The South Africa water resources situation: background and motivation This option is particularly promising for arid South African settlements with limited access to freshwater sources, still in the process of developing their basic infrastructure, in proximity to saline (i.e. brackish or sea) waters, and/or that generate significant volumes of rain water, storm water runoff, sewage, grey-water and/or mine effluent. Grey-water represents household waste­ water from showers, baths, hand basins, laundry tubs, washing machines, dishwashers and kitchen sinks and does not include water from toilets. Uptake of the recommendations of Botha and Pretorius (1998) study in especially arid settlements of South Africa has been limited, despite the fact that the technology surrounding dual systems and non-potable water use has evolved since then, with great strides made on the subject. Some international and local examples of the different categories of dual systems are pre­ sented below. In addition to aridity (discussed above), other factors encour­ aging non-potable water use in South Africa include (Ilemobade et al., 2008): Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Dual water reticulation systems for individual non-potable use • The Growing demands for greener water strategies • The heightened awareness of the potential nutritional ben­ efits of using suitably treated sewage/grey water effluent (henceforth treated effluent) in the agricultural sector Non-potable water generated by a household is collected on-site and then distributed using separate pipes for non-potable uses within the same household. Treatment and storage is dependent on local circumstances and the targeted water use(s).l • The high costs of supplying large quantities of potable water to arid areas. This is especially true for settlements distant from urban centres and with limited access to municipal water infrastructure. • International examples include: residential toilet flushing and garden irrigation in Springfield (Queensland), the Syd­ ney Olympic Park (Homebush Bay, New South Wales), and Mawson Lakes (South Australia), Australia using treated effluent and storm water runoff (Po et al., 2003 and Dimitri­ adis, 2005); Methodology for the social surveys • International examples include: toilet flushing and fire fight­ ing in Majuro (Marshall Islands) and Tarawa (Kiribati) using saline groundwater (Parr et al., 1997); toilet flushing in Hong Kong (China) using sea water (Tang et al., 2007); horticulture irrigation via the Virginia Pipeline Scheme at Bolivar (South Australia) using treated effluent (Po et al., 2003); residential toilet flushing, car washing, garden irriga­ tion and fire fighting in Rouse Hill (New South Wales, Aus­ tralia) and Pimpama Coomera (Gold Coast, Australia) using treated effluent (Po et al., 2003; Po et al., 2005); landscape irrigation in Windhoek (Namibia) using treated effluent (Van der Merwe, 2006); and landscape irrigation and carpet dyeing in the Irvine Ranch Water District (Orange County, USA) using treated effluent (IRWD, 2006) • International examples include: toilet flushing and fire fight­ ing in Majuro (Marshall Islands) and Tarawa (Kiribati) using saline groundwater (Parr et al., 1997); toilet flushing in Hong Kong (China) using sea water (Tang et al., 2007); horticulture irrigation via the Virginia Pipeline Scheme at Bolivar (South Australia) using treated effluent (Po et al., 2003); residential toilet flushing, car washing, garden irriga­ tion and fire fighting in Rouse Hill (New South Wales, Aus­ tralia) and Pimpama Coomera (Gold Coast, Australia) using treated effluent (Po et al., 2003; Po et al., 2005); landscape irrigation in Windhoek (Namibia) using treated effluent (Van der Merwe, 2006); and landscape irrigation and carpet dyeing in the Irvine Ranch Water District (Orange County, USA) using treated effluent (IRWD, 2006) A significant number of the early studies of public perceptions relating to water reuse were undertaken in the US. Most of these studies were limited in their scope which often aimed to increase public acceptance using applied behavioural methods (e.g. incen­ tives). This early approach to implementing water reuse projects often viewed public acceptance as the principal ‘obstacle’ to implementing recycling projects. In the literature, this approach has been shown to be inadequate (Po et al., 2003; Po et al., 2005). Subsequent research following this view involved finding ways to persuade people to accept recycled water. It is now gener­ ally accepted that social marketing or persuasion is ineffective in influencing people to use non-potable water. The approach, however, of involving communities prior to the conception of the project has produced consistent results especially within developing settlements (Po et al., 2003; Po et al., 2005). Methodology for the social surveys Part of this approach, called the demand responsive approach, involves the early mining of trends, data and perceptions relating to the project. The data generated would then indicate to a large extent, the potential for success or failure of a reuse project. The social survey therefore adopted this approach in determining the per­ ceptions of domestic respondents (most of whom were ignorant of plans to implement mine-water recycling in their area by the local authority), institutional consumers (all of whom utilised treated effluent), and decision-makers. • South African examples include: industrial and mining proc­ ess water and landscape irrigation in the Rustenburg Local Municipality (North West), Mokopane and Potgietersrus (Limpopo), and the City of Cape Town, (henceforth CoCT) (Western Cape) using treated effluent (Jimenez and Asano, 2008 and CoCT, 2007b); and paper production in Mondi Paper (eThekweni, KwaZulu-Natal) using treated effluent (Jimenez and Asano, 2008); Methodology Considering the wealth of experience and significant potential for dual systems in South Africa, the aim of this study was to develop a decision-making framework, using robust criteria, for assessing the viability of implementing dual systems in potential South African settlements. Since governmental decision-making is critical when planning centralised schemes, the assessment framework must specifically apply to dual systems for district level, wide area urban/agricultural, and industrial non-potable use. • International examples include: supplying non-potable domestic requirements in the Hockerton housing scheme and the Beddington Zero Energy housing Development (BedZED) (South London), UK using harvested rain water (Heather, 2005) • South African examples include: garden irrigation and toi­ let flushing in the Lynedoch Ecovillage (Stellenbosch) using treated effluent (Sustainability Institute, 2006); and toilet flushing using saline groundwater and landscape irrigation using treated effluent in Garies (Northern Cape) (Mvula Trust, 2006). The aim of this study was achieved through undertaking three tasks, i.e. • Literature surveys, which attempted to garner local and international experiences of dual systems Literature review In arid regions of the world where there has traditionally been scarcity of water, treated effluent reuse has been successfully implemented e.g. Jordan (Al-Jayyousi, 2004), Israel (Friedler et • South African examples include: garden irrigation in Hull street (Kimberley) using grey-water (Webster, 2006); and • South African examples include: garden irrigation in Hull street (Kimberley) using grey-water (Webster, 2006); and 217 garden and crop irrigation in Carnarvon (Northern Cape) using grey-water (Ilemobade et al., 2008). process water (Jimenez and Asano, 2008) South African examples include: mining process water, toilet flushing and landscape irrigation in the Gold Fields gold mine in Driefontein (Gauteng) using treated effluent and recycled dolomite water (Ilemobade et al., 2008); indus­ trial process water in Sasol (Sasolburg), AECI (Modderfon­ tein), Nampak Tissue (Bellville) using treated process water (Jimenez and Asano, 2008). garden and crop irrigation in Carnarvon (Northern Cape) using grey-water (Ilemobade et al., 2008). process water (Jimenez and Asano, 2008) process water (Jimenez and Asano, 2008) South African examples include: mining process water, toilet flushing and landscape irrigation in the Gold Fields gold mine in Driefontein (Gauteng) using treated effluent and recycled dolomite water (Ilemobade et al., 2008); indus­ trial process water in Sasol (Sasolburg), AECI (Modderfon­ tein), Nampak Tissue (Bellville) using treated process water (Jimenez and Asano, 2008). Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Dual water reticulation systems for district non- potable use Non-potable water is collected at a central location from multiple buildings and then distributed using separate pipes for non-pota­ ble uses within the same or other buildings. These may include large housing developments comprising single and/or multiple storey buildings. Treatment and storage is dependent on local circumstances and the targeted water use(s). Dual water reticulation systems for wide area urban/ agricultural non-potable use • Social surveys using questionnaires, on-site visits, and consultations, which collected and analysed perceptions of some non-potable water consumers and water services deci­ sion-makers Non-potable water is collected at a central location from domes­ tic and non-domestic sources and then treated, stored and dis­ tributed using separate pipes for non-potable domestic and/or non-domestic uses elsewhere. These dual systems may incor­ porate treated effluent supply from a sewage treatment works, STWs). • The development of a framework for assessing the viability of implementing dual systems in South Africa. Methodol­ ogy, results and discussion for the 2nd and 3rd tasks are pre­ sented separately in the sections below. Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Dual water reticulation systems for industrial non- potable use Non-potable water which is generated from industrial use is collected on-site, treated, stored and distributed using separate pipes for on-site non-potable uses. The Department of Water Affairs and Forestry, DWAF (2004b) has set a number of objectives against which strategies of water institutions or consumers (to influence water demand and use) should be measured. These are economic efficiency, social development, social equity, environmental protection, • International examples include: sugar and malt production, and beverage bottle washing in Germany using recycled 218 TABLE 1 Distribution of respondents Category Respondents Number of respondents Description of respondents Consumers Households 68 Domestic consumers of potable water produced from recycled mine effluent in Emahlaleni. The Emahlaleni settlement provided the opportunity to evaluate res­ idents’ perceptions on the consumption of potable water from an unconventional source, the use of non-potable water for non-potable water uses, and willingness to adopt dual systems in homes. Three Emahlaleni settlements were surveyed: Extension 14 – 14 respondents Ackerville – 28 respondents Lynville – 26 respondents Institutions 17 Institutional consumers of treated effluent at the Cities of Cape Town (Western Cape) and Lephalale (Limpopo) include petroleum, pulp and paper, textile, con­ struction, mining and irrigation (public landscapes, sports fields, school fields, crop) based organisations. Decision- makers DWAF officials 2 DWAF officials involved with non-potable water use and reticulation in South Africa and based in Pretoria Treated effluent service providers 1 Service providers of treated effluent at the CoCT Potable water service providers 8 Service providers of potable water in Johannesburg, Klerksdorp, Cannon Rocks and Cape Town sustainability of water supply and services, and political accept­ ability. Po et al. (2003) recommends, in addition to the objec­ tives above, some factors that may influence the acceptance of a water reuse project, i.e. the disgust or ‘yuck’ factor; percep­ tions of risk associated with using recycled water; the specific uses of recycled water; the sources of water to be recycled; the issue of choice; trust in the service provider; knowledge of water reuse; attitudes towards the environment; environmental justice issues; the cost of recycled water; and socio-demographics. An in-depth analyses of these issues was expected to not only pro­ vide insights into potential consumers’ and decision-makers’ perceptions concerning non-potable water use, but also provide useful information for decision-making. primarily due to the high costs of installing long-distance distribution pipelines and the recurrent costs of supplying potential consumers with treated effluent. Dual water reticulation systems for industrial non- potable use For domestic respondents, although initial willingness to use treated mine effluent was low at 36%, the supply of treated mine effluent at tariff lower than the potable water tariff significantly influenced respondents’ willingness (71%) to embrace treated mine effluent reuse (Fig. 2). This is further proven by respondents’ response (a decrease in willingness from 71% to 15%) to using the treated mine effluent if the tariff was higher than the potable water tariff. Therefore, for dual systems to be widely accepted in South Africa, non-po­ table tariffs must be significantly lower than potable water tariffs. The questionnaires, which may be accessed from Ilemobade et al. (2008), were developed using the following key issues (which summarise the DWAF (2004b) objectives and Po et al. (2003) factors): i Figure 2 also shows that colour coding and proper labelling of non-potable pipes played a significant role in encouraging domestic respondents to accept dual systems. l Figure 2 also shows that colour coding and proper labelling of non-potable pipes played a significant role in encouraging domestic respondents to accept dual systems. l domestic respondents to accept dual systems. • An important factor that has impacted on treated efflu­ ent reuse amongst institutional respondents is the effluent quality supplied them from the participating STWs. Effluent quality is largely influenced by influent quality and • An important factor that has impacted on treated efflu­ ent reuse amongst institutional respondents is the effluent quality supplied them from the participating STWs. Effluent quality is largely influenced by influent quality and • Economic efficiency • Technical feasibility • Social acceptance • Organisational capacity • Availability of appropriate legislation/regulations and guide­ lines 56% 40% 50% 60% 19% 13% 6% 6% 0% 10% 20% 30% < 500m 500m - 1000m 1000m - 2000m 2000m - 5000m > 5000m Figure 1 Distance of treated effluent from institutional consumers • Public health and safety and public education. The questionnaires were then administered to some technical and non-technical water supply decision-makers and consum­ ers (Table 1). The data generated and analysed was then used to develop the decision-making framework. The development of the framework is discussed in a later section. Economic efficiency and technical feasibility Figure 2 Non-potable water tariff and interest in installing a household dual system Legend i Recycled water is appropriate for non-potable uses if treated appropriately ii Willing to use treated effluent for toilet flushing iii Willing to use treated effluent for car washing iv Willing to use treated effluent for landscape irrigation v Willing to use treated effluent for laundry vi Willing to use treated effluent for vegetable/crop/fruit irrigation vii Willing to use treated effluent for manufacturing viii Willing to use treated effluent for refinery processes ix Willing to use treated effluent for dust suspension Figure 2 Non-potable water tariff and interest in installing a household dual system Figure 3 Consumers’ preferences for reused water Figure 3 Consumers’ preferences for reused water 94% 64% 48% 60% 70% 80% 90% 100% 26% 48% 43% 6% 13% 0% 10% 20% 30% 40% 50% i ii iii iv v vii vi treatment works efficiency. Due to highly toxic influents (especially from industrial sewage) and sub-optimal STWs efficiencies, many of the participating STWs regularly fail to produce treated effluent of the prescribed quality. For this reason, all institutional consumers (excluding those using treated effluent for irrigation) undertook further on-site treatment of the effluent before reuse. • The use of recycled water for applications that may involve human contact or ingestion generally attracts opposition from potential users (Po et al., 2003; Dimitriadis, 2005). Hence, amongst domestic respondents, the most widely accepted options for non-potable water use were those requiring minimal human contact i.e. toilet flushing, car washing and landscape irrigation (Fig. 3). For non-domestic non-potable uses, decision-makers indicated their highest preferences for landscape and crop irrigation. Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Economic efficiency and technical feasibility • A significant percentage (56%) of institutions consuming treated effluent in the CoCT are located within a radius of 500 m from the STWs (Fig. 1). For distances greater than 500 m, fewer institutions use treated effluent. This is g Distance of treated effluent from institutional consumers g Distance of treated effluent from institutional consumers 219 Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) 71% 50% 63% 50% 60% 70% 80% 36% 15% 0% 10% 20% 30% 40% i ii iii iv v Legend i Initial willingness to use treated mine effluent ii Willingness to use treated mine effluent if the tariff is less than the potable water tariff iii Willingness to use treated mine effluent if the tariff is more than the potable water tariff iv Interested in installing a dual system in your house? v Interested in a dual system in your house if it is colour-coded and properly labelled? Economic efficiency and technical feasibility Figure 2 Non-potable water tariff and interest in installing a household dual system 75% 69% 65% 54% 40% 60% 60% 40% 50% 60% 70% 80% Consumers Decision-makers 40% 29% 40% 10% 10% 0% 10% 20% 30% 40% i ii iii iv v vi vii viii ix Legend i Recycled water is appropriate for non-potable uses if treated appropriately ii Willing to use treated effluent for toilet flushing iii Willing to use treated effluent for car washing iv Willing to use treated effluent for landscape irrigation v Willing to use treated effluent for laundry vi Willing to use treated effluent for vegetable/crop/fruit irrigation vii Willing to use treated effluent for manufacturing viii Willing to use treated effluent for refinery processes ix Willing to use treated effluent for dust suspension Figure 3 Consumers’ preferences for reused water 94% 64% 48% 60% 70% 80% 90% 100% 26% 48% 43% 6% 13% 0% 10% 20% 30% 40% 50% i ii iii iv v vii vi Legend i I will recommend recycled water reuse during a drought ii Recycled water is disgusting iii Not willing to use recycled water iv I trust the municipality to supply the appropriate quality v Not willing to use recycled water even if quality assured by the local authority vi I know of disease outbreaks due to recycled water reuse (CoCT respondents only) vii Risks are high when using recycled water (CoCT respondents only) Figure 4 Social acceptance of non-potable water use 75% 69% 65% 54% 40% 60% 60% 40% 50% 60% 70% 80% Consumers Decision-makers 40% 29% 40% 10% 10% 0% 10% 20% 30% 40% i ii iii iv v vi vii viii ix 71% 50% 63% 50% 60% 70% 80% 36% 15% 0% 10% 20% 30% 40% i ii iii iv v Legend i Initial willingness to use treated mine effluent ii Willingness to use treated mine effluent if the tariff is less than the potable water tariff iii Willingness to use treated mine effluent if the tariff is more than the potable water tariff iv Interested in installing a dual system in your house? v Interested in a dual system in your house if it is colour-coded and properly labelled? Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Social acceptance Public education 65% 47% 100% 71% 65% 50% 90% 73% 82% 55% 60% 70% 80% 90% 100% Consumers Decision-makers 47% 31% 45% 0% 10% 20% 30% 40% 50% i ii iii iv vii vii Legend i Consumers have the right to know that the fruits and vegetables they are buying are irrigated with recycled wastewater ii Water is a valuable resource that should be recycled iii Non-potable water use can assist many drought- prone settlements iv Non-potable water use reduces depletion of groundwater and surface water resources v Non-potable water use reduces the quantity of wastewater dis­ charged to the environment vi Considerable fertiliser savings result on farms irrigated with treated effluent Figure 5 Public health and safety concerns and environmental responsibility 48% 31% 21% 20% 25% 30% 35% 40% 45% 50% 0% 0% 5% 10% 15% 20% To conserve potable water and control the effects of water shortages To save money To irrigate and improve soil productivity To return streams to natural conditions Figure 6 Reasons why institutional consumers were using treated effluent 65% 47% 100% 71% 65% 50% 90% 73% 82% 55% 60% 70% 80% 90% 100% Consumers Decision-makers 47% 31% 45% 0% 10% 20% 30% 40% 50% i ii iii iv vii vii L d 65% 47% 100% 71% 65% 50% 90% 73% 82% 55% 60% 70% 80% 90% 100% Consumers Decision-makers 47% 31% 45% 0% 10% 20% 30% 40% 50% i ii iii iv vii vii Legend i Consumers have the right to know that the fruits and vegetables they are buying are irrigated with recycled wastewater ii Water is a valuable resource that should be recycled iii Non-potable water use can assist many drought- prone settlements iv Non-potable water use reduces depletion of groundwater and surface water resources v Non-potable water use reduces the quantity of wastewater dis­ charged to the environment vi Considerable fertiliser savings result on farms irrigated with treated effluent Figure 5 Public health and safety concerns and environmental responsibility 48% 31% 21% 20% 25% 30% 35% 40% 45% 50% 0% 0% 5% 10% 15% 20% To conserve potable water and control the effects of water shortages To save money To irrigate and improve soil productivity To return streams to natural conditions Figure 6 Reasons why institutional consumers were using treated effluent however a decrease in the percentage of consumers unwill­ ing to use the effluent (from 64% to 43%) if the quality is assured by the local authority. Legislation/regulations and guidelines Below are three of the regulatory clauses that briefly and broadly address grey-water and treated effluent quality and reuse in South Africa. In these documents, there is no objection to the reuse of grey water or treated effluent provided it is permitted and monitored by the relevant water services authority. Because of the brevity of these clauses, they do not address dual water reticulation systems and therefore, there are no national guide­ line documents on the implementation of dual systems. g Reasons why institutional consumers were using treated effluent • Government Gazette No. 9225, Regulation 991: Requirements for the purification of wastewater or effluent (EAF, 1984) The DNHPD (1978) guideline is currently more than 30 yrs old and promotes the concept of ‘No potential risk’ when using treated effluent. As a result, it involves high technology and is therefore a high-cost guideline. This guideline may therefore be largely inappropriate for low- to middle-income South African settlements with potential to use non-potable water. • The latest revision of the Water Services Act of 1997 relating to grey-water and treated effluent (DWAF, 2001) • The latest revision of the National Water Act of 1998, 37(1) (DWAF, 2004a) relating to irrigation of any land with waste or water containing waste generated through any industrial activity or by a water works. Public health and safety • One institutional respondent in the CoCT (representing 6% of the respondents from the CoCT) indicated that they know of disease outbreaks due to treated effluent reuse (Fig. 4). This has therefore resulted in 13% of these respondents considering the potential risks to be low. For any dual sys­ tem to gain public confidence and acceptance, the risk of disease must be minimal. In the CoCT, the low incidence of accidental consumption and disease may be attributed to the fact that the use of treated effluent has been restricted to non-domestic purposes with very low potential for human contact. It is likely that perceptions of potential risks will change if treated effluent is considered for domestic use which has higher potential for human contact (Friedler et al., 2006). ) • Domestic respondents expressed concerns about the safety of children when exposed to non-potable water. As a result, acceptance of a dual system may increase if child and gen­ eral safety are assured. • Perceptions of risk related to the consumption of fruits and vegetables irrigated with non-potable water were significant (above 50%) (Fig. 5). This confirms the discussion presented on Figs. 3 and 4 – that the most widely accepted options for non-potable water will be those requiring minimal human contact. Figure 5 Public health and safety concerns and environmental responsibility 48% 31% 21% 20% 25% 30% 35% 40% 45% 50% 0% 0% 5% 10% 15% 20% To conserve potable water and control the effects of water shortages To save money To irrigate and improve soil productivity To return streams to natural conditions Figure 6 Reasons why institutional consumers were using treated effluent • In general, all respondents showed high degrees of responsi­ bility and concern for environmental protection and preser­ vation (Figs. 5 and 6) through non-potable use. This there­ fore reiterates that respondents were generally disposed to use non-potable water under specific circumstances. Public education There are, however, some detailed guideline documents on non-potable water use. Specifically, The South African Guide for the Permissible Utilisation and Disposal of Treated Effluent (DNHPD, 1978) and The South African Water Quality Guide­ lines (DWAF, 1996). The DWAF (1996) guidelines recommend the different water quality parameters required for various industrial, agricultural and aquatic eco-system water require­ ments irrespective of the water source, while the DNHPD (1978) guideline is specific to the use and disposal of treated effluent. Empowering communities through involvement, interaction and education is a valuable step in facilitating that community’s acceptance of a dual system. Social acceptance As a result, it involves high technology and is therefore a high-cost guideline. This guideline may therefore be largely inappropriate for low- to middle-income South African settlements with potential to use non-potable water. Social acceptance • If a period of water shortage were to be experienced, 94% of consumers would consider water reuse (Fig. 4). However, about a third (26%) of consumers thought recycling water was disgusting. A disgust reaction is likely to be generated from people’s perceived ‘dirtiness’ of the water and their fear of contagious diseases from using the water. Despite the high percentage of respondents recommending recy­ cled water use and the low percentage of disgust, 64% of consumers indicated that they were not willing to use the recycled water. This may be because the consumers saw the logic in reuse, but when confronted about their willingness, immediately felt that they could not use the water. It is there­ fore of utmost importance that decision-makers give priority to this issue. Neglect may result in a failed reuse project. • If a period of water shortage were to be experienced, 94% of consumers would consider water reuse (Fig. 4). However, about a third (26%) of consumers thought recycling water was disgusting. A disgust reaction is likely to be generated from people’s perceived ‘dirtiness’ of the water and their fear of contagious diseases from using the water. Despite the high percentage of respondents recommending recy­ cled water use and the low percentage of disgust, 64% of consumers indicated that they were not willing to use the recycled water. This may be because the consumers saw the logic in reuse, but when confronted about their willingness, immediately felt that they could not use the water. It is there­ fore of utmost importance that decision-makers give priority to this issue. Neglect may result in a failed reuse project. influenced by incidences of illnesses and death directly attributable to poor potable water qualities in settlements such as Delmas (Mail and Guardian, 2007) and the Ukha­ hlamba District Municipality (News24.com, 2008). There is to this issue. Neglect may result in a failed reuse project. • Consumers’ trust in the Water Service Provider to supply the appropriate quality of recycled water was 48% (Fig. 4). This response is poor and unfortunate and may likely be 220 Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) The DNHPD (1978) guideline is currently more than 30 yrs old and promotes the concept of ‘No potential risk’ when using treated effluent. Methodology for development of the framework A holistic decision-making framework should ideally incor­ porate the various aspects of the triple bottom line (TBL) of 221 TABLE 2 Ranking of key issues when planning a dual water reticulation system* Key issues Consumers’ ranking Decision-mak­ ers’ ranking Overall ranking Overall weight Public health and safety 1 2 1 1.00 Economics 2 3 2 1.16 Technical / Engineering 5 1 3 2.09 Legislation 3 5 4 2.28 Organisational capacity 4 6 5 2.44 Social acceptance 7 4 6 2.84 Public education 6 7 7 2.85 *1 represents most important while 7 is least important TABLE 2 *1 represents most important while 7 is least important A schematic flow chart of the assessment process using the framework within the context of other potential water supply and/or demand options is shown in Fig. 7. sustainability, i.e. technical and economic; social, institutional and legal; and environmental and public health and safety (Jimenez and Asano, 2008; DWAF, 2004b). Traditional decision- making tools tend to focus on quantifiable factors (especially cost), leaving out equally important, yet mostly non-quantifiable factors that may have a significant influence on the project. The analysis of quantifiable and non-quantifiable factors will assist in casting a wider net to identify important issues that may sig­ nificantly influence or impact a project. The TBL approach is employed in the evaluation of each aspect within the framework. The TBL approach provides a robust structure for evaluating alternatives and is designed to provide decision-makers with a framework to understand costs, benefits, impacts, risks, etc. of different alternatives. In this way, a more balanced view is created rather than one that relies on only quantifiable factors. It also allows decision makers to vary or weigh different items/criteria to discover those criteria that have the greatest influence on differentiating alternatives (CRD, 2007). The 7 key issues employed in the social surveys formed the backbone for the framework, with each key issue generating a list of items to be evaluated. The framework was categorised using the different aspects of the TBL of sustainability. Weights were allocated to each of the key issues based on the weighted average rank allocated by respondents when asked to rank the seven key issues in order of importance when planning a dual system (Table 2). These weights determined the level of impor­ tance given to the key issues within the framework. Methodology for development of the framework From Table 2, it is interesting to note that consumers gave higher priorities to key issues which are traditionally high on decision-makers’ priorities (i.e. public health and safety, economics, etc.). As such, issues that are very important to consumers such as social acceptance and public education were ranked the least impor­ tant. It is important to note that several reuse projects (e.g. the Dublin County Clean Water Revival Project, California) have failed in the past due to the lack of social acceptance (Po et al., 2003) and as such, decision-makers must pay adequate attention to social acceptance and public education especially for a reuse project. The TBL approach utilises the following: goals to be achieved; criteria which determine whether the goals are achieved; evaluation questions/statements by which each criterion is measured; and a range of scores for measuring each evaluation question/statement. Any number of goals and criteria can be selected. In developing the goals and crite­ ria, a number of important rules, which facilitate an objective approach to achieving the goals for each aspect of the TBL, were followed, i.e. each goal and its criteria must be independent; non-duplicative; measurable; and exhaustive. Tables 3, 4 and 5 show the framework developed for assess­ ing the viability of implementing dual systems in South Africa based on the TBL approach and the data generated using the questionnaires, on-site visits, literature and consultations. The framework was employed to practically assess the viabil­ ity of implementing a dual water reticulation system within the Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Assessment of the different aspects of the Triple Bottom Line Yes No Assessing the viability of implementing a dual water reticulation system Social, Institutional and Legal assessment multiplied by weight Environmental, and Public Health and Safety assessment multiplied by weight Technical and Economic assessment multiplied by weight Aggregation of weighted mean of real score from each Bottom Line assessment Infeasible Implement most appropriate option Reassess TBL? No Comparison with other water supply/demand option Feasible? Yes Figure 7 Schematic flow chart for assessing the viability of implementing dual systems Technical and Economic assessment multiplied by weight Figure 7 Schematic flow chart for assessing the viability of implementing dual systems Figure 7 Schematic flow chart for assessing the viability of implementing dual systems Feasible? Reassess TBL? Methodology for development of the framework Comparison with other water supply/demand option No Yes Implement most appropriate option Infeasible 222 TABLE 3 Framework for assessing the technical and economic aspect of the triple bottom line Goal Criteria Evaluation question / statement Score Weight REAL SCORE = Score x Weight 1 2 3 Technical feasibility Increase in total supply Percentage increase in total supply due to non-potable water use (Figure 8 and associated calculations) Significant (> 50%) Moderate (20-50%) Insignificant (<20%) x 2.09 = Potential supply to current demand Ratio of potential non-potable supply to current demand for non-potable water supply Significant (>2) Moderate (1-2) Insignificant (<1) x 2.09 = Distance Average distance between potential supply and demand Insignificant < 0.5 km Moderate 0.5-1.0 km Significant > 1.0 km x 2.09 = Non-potable water use Potential for human contact with the non-potable water Insignificant Moderate Significant x 2.09 = Treatment technology Treatment technology readily available? Locally available Nationally available Must be imported x 2.09 = Retro-fit system Ease to retro-fit a dual system? Significant Moderate Insignificant x 2.09 = Supply reliability Reliability of non-potable water supply (51 weeks a year / 98% of the time)? Significant Moderate Insignificant x 2.09 = Treatment quality reliability Treatment technology meets effluent quality requirements under expected operating conditions? Significant Moderate Insignificant x 2.09 = Operation & Maintenance Level of skill required to operate and maintain the dual system Low Moderate High x 2.09 = Utilise existing infrastructure Potential to utilise existing infrastructure (e.g. a STW)? Significant Moderate Insignificant x 2.09 = Upgradeability Extent dual system can be readily expanded to supply future flows? Significant Moderate Insignificant x 2.09 = Technical sustainability Long-term applicability Period of impact of the system? (short to long term) Significant > 10 yrs Moderate 3-10 yrs Insignificant < 3 years x 2.09 = Flexibility Technology can be adapted to meet more stringent effluent standards in the future? Methodology for development of the framework Significant Moderate Insignificant x 2.09 = Future supply to current demand Ratio of future non-potable supply to future demand for non-potable water supply Significant (>2) Moderate (1-2) Insignificant (<1) x 2.09 = Economical feasibility Cost difference Difference in the overall cost of supplying potable and non-potable water Significant Moderate Insignificant x 1.16 = Savings Extent of cost savings for non-potable use Significant Moderate Insignificant x 1.16 = Financial help Financial assistance/incentives for non-potable use Significant Moderate Insignificant x 1.16 = Job creation Potential for job creation Significant Moderate Insignificant x 1.16 = Weighted mean of real scores (ΣReal Score/ΣNumber of items) (Range: 1.9 – 5.7) 223 Urban water system R R L S F R where F = Potable water supply R = Recycled water supply L = Losses (e.g. leakage and evaporation) S = Effluent discharge Figure 8 A schematic mass balance of an urban water system incorporating reuse (Grobicki and Cohen, 1999) Figure 8 A schematic mass balance of an urban water system incorporating reuse (Grobicki and Cohen, 1999) L where F = Potable water supply R = Recycled water supply L = Losses (e.g. leakage and evaporation) S = Effluent discharge where Gold Fields Gold Mine in Driefontein (Gauteng). Detailed results and discussion of the assessment are presented in Ilemobade et al. (2008). In the exercise, the framework facilitated a holistic assessment of the different criteria that are necessary to be con­ sidered prior to the implementation of a dual system. Summing the different values and rearranging, Eq. (2) becomes: Summing the different values and rearranging, Eq. (2) becomes: L = 616.93 (i.e. 48.94% of F + R) (3) L = 616.93 (i.e. 48.94% of F + R) (3) With L = 49% of F + R and assuming that all effluent is recycled (i.e. effluent discharge, S = 0), Eq. (1) becomes (3) (3) With L = 49% of F + R and assuming that all effluent is recycled (i.e. effluent discharge, S = 0), Eq. (1) becomes With L = 49% of F + R and assuming that all effluent is recycled (i.e. effluent discharge, S = 0), Eq. (1) becomes Percentage increase in total supply due to non-potable water use R = 1.04 F (4) R = 1.04 F (4) The first evaluation statement in Table 3 requires an assess­ ment of the percentage increase in total supply due to non- potable water use. This section provides a guide to calculating this percentage. By substituting Eq. (4) into the left-hand side of Eq. (1), Eq. (1) becomes By substituting Eq. (4) into the left-hand side of Eq. (1), Eq. (1) becomes 2.04F = L + R (5) (5) 2.04F = L + R Grobicki and Cohen (1999) proposed an urban water-demand model for water reuse potential in South Africa (Fig. 8). Equation (5) implies that with Losses, L equal to 49% of total supply and all effluent recycled (i.e. effluent discharge, S = 0), the percentage increase in total supply due to non-potable water use would be 104%. A water balance equation for the urban water system repre­ sented in Fig. 8 is: F + R = L + S + R (1) (1) F + R = L + S + R Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Conclusion Prior to calculating the percentage increase in total supply due to non-potable water use, the quantities of each of the vari­ ables in Eq. (1) must be determined for the water system being considered. For several reasons including the dearth of relevant regulatory and guideline documents, consumer and decision-maker percep­ tions, ignorance, and the lack of appropriate decision-making tools, the prevalence of dual water reticulation systems has been limited in South Africa. Considering the wealth of international experience and the significant potential for implementing dual systems in South Africa, the aim of this study was to develop a In the CoCT for example (CoCT, 2007a), F = 1180 Mℓ/d, S = 563.07 Mℓ/d and R = 80.50 Mℓ/d. For the CoCT therefore, Eq. (1) becomes: 1180 + 80.50 = L + 563.07 + 80.50 (2) (2) TABLE 4 Framework for assessing the social, institutional and legislative aspect of the triple bottom line Goal Criteria Evaluation question / statement Score Weight Real score = Score x weight 1 2 3 Social feasibility Disgust Extent of ‘disgust’ to non-potable water use Insignificant Moderate Significant x 2.84 = Acceptance** Acceptance of the dual system by the community Significant Moderate Insignifi­ cant x 2.84 = Aesthetics Unpleasant sight, noise and/or odour emissions from the system Insignificant Moderate Significant x 2.84 = Trust/confi­ dence in serv­ ice provider Consumers’ level of trust and confidence in the potable water service provider High Moderate Low x 2.84 = Institutional feasibility Local capacity Availability of Institutional capacity to operate the system Significant Moderate Insignifi­ cant x 2.44 = Acceptance** Acceptance of the dual system by decision makers Significant Moderate Insignifi­ cant x 2.44 = Legislative availability Legislation / Regulation Municipal Regulations/by-laws available to guide system planning and operation Significant Moderate Insignifi­ cant x 2.28 = Weighted mean of Real Scores (ΣReal Score/ΣNumber of items) (Range: 2.7 – 7.9) **A score of 1 for this evaluation statement may likely render the project infeasible TABLE 4 224 Table 5 Framework for assessing the environmental, and public health and safety aspect of the triple bottom line Goal Criteria Evaluation question / statement Score Weight Real score = Score x weight 1 2 3 Environmental feasibility Erosion and scouring Anticipated increase in erosion and scouring in the receiving water course? Insignificant Acceptable Significant x 1.00 = Flow regimes Anticipated unnatural alterations to the flow regime in the receiving water course? Conclusion In order of priority dictated by the respondents, these objectives and factors are: CRD, Capital Regional District (2007) Core Area and West Shore Sewage Treatment. Discussion Paper No. 1-4. http://www.crd. bc.ca/ (Accessed 21 March 2007). Dimitriadis S (2005) Issues encountered in advancing Australia’s water recycling schemes. Research Brief, Parliamentary Library, Parliament of Australia, No. 2. Department of Parliamentary Serv­ ices, Australia. ISSN 18322883. Dixon A, Butler D and Fewkes A (1999) Water saving poten­ tial of domestic water reuse systems using greywater and rainwater combination. Water Sci. Technol. 39 25-32. DNHPD (DEPARTMENT OF NATIONAL HEALTH AND POPULA­ TION DEVELOPMENT) (1978) Guide: Permissible Utilization and Disposal of Treated Sewage Effluent. Report No. 11/2/5/3. Depart­ ment Of National Health And Population Development, Pretoria, South Africa. • Public health and safety (i.e. the potential for ill-health due to non-potable water use) • Economics (i.e. non-potable water tariffs in comparison to potable tariffs) DWAF (Department of Water Affairs and Forestry) (1996) South African Water Quality Guidelines (3-7. 2nd edn.). Pre­ toria, South Africa. • Technical feasibility (i.e. the aridity of the area, potential consumers distance from the non-potable water source, the quality of the raw non-potable water, proper labelling of the different components of the dual system, and the potential non-potable water uses) DWAF (Department of Water Affairs and Forestry) (2001) Regulation Gazette No. 7079. http://www.info.gov.za/gazette/ l ti /2001/22355 df (A d 29 N 2006) regulation/2001/22355.pdf (Accessed 29 Nov 2006). DWAF (Department of Water Affairs and Forestry) (2004a) Revision of General Authorisations in Terms of Section 39 of the National Water Act 1998 (Act No 36 of 1998) – Engaging in a Controlled Activity. Government Gazette No. 26187. Pretoria, South Africa. • Availability of appropriate legislation/regulations and guide­ lines • Organisational capacity • Social acceptance (i.e. willingness to use non-potable water and potential consumers’ trust in existing water services providers) DWAF (Department of Water Affairs and Forestry) (2004b) DWAF’s framework and Checklist for the Development of Water Services Development Plans. Department of Water Affairs and Forestry, Pretoria, South Africa. • Public education (i.e. empowerment of potential users). DWAF (DEPARTMENT OF WATER AFFAIRS AND FORESTRY) (2004c) National Water Resource Strategy. Department of Water Affairs and Forestry, Pretoria, South Africa. The 7 key issues above, categorised using the different aspects of the TBL of sustainability (i.e. economic, environmental and social), formed the basis for the developed framework. Conclusion Insignificant Acceptable Significant x 1.00 = Water quality Anticipated negative changes in water quality in the receiving water course? Insignificant Acceptable Significant x 1.00 = Wetlands Extent wetlands will be negatively affected and/or wetland value diminished? Insignificant Acceptable Significant x 1.00 = Habitats Extent to which habitats in the downstream water course will be disrupted? Insignificant Acceptable Significant x 1.00 = Downstream availability Anticipated decrease in downstream water availability for users due to upstream reuse? Insignificant Acceptable Unacceptable x 1.00 = Energy efficiency Application of the technology results in greenhouse gas emissions? Insignificant Acceptable Significant x 1.00 = Public health and safety Monitoring and control Monitoring and control systems in place to minimise public health hazards? Significant Acceptable Insignificant x 1.00 = Risks Health risks to O&M staff or consumers? Low Acceptable High x 1.00 = Liability Insurance cover in case of system failure? Significant Acceptable Insignificant x 1.00 = Public education Education / Awareness Current level of education/awareness about non-potable water use High Moderate Low x 2.85 = Public education System implementation enables public education opportunities to be maximised Significant Acceptable Insignificant x 2.85 = Weighted mean of Real Scores (ΣReal Score/ΣNumber of items) (Range: 1.3 - 3.9) AGGREGATION OF THE WEIGHTED MEAN OF REAL SCORES FOR THE TRIPLE BOTTOM LINE ASPECTS (RANGE: 5.9 – 17.5) ASSESSMENT RESULT BASED ON THE AGGREGATED WEIGHTED MEAN OF REAL SCORES FOR THE TBL ASPECTS 5.9 - 8.6 : Very high potential to be viable 8.6 - 11.4 : High potential to be viable 11.4 - 14.2 : Middle to low potential to be viable 14.2 - 17.5 : Unlikely to be viable 225 CoCT, The City of Cape Town (2007b) Water Services Develop­ ment Plan 2007. The City of Cape Town, South Africa. decision-making framework, using robust criteria, for assessing the viability of implementing dual systems in potential South African settlements. In achieving this aim, this study reviews literature on the subject and investigates, using questionnaires, the perceptions of some households in Emahlaleni and institu­ tions in the CoCT and Lephalale (non-potable water consumers), and water and wastewater services providers and DWAF offi­ cials (decision-makers) in several South African local authori­ ties. The questionnaires were developed using the objectives and factors recommended by DWAF (2004b) and Po et al. (2003) respectively. Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) Acknowledgements The authors appreciate financial support from the Water Research Commission (Project No. K5/1701) The authors appreciate financial support from the Water Research Commission (Project No. K5/1701) Heather L (2005) Supply and Demand for Low Energy Housing in the UK: Insights from a Science and Technology Studies Approach. Housing Stud. 20 (5) 815-829. Conclusion Each key issue was represented by a list of items which were generated dur­ ing the surveys. Weights, which indicated level of importance, were assigned to each issue and were based on the weighted average rank allocated by respondents. The TBL approach was then employed in the assessment of the different items. A scale of the aggregated weighted mean of real scores was provided to assist in the overall assessment of the viability of implementing a dual system with 5.90 representing very high potential to be viable and 17.50 representing unlikely to be viable. EBERHARD R and ROBINSON P (2003) Guidelines for the Devel­ opment of National Water Policies and Strategies to support IWRM. Draft. SADC Water Sector Co-ordination Unit, Gaborone, Botswana. EAF, Ministry of Environmental Affairs AND Fisher­ ies (1984) Requirements for the purification of waste water or efflu­ ent. Government Gazette No. 9225, Regulation 991. Pretoria, South Africa. Friedler E, Lahav E, Jizhaki H and Lahav T (2006) Study of urban population attitudes towards various wastewater reuse options: Israel as a case study. J. Environ. Manage. 81 360-370. Grobicki A and Cohen B (1999) Water Reclamation for Direct Re- Use in Urban and Industrial Applications in South Africa and its Projected Impact upon Water Demand. WRC Report No. KV 118/99. Water Research Commission, Pretoria, South Africa. References Health Stream (2003). Setback for Netherlands dual supplies. Cooperative Research Centre for Water Quality and Treatment. Issue 30. June. http://www.waterquality.crc.org.au/hsarch/HS30d. htm (Accessed 13 Oct 2006). Al-Jayyousi OR (2004) Greywater reuse: knowledge management for sustainability. Desalination 167 27-37. Ilemobade AA, Adewumi JR and Van Zyl JE (2008) Assess­ ment of the Feasibility of using a Dual Water Reticulation System in South Africa. WRC Report No. K5/1701. Water Research Commis­ sion, Pretoria, South Africa. Basson MS, Van Niekerk PH and Van Rooyen JA (1997) Overview of Water Resources Availability and Utilisation in South Africa. Department of Water Affairs and Forestry, DWAF Report P RSA/00/0197. CTP Book Printers, Cape Town, South Africa. IRWD, Irvine Ranch Water District (2006) Water Reclama­ tion. http://www.irwd.com/Reclamation/index.php (Accessed 22 Nov. 2006). Botha J and Pretorius WA (1998) Die Uitvoerbaarheid van Dubbel­watervoorsieningstelsels. WRC Report No. KV 113/98. Water Research Commission, Pretoria, South Africa. JIMENEZ B and ASANO T (2008) Water Reuse. An International Sur­ vey of Current Practice, Issues and Needs. Technical report No. 20. International Water Association (IWA). ISBN 1843390892. Business and Economy (2003) Quenching a Global Thirst. Japa­ nese Researchers Help to Combat Water Shortages. June 26. http:// web-japan.org/trends/business/bus030626.html (Accessed 24 Feb 2006).l Junying C, Jining C, Can W and Ping F (2004) Wastewater reuse potential analysis: implications for China water resources manage­ ment. Water Res. 38 2746-2756. CoCT, The City of Cape Town (2007a) Treated Effluent Re-Use Strategy and Master Planning within the City of Cape Town. BVi Report No. C1500/1.1. The City of Cape Town, South Africa. 226 Mail and GUARDIAN (M & G) (2007) Over 500 Suffering from Diarrhoea in Delmas. Nov 9. http://www.mg.co.za/article/2007-11 -09-over-500-suffering-from-diarrhoea-in-delmas (Accessed 12 September 2008). Parr J, Smith MD and Stear RM (1997) Decreasing freshwater demand: dual supply systems. Proc. of the 23rd WEDC Conference. September 1997, Durban, South Africa. 404-406. Po M, Kaercher JD and Nancarrow BE (2003) Literature Review of Factors Influencing Public Perceptions of Water Reuse. Technical Report 54/03. CSIRO Land and Water. March JG, Gual M and Orozco F (2004) Experience on grey­ water reuse for toilet flushing in a hotel. Desalination 164 241-247. Po M, Nancarrow BE, Leviston Z, Porter NB, Syme GJ and Kaercher JD (2005) Predicting Community Behaviour in Relation to Wastewater Reuse: What Drives Decisions to Accept or Reject? Water for a Healthy Country National Research Flagship. CSIRO Land and Water. Perth, Australia. Available on website http://www.wrc.org.za ISSN 0378-4738 = Water SA Vol. 35 No. 2 (Special WISA 2008 edition) 2009 ISSN 1816-7950 = Water SA (on-line) References Sustainability Institute (2006) L d h E ill h // MUKHEIBIR P (2005) Local water resource management strategies for adaptation to climate induced impacts in South Africa. Proc. Work­ shop on Rural Development and the Role of Food, Water and Bio­ mass: Opportunities for Development and Climate. 14-16 November 2005, Dakar, Senegal. Mvula Trust (2006) Prioritising Planning: The Importance of Water Services Development Plans and Integrated Plans. http:// www.mvula.co.za/page/467 (Assessed 19 March 2007). Sustainability Institute (2006) Lynedoch Ecovillage. http:// www.sustainabilityinstitute.net/index.php. Accessed 13 August 2006. Tang SL, Derek PTY and Damien CCK (2007) Engineering and Costs of Dual Water Supply Systems. IWA, UK. ISBN 1843391325. NEWS24.COM (2008) Unsafe water kills 80 kids. 22 April. http://www. news24.com/news24/south_africa/news/0,,2-7-1442_2310451,00. html (Accessed 12 September 2008).l Van der Merwe B (2006) Water reuse in Windhoek through a dual pipe system and artificial recharge of the aquifer. Proc. Int. Water Reuse Workshop organized by the Water and Wastewater Agency. 17-19 May, Tijuana, Baja California, Mexico. Nolde E (1999) Greywater reuse systems for toilet flushing in multi- storey buildings – over ten years experience in Berlin. Urban Water 1 275-284. 17-19 May, Tijuana, Baja California, Mexico. Okun DA (1996) Distributing reclaimed water through dual systems. J. Am. Water Works Assoc. 89 52-64. Webster E (2006) Life on Hull Street: sorting out sanitation. Dry sanitation and grey water. WASE (Water Sewage and Effluent) Africa 26 (2) 18-23. 227

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Pareja con pareja. Elección del test estadístico.

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ReaR REV ELECT ANESTESIAR- VOL 11 (12) :4 ISNN 1989 4090 Revista electrónica de AnestesiaR Diciembre 2019 FORMACIÓN MÉDICA Pareja con pareja. Elección del test estadístico. Molina Arias M. Hospital Infantil Universitario La Paz. Resumen El contraste de hipótesis requiere de la correcta elección del test estadístico para cada situación. Esta elección dependerá del tipo de variable dependiente e independiente a comparar, de que se trate de muestras independientes o apareadas y de la necesidad de utilizar pruebas paramétricas o no paramétricas. Introducción Todos conoceréis el caso de alguien que, tras realizar un estudio y recoger varios millones de variables, se ha dirigido al estadístico de su centro de trabajo y, demostrando de forma fehaciente su claridad de ideas respecto a su trabajo, le ha dicho: por favor (hay que ser educados), crúzalo todo con todo, a ver qué sale. El contraste de hipótesis requiere de la correcta elección del test estadístico para cada situación. Esta elección dependerá del tipo de variable dependiente e independiente a comparar, de que se trate de muestras independientes o apareadas y de la necesidad de utilizar pruebas paramétricas o no paramétricas. Llegados a este punto te pueden ocurrir varias cosas. Si el estadístico es un desalmado sin escrúpulos te dirigirá una media sonrisa y te dirá que vuelvas al cabo de unos días. Entonces te dará varios centenares de hojas con gráficos, tablas y números que no sabrás por dónde coger. Otra cosa que te puede ocurrir es que te mande a paseo, cansado como estará de que le hagan peticiones semejantes. Los estudios ecológicos son estudios observacionales que tienen la peculiaridad de que la población de estudio no son sujetos individuales, sino sujetos agrupados, por lo que el nivel de inferencia de sus estimaciones es también agregado. Para conocer el valor de sus conclusiones será importante estudiar su validez interna, la importancia clínica de sus conclusiones y si son aplicables y de utilidad en nuestro entorno clínico. Pero puedes tener suerte y encontrar un estadístico competente y paciente que, de forma abnegada, te explicará que la cosa no debe funcionar así. Lo lógico es que tú, antes de recoger ningún dato, hayas elaborado una memoria del proyecto en la que esté previsto, entre otras cosas, qué hay que analizar y qué variables hay que cruzar entre sí. Incluso, te puede sugerir que, si el Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro. análisis no es muy complicado, intentes hacerlo tú mismo. Esto último te puede parecer el desvarío de una mente trastornada por las matemáticas pero, si lo piensas un momento, no es tan mala idea. Si nosotros hacemos el análisis, al menos el preliminar, de nuestros resultados, nos puede ayudar a entender mejor el estudio. Además, ¿quién mejor que nosotros mismos puede saber lo que queremos? Con los paquetes estadísticos actuales, la estadística bivariante más sencilla puede estar a nuestro alcance. Únicamente tenemos que tener buen cuidado en saber elegir el test de contraste de hipótesis adecuado, para lo cual habremos de tener en cuenta tres aspectos: el tipo de variables que queremos comparar, si los datos son apareados o independientes y si tenemos que utilizar test paramétricos o no paramétricos. Veamos estos tres aspectos. En cuanto al tipo de variables, existen múltiples denominaciones según la clasificación o el paquete estadístico que utilicemos pero, simplificando, diremos que hay tres tipos de variables. En primer lugar, están las continuas o de escala. Como su nombre indica, recogen el valor de una variable continua como puede ser el peso, la talla, la glucemia, etc. En segundo lugar, están las variables nominales, que constan de dos o más categorías que son mutuamente excluyentes. Por ejemplo, la variable color de pelo puede tener las categorías “moreno”, “rubio” y “pelirrojo”. Cuando estas variables tienen dos categorías, las llamamos dicotómicas (sí/no, vivo/muerto, etc.). Por último, cuando las categorías están ordenadas por rango, hablamos de variables ordinales: “no fuma”, “fuma poco”, “fuma moderadamente”, “fuma mucho”. Aunque a veces puedan usar números, estos indican la posición de las categorías dentro de la serie, sin implicar, por ejemplo, que la distancia de la categoría 1 a la 2 sea la misma que la de la 2 a la 3. Por ejemplo, podemos clasificar el reflujo vesicoureteral en grados I, II, III y IV (tener un grado IV es más que un II, pero no significa que se tenga el doble de reflujo). Saber qué tipo de variable tenemos entre manos es sencillo. Si tenemos duda, podemos seguir el siguiente razonamiento basado en la respuesta a dos preguntas: 1. ¿Tiene la variable valores teóricos infinitos? Aquí hay que abstraerse un poco y fijarse en los de “valores teóricos”. Por ejemplo, si recogemos el peso de nuestros participantes, los valores teóricos serán infinitos aunque, en la práctica, esto estará limitado por la precisión de nuestra báscula. Si la respuesta es sí estaremos antes una variable continua o de escala. Si es no, pasamos a la siguiente pregunta. 2. ¿Los valores están ordenados en algún tipo de rango? Si la respuesta es sí, nos encontraremos ante una variable ordinal. Si la respuesta es no, tendremos una variable nominal. El segundo aspecto es el de las medidas apareadas o independientes. Dos medidas están apareadas cuando se mide una variable en dos ocasiones tras haber aplicado algún cambio, habitualmente en el mismo sujeto. Por ejemplo: presión arterial antes y después de un test de esfuerzo, peso antes y después de una intervención nutricional, etc. Por su parte, las medidas independientes son aquellas que no tienen relación entre sí (son variables diferentes): peso, talla, género, edad, etc. Por último, hemos mencionado lo de poder utilizar test paramétricos o no Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro. REV ELECT ANESTESIAR- VOL 11 (12) :4 paramétricos. No vamos a entrar ahora en detalle, pero para poder utilizar un test paramétrico la variable debe cumplir una serie de características, como seguir una distribución normal, tener un determinado tamaño muestral, etc. Además, hay técnicas que son más exigentes que otras a la hora de tener que cumplir estas condiciones. Ante la duda, es preferible utilizar técnicas no paramétricas sin necesidad (el único problema es que es más difícil conseguir significación estadística, pero el contraste es igual de válido) que usar una prueba paramétrica cuando no se cumplan los requisitos necesarios. Una vez que ya hemos dado respuesta a estos tres aspectos, solo nos queda hacer las parejas de variables que vamos a comparar y elegir el test estadístico apropiado. Lo podéis ver resumido en la tabla adjunta. En las filas está representado el tipo de variable independiente, que es aquella cuyo valor no depende de otra variable (suele estar en el eje x de las representaciones gráficas) y que suele ser la que modificamos en el estudio para ver el efecto sobre otra variable (la dependiente). En las columnas, por su parte, tenemos la variable dependiente, que es aquella cuyo valor se modifica con los cambios de la variable independiente. De todas formas, no os lieis: el programa estadístico hará el contraste de hipótesis sin tener en cuenta cuál es la dependiente y cuál la independiente, solo tendrá en cuenta los tipos de variables. La tabla se explica sola, así que no le vamos a dar muchas vueltas. Por ejemplo, si hemos medido la presión arterial (variable de escala) y queremos saber si hay diferencias entre hombres y mujeres (género, variable nominal dicotómica), el test adecuado será el de la t de Student para muestras independientes. Si quisiéramos ver si hay diferencia en la presión antes y después de un tratamiento, utilizaríamos el mismo test de la t de Student pero para muestras apareadas. Otro ejemplo: si queremos saber si hay diferencias significativas en el color de pelo (nominal politómica: “rubio”, “moreno” y “pelirrojo) y si el participante es del norte o sur de Europa (nominal dicotómica), podríamos emplear un test de la Ji-cuadrado. Y aquí lo vamos a dejar por hoy. No hemos hablado nada de las peculiaridades de cada test que debemos tener en cuenta, sino que solo hemos mencionado el test en sí. Por ejemplo, la ji-cuadrado tiene que cumplir unos mínimos en cada casilla de la tabla de contingencia, en el caso de la t de Student debemos considerar si las varianzas son iguales (homocedasticidad) o no, etc. Pero esa es otra historia… Bibliografía – Ochoa Sangrador C. Contraste de hipótesis. Elección del test estadístico. En: Ochoa Sangrador C, ed. Diseño y análisis en investigación. International Marketing & Communication, Madrid; 2019: 113-32. – Peró Cebollero M, Leiva Ureña D, Guàrdia Olmos J, Solanas Pérez A, eds. Estadística aplicada a las ciencias sociales mediante R y RCommander. Grupo editorial Garceta. Madrid, 2012. – Bowers D, ed. Medical statistics from scratch. An introduction for health professionals, 2nd ed. John Wiley & Sons, Ltd. West Sussex, UK, 2008. (PDF) Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro. – Martínez González MA, Sánchez Villegas A, Toledo Atucha EA, Faulin Fajardo J, eds. Bioestadística amigable, 3ª ed. Elsevier España SL. Madrid, 2014. Correspondencia al autor Manuel Molina Arias [email protected] Servicio de Gastroenterología. Hospital Infantil Universitario La Paz. Madrid. España. Aceptado para el blog en junio de 2019. Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro. 

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Review of Energy Storage Systems in Regenerative Braking Energy Recovery in DC Electrified Urban Railway Systems: Converter Topologies, Control Methods &amp; Future Prospects

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anlami Sadiq1, Muhamad Mansor1, Yong Jia Ying1, Vigna K. Ramachandaramurthy1, M.A annan1, Mohd. Azrin Mohd Azau1, Muhamad Safwan Abd Rahman1, Azri Husni bin Hasa ur A. Salim3. Danlami Sadiq1, Muhamad Mansor1, Yong Jia Ying1, Vigna K. Ramachandaramurthy1, M.A Hannan1, Mohd. Azrin Mohd Azau1, Muhamad Safwan Abd Rahman1, Azri Husni bin Hasani2, Nur A. Salim3. 1Dept of Electrical & Electronics Engineering, College of Engineering, Universiti Tenaga Nasional, Kajang 43000, Selangor, Malaysia. 2URND Sdn Bhd, Universiti Tenaga Nasional, Kajang, Malaysia. 2URND Sdn Bhd, Universiti Tenaga Nasional, Kajang, Malaysia. 3Faculty of Electrical Engineering, Universiti Teknologi MARA, Shah Alam, Shah Alam, Malaysia. Corresponding Author: Danlami Sadiq ([email protected]) Funding information: Long Term Research Grant Scheme (LRGS) under the Ministry of Education, Malaysia, Grant Number: LRGS/1/2018/UNITEN/01/1/2 Review of Energy Storage Systems in Regenerative Braking Energy Recovery in DC Electrified Urban Railway Systems: Converter Topologies, Control Methods & Future Prospects Review of Energy Storage Systems in Regenerative Braking Energy Recovery in DC Electrified Urban Railway Systems: Converter Topologies, Control Methods & Future Prospects Danlami Sadiq1, Muhamad Mansor1, Yong Jia Ying1, Vigna K. Ramachandaramurthy1, M.A Hannan1, Mohd. Azrin Mohd Azau1, Muhamad Safwan Abd Rahman1, Azri Husni bin Hasani2, Nur A. Salim3. SUBMISSION DATE / POSTED DATE SUBMISSION DATE / POSTED DATE 29-09-2021 / 30-09-2021 SADIQ, DANLAMI (2021): Review of Energy Storage Systems in Regenerative Braking Energy Recovery in DC Electrified Urban Railway Systems: Converter Topologies, Control Methods & Future Prospects. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.16699942.v1 SADIQ, DANLAMI (2021): Review of Energy Storage Systems in Regenerative Braking Energy Recovery in DC Electrified Urban Railway Systems: Converter Topologies, Control Methods & Future Prospects. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.16699942.v1 10.36227/techrxiv.16699942.v1 10.36227/techrxiv.16699942.v1 1 Introduction Electric railway system has become a major means of transporting people and goods, especially in the urban and suburban areas, and as such contributes greatly to the reduction of traffic and environmental pollution (CO2 emission in particular) [1], [2]. Therefore, the electric traction system has uncompromised advantages more especially in densely populated areas like in the urban and suburban cities where the rapid transit systems are found and where the safety, protection, high level of performances, environmental protection, and cost effectiveness of service must be assured [3]. Currently, there is a fast development of electric railway transport system because of the rapid growth of the worldwide populations. As a result, this has led to high increase of energy consumption as well as the environmental pollutions. Therefore, increasing the energy efficiency of rail transit systems is crucial in order to achieve high reduction in energy consumption and CO2 emissions, [3]–[5]. Generally, in urban railway systems, electric rail vehicle has four working conditions, those are acceleration, moving with constant speed (coasting), braking process, and stopping mode, [6]–[9]. In the acceleration process, the train draws large amount of electric energy from the railway substation via the catenary line or third rail. This is the power required to speed up the vehicle to reach the constant speed. During the coasting mode, negligible energy is consumed. Whereas in the braking mode, the vehicle continuously slows down its speed to a final stop point at a station (deceleration process). 2 Nowadays, most electric trains are equipped with dynamic system of braking which enable them to convert their kinetic energy developed during motion into electricity. This is achieved in deceleration process during which, the traction of the electric motors behave as generators, [6], [10]–[13]. The electrical energy produced is called regenerated braking energy which is fed back into the traction network (DC-link). During the braking process, the regenerated energy is directly supplied to the train’s auxiliaries onboard via the traction network for direct consumption while the excess is sent back to the catenary line or third rail for other vehicles in the same power supply network to utilize during their acceleration process. Otherwise, this surplus energy is lost in the form of heat by dissipating it into a bank of resistors located onboard of the train or at the substation (this is called rheostatic braking), [4], [10], [14]–[17]. Abstract Electrified urban railway systems are large consumers of energy in urban areas and thus, there is a need for energy saving measures in this transportation sector. Recuperation of train’s regenerative braking energy (RBE) is one of the best ways for attaining high levels of energy efficiency in this area. Energy Storage Systems (ESSs) prove to be the most practical and viable solution for maximizing the RBE utilization in urban railway systems. In the existing related reviews of ESSs, few papers covered the review of ESS technologies or converter interface. However, a comprehensive review is needed in this regard. This paper discusses an overview of urban railway electrification, and detail review for the three ESS components – ESS Technologies, Bidirectional DC-DC Converters (BDCs), and Controller Unit. This study concludes that among the storage technologies, supercapacitor ESS appears to be the most suitable followed by Lithium-ion batteries and flywheels. For BDC, cascaded BDC is the most suitable followed by dual active bridge. For control methods, fuzzy logic and artificial intelligent are recommended among other control strategies. Thus, the key contribution of this paper is the comprehensive review and analyses of the ESS’s components in the recovery of RBE in urban railways. Keywords: Energy Saving; Energy Storage Technologies; Control System; Energy Management; DC- DC Power Converters; Bidirectional Power Flow; Regenerative Braking Energy; Urban Railway Systems. 1 1 1 Introduction Note that, there is an issue with regard to feeding back the regenerated braking energy 2 to the catenary line in DC supply substation (non-inverting substation) if there are no simultaneous accelerating vehicles to consume such energy, [18]. Hence, this result into catenary line/third rail overvoltage which has the potential to damage the traction network equipment such as the electric motors’ power converters and the others [6], [10], [14]. It is for this reason that the urban railway DC distribution network is said to be non-receptive, which means not all the times the regenerated energy is fully consumed by other accelerating vehicles. Therefore, the fundamental solution being only to send the regenerated braking energy to the catenary line network on a condition that there exists another accelerating vehicles in the same power supply network to consume it, otherwise rheostatic braking is applied to waste the energy in the form of heat [6]. However, rheostatic braking method has some negative effects especially in the underground metro systems or tunnels where the excess heat produced by the onboard resistors causes excessive warmness within the surrounding whose level needs to be controlled by additional ventilation facilities (hence, additional cost involved), [10]. In order to overcome the above challenges, the regenerated braking energy needs to be maximized while at the same time the number of the onboard resistors is reduced. To achieve this, three major solutions have been studied and proposed regarding the utilization of regenerative braking energy as mostly found in the literature [5], [6], [10], [19]–[25]. The solutions are: i. Timetable Optimization ii. Reversible Substation iii. Energy Storage System The first solution being improving the DC distribution network receptivity by bringing in more loads into the system network demanding excess energy for their acceleration process while at the same time the braking processes of other trains are occurring. This solution is called Timetable Optimization as shown in Fig.1 [5], [26]–[29]. 3 3 Fig. 1. Timetable Optimization Method (Exchange of regenerative braking energy between trains), [10] Traction Substation Catenary Line Train 1 (Braking) Train 2 (Accelearting) STATION GND Regenerative braking energy flow Regenerative braking energy flow Train 1 (Braking) Train 2 (Accelearting) Fig. 1. Timetable Optimization Method (Exchange of regenerative braking energy between trains), [10] This method allows a simultaneous exchange of the regenerated braking energy among the vehicles. 1 Introduction Research studies in this field have confirmed that energy consumption reduction of up to 14% could be achieved with this method [30]. This method is regarded to be the cheapest way of utilizing regenerative braking energy compared to ESS and reversible methods. However, it requires reliable driving strategies and control systems to achieve schedule strategies and an unplanned occurrence of events for example accidents or delays. Moreover, this solution suffers difficulties in synchronizing accelerating and decelerating vehicles due to many uncertainties which occur in the system schedules. Hence in some cases, the energy is not being fully recovered and therefore wasted in the onboard resistors [6], [10]. The second solution is to improve the receptivity of the DC distribution network. This can be achieved by making the DC substation to become an inverting one i.e. to connect DC – AC inverter in parallel with the unidirectional power supply rectifier circuit so that the regenerative braking energy could be fed directly to the AC grid as shown in Fig. 2 [5], [6], [19], [31]–[37]. Fig. 2. Reversible Substation (Sending back regenerated energy to power grid) Catenary Line Braking Train STATION GND Regenerative braking energy flow Reversible Substation DC AC Reversible Substation Regenerative braking energy flow Braking Train Fig. 2. Reversible Substation (Sending back regenerated energy to power grid) 4 This method is called reversible or inverting substation. This method presents a relatively high energy efficiency compared to ESSs because of lower losses involved [6]. Moreover, minimal maintenance and space are required, and high reliability in terms of safety. However, reversible substations do not allow for vehicle’s autonomous driving (driving freely without power supply from the substations as ESSs do). This method cannot provide catenary line voltage regulation as well as reduction in power peaks and demand. They have inherent harmonics on both sides of the bidirectional inverter, [10]. With this method, the energy consumption reduction of around 11% could be achieved [38]. The third solution is the use of Energy Storage Systems (ESSs) placed onboard of the vehicle or at the substation/ trackside in order to accumulate the excess regenerated braking energy and release it later during the vehicle’s acceleration process as shown in Fig. 3, [14], [19], [39]–[46]. Fig. 3: Energy Storage System Method. 1 Introduction (a) Green line for Wayside ESS (b) Red Line for Onboard ESS Traction Substation Catenary Line Braking Train (Regenerated Energy) STATION ESS (Onboard) Trackside ESS GND Trackside ESS Accelerating Train (Absorbing Stored Energy) ESS (Onboard) Trackside ESS Trackside ESS Traction Substation Catenary Line ESS (Onboard) ESS (Onboard) Accelerating Train (Absorbing Stored Energy) Braking Train (Regenerated Energy) g ( Energy) Energy) Fig. 3: Energy Storage System Method. (a) Green line for Wayside ESS (b) Red Line for Onbo 5 With this solution, the total energy consumption required by the vehicle from the substation during the acceleration process is greatly reduced. The benefits of using energy storage systems in storing the regenerated energy are not only for total energy consumption reduction but also for providing reduction in peak power and demand, catenary-line voltage stabilization, compensation of power and energy, and enables the vehicles to have autonomous driving (called catenary-free driving) where a vehicle can continue to run freely without having to connect to the catenary line for power supply [14], [47], [48]. Autonomous driving is advantageous in urban cities where it is not possible to install the catenary lines network because of the existence of aesthetic or historic structures. With this method, as confirmed by a few studies, an energy saving of up to 35% could be achieved [49]–[54]. The most widely used energy storage devices for such applications include batteries, supercapacitors, flywheels, and hybrid energy 5 5 storage systems. It could be observed that, a more practical and viable solution to maximize the regenerative braking energy is by the use of ESS especially in the urban railway system where acceleration and braking processes of trains occur at high frequency and in excess [10]. The aim of this paper is to provide a literature review on the applications of the most widely used energy storage technologies in recovering regenerative braking energy in urban rail transit system, and to review various topologies of bidirectional DC-DC power converters, and control strategies/energy management applied to both the converters and the energy storage devices. Various applications and detail descriptions of the technologies have been reviewed as presented by research studies and industrial implementation in maximizing the utilization of the regenerative braking energy. Moreover, advantages and disadvantages of the energy storages technologies, power converter topologies and control methods have been presented and analysed for various applications. 1 Introduction The organization of the remaining part of the paper is as follows. In section 2, an overview of railway electrification system, and urban rail transit system have been presented. In section 3, detail description of energy storage technologies, advantages/disadvantages and their applications in urban railway systems are presented and analysed. The techno-economic comparative assessment/analysis and the use of Ragone plot are also presented. Moreover, the ESSs installation configurations are discussed and analysed as presented commercially by the industries and academic research efforts. In section 4, detail descriptions, uses and advantages/disadvantages of various topologies of bidirectional DC-DC power converters as applied in the recovering process of the regenerative braking energy are presented. In section 5, the descriptions and usage of control methods and energy management as applied to the ESSs and bidirectional DC-DC converters in various application types are presented. In sections 6, discussion and recommendation of the most suitable energy storage technology, dc-dc power converter and control method are presented based on the comparative analysis and assessment. In section 7, challenges and future research direction are outlined. Finally, section 8 concludes the paper. 6 6 2.1 Railway Electrification with System Integration Generally, railway electrification systems are of two types which are AC and DC electrification systems and each with its different voltage amplitudes and frequencies [55], [56]. The AC system supply voltages include 15kV at 16.7Hz and 25kV at 50Hz or 60Hz while for the DC systems supply voltages are 600V, 750V, 1500V and 3000V. Depending upon the speed profile requirements and travelling distance, AC electrification system is usually employed for long distance travel and high speed profiles electric railway vehicles [57]–[60]. One good advantage of the AC systems is that it exhibits very low power losses compared to the DC system due to the smaller value of currents that can be transmitted over a long distance [61]. In AC systems, as shown in Fig. 4, the power is mostly supplied to the vehicle via overhead catenary line and the examples of vehicles used for this application include high-speed trains and locomotives. Single Phase AC Transformer (16.7/50/60Hz) Four Quadrant Single-Phase Two-level Converter Three Phase Two-level Converter Traction Motor 15KV (16.7Hz)/25KV (50Hz/60Hz) (AC) AC Traction Substation Catenary Line Pantograph 35KV Medium Voltage Power Grid Supply Fig. 4: AC Railway Electrification System with Train System integration [62]. 15KV (16.7Hz)/25KV (50Hz/60Hz) (AC) Pantograph Fig. 4: AC Railway Electrification System with Train System integration [62]. On the other hand, DC electrification system as shown in Fig. 5 is employed for low-speed profiles and shorter distances, and for this reason it is the most preferred option in urban railway system. 7 Three Phase Two-level Converter Traction Motor 600V/750V/1500V/3000V (DC) DC Traction Substation Catenary Line Pantograph Medium Voltage Power Grid Supply 10/35KV AC Fig. 5: DC Railway Electrification System with Train System integration Medium Voltage Power Grid Supply 1 600V/750V/1500V/3000V (DC) Pantograph Fig. 5: DC Railway Electrification System with Train System integration Examples for this application are trams, light rail vehicles, and metro systems. For DC systems, the power is conveyed by either the overhead line catenary or the third rail [55]–[58]. Examples for this application are trams, light rail vehicles, and metro systems. For DC systems, the power is conveyed by either the overhead line catenary or the third rail [55]–[58]. In an urban rail transit system, the main load is the electric train with electric motors attached to the train. 2.1 Railway Electrification with System Integration The power required to drive the electric motors is supplied from the catenary line or third rail through a bidirectional power electronic converter (three phase two level converter). In both AC and DC supply systems, the power is conveyed to the traction network (DC-link) via a device called pantograph which is located on top of the train. A high-speed circuit breaker (HSCB) is connected in between the pantograph and the traction network for protection purposes [3], [6]. For DC systems, the DC supply goes straight to the traction motors via the power converter and to the train’s auxiliaries. In the case of AC supply, the power passes through a step-down AC transformer located inside the train, then through a four quadrant two level rectifier, and then to the traction network. Normally a filter consisting of an inductor and a capacitor is placed between the traction network and input supply for filtering the high frequency harmonics generated by the power converter due to its high switching frequencies [3], [59], [62]. The return (ground path) of the supply is connected by one of the rails. The electric motors produce the required torque and speed in order to provide the traction force to drive the train along the rails and being controlled by the power converter with a proper control circuit. Typical power converters applicable in DC railway traction system are the DC-AC converter (inverter) and DC- DC converters. Two types of electric motors are used to provide the required traction force and these 8 8 are the DC motors and AC induction motors (three-phase). The former is usually controlled by a resistor bank together with a DC-DC converter while the latter is by a DC-AC converter (inverter) only. The traction force produced by the electric motors is transmitted to the vehicle wheels on the traction rails via a gearbox in order to move the vehicle along the rail track at a required speed, [6], [61]–[68]. 2.2 Overview of Urban Railway Systems Worldwide, in most of the major cities especially in the developed countries, there are urban railway networks and city bus services that serve as public transportation systems. The urban rail transit network consists of suburban and regional rail networks, light rail transit systems, metro systems and trams. Nowadays, almost all the urban railway networks run wholly on electricity supply and the power is taken from various distributed traction power supply substations (TPSSs) located at specified intervals, [69]. [69]. The urban or suburban rail transport system usually spans a distance of up to 50km from the city centres and utilizes the railway line routes connecting both the urban and suburban areas. The regional railway transport system spans a distance of up to 150km from the urban city centres and utilizes the railway lines routes connecting urban and peri-urban areas. The tramway transport systems consist of a tram also known as streetcar, trolley car, tramcar or simply trolley. Tram is a rail car that moves or runs alongside public transport streets and in some countries along separate ways. Tramways provides services of transporting people or goods in urban and suburban areas. The approximate length of a typical tramcar is around 30m and can carry up to 300 people at a time. The metro transport system also known as subway, rapid transit system, underground or tube is a rail transit system with higher passenger capacity and frequency of operation and they run on independent rails/guides that are wholly separated from all other forms of traffics. The light rail transit system (LRT), as the name implies, is a type of urban and suburban railway transport service that uses equipment and infrastructures that are less heavier compared to those for metro systems and other heavier rail vehicles such as high-speed rail vehicles or locomotives. It provides benefits of higher flow rates and revenue speed and minimizes interference with other vehicles movement and pedestrians. It has a moderate passenger capacity compared to metro system and trams [55]–[59], [70]–[74]. 9 3 Energy Storage Solutions in Recuperation of Regenerative Braking Energy The recent developments in power electronic converters and energy storage technologies have enable energy storage systems to become one of the most practical and viable solutions for recovering the regenerative braking energy in electric railway vehicles and road electric vehicles [10], [39], [40], [75]– [77]. Depending on the electric railway applications, in most cases, they are considered as the most accepted solution for increasing energy efficiency and reliability of the electrified urban railway systems [39]. As explained earlier, in addition to the total energy consumption reduction, the advantages of the ESSs that make them have such outstanding performances in recuperating regenerative braking energy are the peak power demand reduction in both urban railway and the power utility systems, catenary-line voltage stabilization, compensation of power and energy, and catenary-free driving. Since the ESSs provide an excellent recovery of the braking energy, the heat being wasted in the onboard or wayside braking resistors is highly minimized and hence the additional costs of equipment required for ventilation systems is greatly reduced, [78]–[81]. Two ways of using the ESSs in the urban rail system are by installing them either in mobile form or at stationary. The former, which is generally called onboard configuration is the one in which the ESSs are being conveyed along with the vehicle either at the roof top or under the floor of the train. This gives the opportunity for the trains to accumulate their regenerative energy directly and re-use the energy later during their acceleration processes. On the other hand, the stationary ESSs are placed along trackside or at substations (also called wayside configuration) and they are used to recover and store the excess regenerated braking energy from the catenary line and deliver back to the other trains during their acceleration processes [6], [10], [39]. The following sub-sections give the details of the content under this main section. To start with, the detail descriptions of ESS’s components including their functions are presented. Next, is the detail discussions of the most widely used energy storage technologies in recovering regenerative braking energy in urban railway system including Ragone plot and their techno-economic comparisons. Finally, the two installation configurations of the ESSs are presented together with their application examples in urban railway systems. 10 3.1 The Energy Storage System Components Generally, as can be found in both industrial applications and academic research studies, the energy storage system comprises three major components namely an energy storage device (ESD), an electronic power converter, and a controller (control unit) [10]. The combination of these three components forms the electrical energy storage system and together they are used to perform several functions. The energy storage device converts electrical energy from a power source in order to store it into another form (electrochemically, mechanically, thermal, electromagnetically, and etc), and converts it back to its original form (electrical energy) whenever required [10], [82]–[85]. The power converter primarily performs the function of providing efficient flow of electrical energy between the power source (in this case the DC-link/Traction Network) and the energy storage device. It also serves as an interface between the two [39]. Fig. 6 below shows the ESS components. Bidirectional DC-DC Converter Energy Storage Device Energy Storage System (ESS) Energy Flow Controller SOC Voltage - Current - Waveform - Voltage - Waveform - Current - Storage Device & Converter Control Strategies Regenerated Braking Energy Flow Fig. 6: Energy Storage System (ESS) Components [10]. Energy Storage System (ESS) Regenerated Braking Energy Flow Bidirectional DC-DC Converter Voltage - Waveform - Current - Fig. 6: Energy Storage System (ESS) Components [10]. It should be noted here that for applications in recovering of regenerative braking energy, the converter has to provide a bidirectional energy flow (to and from the energy storage device) and also ensures that the DC-link electrical parameters are aligned with those for the energy storage device parameters such as the voltage and current. For this type of application, the power converter being used is called a bidirectional DC-DC converter for all battery and supercapacitors energy storage devices while it is a bidirectional AC-DC converter in the case of a flywheel energy storage system. It should be noted here 11 that from now henceforth, bidirectional DC-DC converter will be considered because for most applications in railway recuperation of regenerative braking energy, batteries and supercapacitors are the most commonly used energy storage devices. The controller which is the control unit for the system performs the functions of managing the energy storage device’s charging and discharging processes during the energy flow conditions. This is done in an efficient manner in order to minimize losses. 3.1 The Energy Storage System Components Moreover, the controller carries out this function according to the energy storage device’s major working parameter called the state of charge (SOC – minimum, maximum and nominal values) with respect to the traction network (DC-link) voltage. The charging and discharging of the energy storage device are based on specified threshold values of the DC-link voltages [10], [39], [62]. The details of the mentioned components with their various functions, applications, topologies, types, technologies, and etc are covered in section 3.2 (Energy Storage Technologies), section 4 (DC-DC Power Converter Topologies) and section 5 (Control Strategies/Energy Management and bidirectional Converter Control). 3.2 Energy Storage Technologies and their Applications in Urban Railway System In this section, descriptions and applications of the most commonly used energy storage technologies (batteries, supercapacitors, flywheels and hybrid energy storage systems) applied to urban rail transit system in regenerative braking energy recovery are discussed. The techno-economic comparisons among the technologies are presented, as well as their pictorial graph comparisons in terms of power and energy densities called Ragone plot. The electrical energy storage technologies are the means in which electrical energy can be stored by converting it into electrochemically, potentially, kinetically, electromagnetically, and etc as it is not possible to store the energy in its original form. When the electrical energy is required from the storage device, the stored energy is then converted back into electrical form in order to perform a required operation, [39], [75]. The examples of energy storage technologies include batteries, flywheels, supercapacitors, superconducting, hydrogen fuel cells, and etc. Generally, ESSs are basically required for back-up power supply i.e. when there is a need to supply electrical loads in the absence of mains 12 12 power supply or in the event of system maintenance/fault occurrence or during off-peak periods in renewable energy microgrids or when there is a need for short-term free movement of vehicles with onboard ESS or when it is necessary to get additional power supply if the available primary power supply cannot meet a certain demand [10], [75], [83], [86]. Therefore, in order to get ESS optimal performance for a particular application, selection of the best energy storage technology is crucial. To realize this, some factors are considered in aiding the ESSs design which consists of the number of charging and discharging cycles that a storage device can withstand during its lifetime; the device storage capacity in terms of energy capacity and energy density and power rating of the storage device i.e. the maximum current that it can handle from the mains supply, and the others [10], [39]. 3.2.1 Batteries Energy Storage System Technologies (BESS) Batteries are the earliest energy storage devices which store electrical energy by reversible electrochemical reactions between an anode and cathode within an electrolyte solution. With this, a reversible means of converting energy from one form (electrical) to another (chemical) is achieved [75], [86]. Batteries features are known for many decades and depending on the chemistry involved (types of electrodes and electrolyte), they offer different operational characteristics. Brief descriptions of battery technologies that are most commonly used in urban railway system applications are outlined. 3.2.1.1 Lead-acid Batteries (PbA) This battery technology has been the oldest among all the electrochemical (rechargeable) batteries [87]– [89]. These lead-acid batteries are the most widely used for different applications. They have both the highest operational efficiency (70% – 90%) and cell voltage [90], [91]. The batteries have two types of electrodes namely cathode (PbO2 - Lead Oxide) and anode (Pb - Lead metal). They are immersed in a diluted electrolyte solution called sulfuric acid (H2SO4). Fig. 7 shows the charging and discharging chemical reactions of a Lead-acid battery. During the discharging condition, both cathode and anode become lead sulphate while the solution turns into water during charging process [92], [93]. 13 13 4H+ SO4 2- SO4 2- 2e- Charging 2e- PbO2 Pb2+ PbSO4 2H2O Pb2+ PbO2 PbSO4 2H2SO4 PbO2 Pb2+ Pb PbSO4 Pb2+ PbSO4 2H2O 2H2SO4 4H+ SO4 2- SO4 2- 2e- Discharging 2e- Charger Load Fig. 7: Lead-Acid Battery (chemical reactions during charging and discharging conditions) [86]. Fig. 7: Lead-Acid Battery (chemical reactions during charging and discharging conditions) [86] Advantages and Disadvantages: The advantages of this type of batteries are, they have high power density over other types, high operational reliability, low cost in terms of watt-hour (Wh), long history with wide development and high efficiency. The disadvantages are, the batteries have poor performance at low temperature, limited life-time, low energy densities, contribute to environmental pollution, and etc. [87]–[91]. Applications: The lead-acid batteries have a long history of applications in power utilities and other applications for back-up power supplies. Due to their poor energy densities, short lifetime, cost of maintenance, and etc, they are generally being used in applications where low energy density and short lifetime are not major concerns. However, in the early 80’s, they were used in a railway system in Japan where the Japanese National Railway installed lead-acid batteries with total voltage of almost 1600V at Nakajima substation. The purpose of such installation was to compensate for a voltage drop along the supply line from the substation, and this project was successful for a duration of three years [94]. In order to further upgrade the lead-acid batteries to enable them to fit in traction systems, both their power and energy densities need to be increased. In that regard, some research studies are ongoing to change the lead electrodes with carbon material [10]. 14 14 3.2.1.2 Nickel Metal Hydride Batteries (Ni-MH) In 2012, a light rail vehicle called SWIMO, Japan was tested by Kawasaki in order to enable the vehicle to have an autonomous driving (catenary-free). The vehicle’s ESS was charged to maximum from the catenary line voltage (600V) for 5 minutes and the vehicle covered a distance of 10km freely [94]. Applications: Ni-MH had first been implemented in the nineties in hybrid electric cars. Unlike lead- acid batteries, in 2006 Ni-MH batteries were applied in traction rail vehicles in terms of energy consumption reduction where they were used in a tramcar called Citadis in Nice, France. A railway manufacturing company known as Alstom did the installation of such batteries as an onboard energy storage system which enabled the tramcar to a have catenary-free driving with a speed of 32.19Km/h and covered a distance of a half of a kilometre [100]. the batteries were also used at a substation in Japan called Komagawa installed by Osaka Municipal Transportation Bureau (OMTB) with power and energy densities of 5.5 MW and 576kWh respectively. The essence of this batteries installation was for energy consumption reduction, [101]. In 2012, a light rail vehicle called SWIMO, Japan was tested by Kawasaki in order to enable the vehicle to have an autonomous driving (catenary-free). The vehicle’s ESS was charged to maximum from the catenary line voltage (600V) for 5 minutes and the vehicle covered a distance of 10km freely [94]. 3.2.1.2 Nickel Metal Hydride Batteries (Ni-MH) The Ni-MH battery uses nickel hydroxide as its positive electrode, metal alloy as the negative electrode and both of them are immersed in an electrolyte solution of alkaline. These batteries are the earliest advanced rechargeable batteries with low maintenance and high reliability [95], [96]. From 1991 to date, the energy densities of Ni-MH increased from around 55 Wh/kg to about 100 W/kg whereas the power densities have increased from about 200 to 1300 W/kg over the last two decades [10], [97]. Fig. 8 shows the chemical reaction during charging and discharging operations. X(Cd/Zn/ Fe) X(OH)2 e- e- NiOOH H2O OH- OH- H2O Ni(OH)2 Discharging Separator Electrolyte X(Cd/Zn/ Fe) X(OH)2 e- e- NiOOH H2O OH- OH- H2O Ni(OH)2 Charging Separator Electrolyte Load Charger Fig. 8: Ni-MH Battery (chemical reactions during charging/discharging conditions) [86]. Load Charger Discharging Separator Fig. 8: Ni-MH Battery (chemical reactions during charging/discharging conditions) [86]. Advantages and Disadvantages: The advantages of Ni-MH batteries are as the followings. Compared to Lead-acid batteries, they have high power and energy densities and also with longer lifetime. Moreover, they have higher charging and discharging current, and low impact to environmental pollution. The disadvantages include low efficiency, costlier in terms of Watt-hour (Wh) and maintenance compared to lead-acid batteries. They also have a low resistance and present high rate of self-discharge. Hence, their terminal voltage is 1.2V. For this reason, they have to be connected in series to build up an ESSs with high nominal voltage, [62], [95]–[99] In order to overcome the Ni-MH high discharge rate, some novel separators are recommended in their formation [10]. 15 Applications: Ni-MH had first been implemented in the nineties in hybrid electric cars. Unlike lead- acid batteries, in 2006 Ni-MH batteries were applied in traction rail vehicles in terms of energy consumption reduction where they were used in a tramcar called Citadis in Nice, France. A railway manufacturing company known as Alstom did the installation of such batteries as an onboard energy storage system which enabled the tramcar to a have catenary-free driving with a speed of 32.19Km/h and covered a distance of a half of a kilometre [100]. the batteries were also used at a substation in Japan called Komagawa installed by Osaka Municipal Transportation Bureau (OMTB) with power and energy densities of 5.5 MW and 576kWh respectively. The essence of this batteries installation was for energy consumption reduction, [101]. 3.2.1.3 Lithium-based batteries (Li-ion) The lithium-ion batteries have excellent characteristics over all the other types of batteries, and these are because of their high energy and power densities. As shown in Fig. 9, a Li-ion battery is formed from a lithium metal oxide being the positive electrode, and a titanate/carbon material as the negative electrode and both are placed in an electrolyte of liquid/gel/solid. Li+ Separator Electrolyte Charge Discharge Li+ Cathode Anode Aluminium Collector Copper Collector Load Discharging through Load Charging by Power Source Charger Fig. 9: Lithium-ion Cell internal components [62]. Charger Charging by Power Source Discharging through Load Load Anode Cathode Charge Aluminium Collector Fig. 9: Lithium-ion Cell internal components [62]. 16 The electrolyte is made from lithium salts and organic solvents, [62], [102], [103]. There are different types of Li-ion batteries and the types depend on the compositions of the electrolyte and the different combinations of the metal oxides that form the two electrodes (anode and cathode), [102], [104]–[106]. Advantages and Disadvantages: The advantages of Li-ion batteries compared to other types of batteries are, they have higher relative energy and power densities, smaller in sizes and weigh lighter, wider range of operating temperature (-20oC to 60oC), higher efficiency, longer operating cycles (longer lifetime), they cost lesser in terms of maintenance and they have zero memory effect. In addition, their cells have a high nominal voltage at3.6V hence high voltage ratings of energy storage systems can be designed with just few numbers of the cells [62], [102]–[105], [107], [108]. The disadvantages are, the batteries have higher relative cost per watt-hour (Wh) due to the costs of the protection circuits and packaging, the battery pack requires a battery management system to maintain the safe operating temperatures, state of charge (SOC) and nominal voltages [106], [108]. It should be noted that even with the mentioned excellent characteristics that make Li-ion the best among all other battery chemistries, there are ongoing research studies to improve their performances and to reduce the cost by combining nanostructure and electrochemical materials [108]. Applications: Generally, because of their excellent characteristics, Li-ion batteries are being used in many areas such as aerospace, hybrid and electric cars, power utilities (for power quality support) and in some devices like mobile phones, laptops, tables, and etc [62]. In electrified railways, Li-ion batteries have profound applications in areas like energy saving, voltage drop, autonomous driving (catenary-free), and etc. 3.2.1.3 Lithium-based batteries (Li-ion) In energy saving area, Li-ion batteries were installed in electric railway systems in Japan for energy saving purpose as discussed in [94] and [109]. The authors outlined the installation modes which took place in two stages. The first stage called temporary batteries while the second as permanent batteries. Moreover, for catenary line voltage stabilization (due to voltage fluctuations) and voltage drop compensation, stationary lithium-ion batteries were installed in 2006 in Shin-Hikida substation. In 2013, lithium ion batteries with a total power rating of 2000 kW 17 17 and rated capacity of 76 kWh were also installed at Haijima substation for voltage stabilization and voltage drop compensation [110]. For catenary-free applications, Li-ion batteries are the best solution because of their high energy density as they allow the train to travel for a long distance before the batteries reach their safe minimum state of charge (SOCmin). Li-ion batteries allow tramcars to run freely (without having power from substations) and pass through all areas in urban city centres where it is not possible to run overhead catenary lines power supply because of aesthetic and historic structures. For this type of application, the batteries must be carried onboard of the tramcar, [10]. 3.2.1.4 Sodium-Sulphur Batteries (NaS) The Sodium Sulphur battery operation depends on the movement of sodium ions between its electrodes as can be seen in Fig. 10 below. The NaS battery has sulphur in liquid form (molten) as its positive electrode while sodium also in liquid form (molten) as its negative electrode, and both electrodes are kept apart by an electrolyte called beta alumina ceramic [111]. Na2SX e- e- Na+ Charging Discharging Na POWER SUPPLY/LOAD S Electrode + S Electrode - e- e- Fig. 10: NaS Battery (chemical reactions during charging and discharging conditions) [86]. Fig. 10: NaS Battery (chemical reactions during charging and discharging conditions) [86]. During the discharge operation of NaS battery, Na+ ions pass through the electrolyte alumina ceramic (the electrolyte) thereby combines with the sulphur to form polysulphides (Na2S4). This reaction is reversible during the charging period. When connected to an external circuit during discharging period, a 2V potential difference is obtained at its terminal [112]. 18 Advantages and Disadvantages: The advantages of NaS batteries are, they have high energy density (150Wh/kg – 240Wh/kg) and power density (90W/kg – 230W/kg), high efficiency in the range of 75% – 90 %, and they have longer relative lifetime. They exhibit some disadvantages, these are, their reaction processes work at a high temperature typically between 300oC and 350oC which consequently develop a high self-discharge rate, they have relatively high cost and environmental issues compared to some other types of batteries. Moreover, a ventilating equipment is required to cool the battery during operation due to high operating temperature [112]–[115]. It is also worth to mention the other type of sodium-based battery, sodium nickel chloride battery (NaNiCl2), also known as ZEBRA cell. The detail of this battery is not explained here as it shares some similar operating characteristics and applications with the NaS but with few differences for example, it has a positive electrode that is nickel chloride and with electrolyte that is sodium chloroaluminate. However, both of the cells have similar kind of negative electrodes (liquid sodium metal) [112], [116], [117]. Applications: NaS batteries produced by a company named ShangHai NaS are for commercial purpose to be used for grid services. However, their applications in electrified railway systems are limited since they have a drawback of producing heat while in operation that lowers their performances in terms of peak power demand reduction and for power quality applications [112]. 3.2.1.5 Other Potential Battery Technologies In addition to the previously discussed battery technologies, there are some other types of batteries that could be used in electrified urban railway systems such as, flow batteries (redox flow batteries) and metal-air batteries. The flow batteries have excellent characteristics such as negligible self-discharge with a long lifetime of operation but they still have some drawbacks such as high relative cost and with low energy density. On the other hand, the metal-air batteries have some reasonable advantages like having high energy density at relatively lower cost but the disadvantage is, they present low energy efficiency. The metal-air batteries are used in some applications for examples in electric cars and mobile electronic devices [112], [113]. 3.2.1.4 Sodium-Sulphur Batteries (NaS) A summary of comparisons in terms of the advantages, disadvantages and applications for each type of battery energy storage technologies is presented in Table 1. A summary of comparisons in terms of the advantages, disadvantages and applications for each type of battery energy storage technologies is presented in Table 1. 19 3.2.2 Supercapacitors Energy Storage System Technologies (SESS) These useful features enable them to have wide range of applications in hybrid power systems, electric transportation systems such as electric cars and trains for harvesting of regenerative braking energy which occurs at high power levels and for short periods (few seconds). Moreover, they are used in line voltage regulation due to the intermittent fluctuations caused by the regenerative braking energy (10 – 25 sec) [62], [121]–[123]. Applications: The reason why supercapacitors become highly required in energy storage systems is because of their excellent characteristics of being able to withstand high rate of charging and discharging cycles without affecting their efficiency. Moreover, because of their high-power density coupled with the fact that they can be discharged and charged very fast, they can therefore supply high power peak demand. These useful features enable them to have wide range of applications in hybrid power systems, electric transportation systems such as electric cars and trains for harvesting of regenerative braking energy which occurs at high power levels and for short periods (few seconds). Moreover, they are used in line voltage regulation due to the intermittent fluctuations caused by the regenerative braking energy (10 – 25 sec) [62], [121]–[123]. It should be noted that, in regenerative braking energy applications, the supercapacitors are interfaced with a DC/DC power converter (bi-directional) that controls the power flows between them and the DC-link. A controller manages the operations of the supercapacitor and the converter [62]. The subsequent paragraph discusses some applications of supercapacitors energy storage technologies in electrified railway systems. 22 Supercapacitors can be found in many applications ranging from high power applications at power grid, electric vehicles to electrified railway systems. Some of the applications in electrified railway system for recuperating of regenerative braking energy are: In [124], a siemens SITRAS wayside ESS was used in Toronto LRT for the purpose of energy consumption reduction where the ESS voltage rating was at 600V. In another Siemens SITRAS application, a wayside supercapacitor ESS was used in Cologne public transport, Germany for the purpose of energy saving and voltage regulation. An energy saving of up to 37% was achieved [125]. 3.2.2 Supercapacitors Energy Storage System Technologies (SESS) A supercapacitor is a device which operates based on electrostatic field principle to store energy in the same way a conventional electrolytic capacitor does. However, it has a very high energy density compared to electrolytic capacitors [62]. It is also called ultracapacitor – UC, pseudo capacitor or electrochemical double layer capacitor – EDLC. As shown in Fig. 11, it consists of three main components which are two electrodes, a separator and electrolyte solution. It stores energy using 20 electrostatic charge transfer between the electrolyte and the two electrodes without chemical reactions taking place (unlike batteries). Porous Carbon Electrode Electrode Porous Paper Separator Electrolyte Porous Carbon Electrode Charged Ions Fig. 11: Schematic of Supercapacitor Energy Storage Technology, [83]. Porous Paper Separator Charged Ions Porous Carbon Electrode Fig. 11: Schematic of Supercapacitor Energy Storage Technology, [83]. A typical supercapacitor consists of two double layer capacitors adjoined to one another in a package. The complete cell has two porous electrodes made of carbon deposited aluminium with a separator between them (foil paper) and all are immersed in an electrolyte solution called organic acetonitrile,[10], [118]–[120]. Advantages and Disadvantages: The major advantages of supercapacitors are, they have a very high lifecycle (typically 106 charging and discharging cycles), they have a high-power density above 4000W/kg; having high rate of charging and discharging; high efficiency (approximately 95%) due to the very low internal resistance, they can operate between wide temperature range (– 40oC to + 65oC). Moreover, their state of charge (SOC) is easily obtained from the potential difference between its two terminals. On the other hand, the drawbacks are, the supercapacitors have a low energy density compared to other batteries that is 0.5Wh/kg – 10Wh/kg where the lowest energy density in batteries being the lead-acid battery has 30Wh/kg – 40Wh/kg. They also suffer from high self-discharge rates due to leakage currents sensitive to overcharging which damage them easily, frequent vibrations, poor storage, high cycling current, and etc which actually affect their lifecycle [6], [10], [120]. 21 21 Applications: The reason why supercapacitors become highly required in energy storage systems is because of their excellent characteristics of being able to withstand high rate of charging and discharging cycles without affecting their efficiency. Moreover, because of their high-power density coupled with the fact that they can be discharged and charged very fast, they can therefore supply high power peak demand. 3.2.2 Supercapacitors Energy Storage System Technologies (SESS) In [126], two supercapacitor ESSs of the same energy density of 2.8 x 10-4 MWh were placed under the floor of a railway passenger car for the purpose of minimizing the use of the conventional friction braking in the event of failure of the train to send back regenerative braking energy to the supply substation. The result confirmed that the supercapacitors were both able to save regenerative braking energy of 8% from the electric drives of the train. In another application, modules of supercapacitors were used in a Korean metro system, Daejeon Rapid Transit, for the purpose of energy consumption reduction and voltage regulation. Both of the supercapacitors’ modules were 22 22 having an energy and power densities of approximately 10 kWh and 1.9 MW respectively. The results of this application confirmed that the catenary line voltage was stabilized at a nominal of 1.5 kV DC [127]. A MITRAC energy saver being an onboard supercapacitors ESS installation is used mainly for energy saving and autonomous driving [128]. In one of the applications, it was installed on an LRT vehicle in Mannheim, Germany and used for up to five years. In such application, an energy consumption was reduced by 30%. Moreover, the result of the installation test revealed that up to half of the current peak demand and voltage drop had been curtailed [129]. 3.2.3 Flywheels Energy Storage System (FESS) Flywheel ESS is a type of mechanical energy storage system consists of an electrical machine that has a stator and a rotor, friction bearings, rotating cylindrical mass, AC/DC power electronic converter (bi- directional) and a rugged housing as shown in the Fig. 12. Bidirectional AC – DC Converter Motor/Generator Unit Vacuum Pump Magnetic Bearing (Upper) Magnetic Bearing (Lower) Rotor (Flywheel) Housing DC-Link Axle Shaft for Rotation Fig. 12: Flywheel Energy Storage System (FESS) [114], [130]. Housing Magnetic Bearing (Upper) Bidirectional AC – DC Converter Motor/Generator Unit Motor/Generator Axle Shaft for Rotation Rotor (Flywheel) Vacuum Pump Magnetic Bearing (Lower) Fig. 12: Flywheel Energy Storage System (FESS) [114], [130]. Essentially, it operates based on the principle of converting electrical energy into kinetic energy and vice-versa. The cylindrical mass is attached to the rotor while the rotor is suspended by the bearings. The flywheel rotor is made of combination of carbon and fibre materials. Although the composite materials reduce the flywheel’s inertia, a very high rotational speed of the rotor is attained. In order to 23 reduce the flywheel’s internal losses by providing low frictions, the bearings are made from magnetic material, [75], [86], [131], [132]. During the flywheel ESS operation, the electric machine acts as a motor by receiving an electrical energy via the power converter interface thus spinning the cylindrical mass at a very high speed hence enabling it to acquire the kinetic energy in proportion to the amount of electrical energy received [130]. During discharging state, however, the stored kinetic energy is converted back into electrical energy by decelerating rotor torque and at this instant the electrical machine behaves as a generator (i.e. supply electrical power back through the power electronic converter) [10], [39], [132], [133]. Advantages and Disadvantages: The flywheel ESSs have high relative efficiency with high power and energy densities Similar to supercapacitors, flywheel ESSs have a high charging and discharging frequency, they have minimal adverse effects to the environment, their SOC can be obtained easily by determining the flywheel’s angular velocity, and they can operate over a wide temperature range. The first major drawback of using flywheels is that they are very much heavier compare to all other ESSs. They are sensitive to overloading which can easily cause them to explode into pieces although advanced rotors are made from fibre materials which are less risky compared to those made from metal materials. 3.2.3 Flywheels Energy Storage System (FESS) Their internal friction make them to have high rate of self-discharge, [39], [75], [86], [132], [134]. Advantages and Disadvantages: The flywheel ESSs have high relative efficiency with high power and energy densities Similar to supercapacitors, flywheel ESSs have a high charging and discharging frequency, they have minimal adverse effects to the environment, their SOC can be obtained easily by determining the flywheel’s angular velocity, and they can operate over a wide temperature range. The first major drawback of using flywheels is that they are very much heavier compare to all other ESSs. Applications: Flywheel ESSs can be considered equal or next best to supercapacitors in terms of suitability in high power applications and electrified transportation systems especially in railway systems due to some of their excellent characteristics. However, when they are carried onboard, they easily develop a high rate of self-discharge when the train changes in its direction. Moreover, flywheel ESSs have heavier weight and occupy much space, hence these factors become major issues in railway applications especially when they are carried onboard. Another big threat in their applications for railways system is, they pose the risk of explosion [10], [131], [133], [134]. Nevertheless, flywheels can be found in some applications as discussed in reference [135] where the flywheel energy storage system was used in a light rail transit (LRT) system for the purpose of energy consumption reduction. The results showed that an energy saving of 31% was achieved with an energy 24 capacity of 2.9 kWh and rated power of 725 kW respectively. In another application, a flywheel storage system was tested in Hanover, Germany by Pillar for the purpose of energy consumption reduction. In this test, an energy saving of 462 kWh per annum was achieved at the flywheel rated power capacity of 2 x 10-1 MW and energy capacity of 1500Wh [136]. In [137], a flywheel energy storage system was applied to achieve a voltage stabilization at Kehin electric railway system in Japan whose rated power was 3000 kW and energy capacity of 0.025 MW. In the USA, the flywheel energy storage system was used at Los Angeles metro system for energy saving purpose. The storage rated power was at 2000 kW, and energy capacity of around 8 kWh [131]. 3.2.3 Flywheels Energy Storage System (FESS) A flywheel energy storage system with rated power of 1000 kW was installed by Urenco Power Utility company at Far Rockaway, New York, USA for the purpose of power peak shaving. An energy saving of up to 25% was achieved in this system, [130]. 3.2.4.1 Hybrid Electrical Energy Storage System (HEESS) The idea behind hybridization of two or more energy storage systems is to derive the best benefits of each individual storage technology so as to achieve the overall combined performances in a single system. This is called hybrid energy storage system (HESS). It is known that no any single ESS can provide the best characteristics required in a particular application, hence the reason for using HEESs technology. In HEESs, a combined high power and energy densities, high efficiency, long lifecycle of operations, fast response, and etc can be achieved altogether [138]. Different combinations of hybrid electrical energy storage systems can be achieved by using combinations of either Supercapacitor/Battery or Supercapacitor/Flywheel or Battery/Flywheelor Supercapacitor/Battery/Flywheel. However, the most common HEES in many applications is the hybrid of Supercapacitor/Battery [101], [138]. Advantages and Disadvantages: The advantages of HEESs are high energy saving of braking energy, line voltage stabilization, and the combined benefits such as high efficiency, high energy and power densities, long lifetime, and etc. There is an improvement in safety and protection of one of the ESSs. For instance, in supercapacitor/battery HEES, the battery lifetime is extended due to protection of their 25 cells against damage as a result of having lower charging current. The performance of HESS is always better compared to a single ESS. However, HEESs also have some drawbacks such as having a complex control strategy as two or more different ESSs are used thus increased in maintenance and cost are inevitable, [139]–[141]. Applications: There are some applications of HEESs in electrified railway systems as discussed in the following references. In [142], Sitras hybrid energy storage by Siemens was used to reduce a tramcar energy consumption during acceleration and also enabled it to have autonomous driving. In such application, the HESS was installed on the tramcar consisted of NiMH battery and a module of supercapacitors in order to obtain both high energy and power densities. The design of the HESS was done in such a way that the NiMH battery ESS with the capacity of 18kWh was used to provide the tram with a long-distance catenary-free driving and to supply power to the cooling and heating systems required for the passengers. While the supercapacitors ESS with the lowest capacity of around 2kWh was used to provide the stored energy to the tram during its acceleration mode. 3.2.4.1 Hybrid Electrical Energy Storage System (HEESS) The recharging of the Sitras energy storage system was both done during the tram braking or at the substation charging point. In [143], an onboard Sitras energy storage system was used in Portugal on a tram known as Combino plus MTS in the year of 2008. The HESS enabled the tram to have autonomous driving for a distance of about 2.4km and the system has a minimal adverse impact to the environment. A summary of comparisons in terms of the advantages, disadvantages and applications for each type of energy storage technologies are presented in Table 2. 26 3.2.4.1 Lithium-ion Capacitor (LiC) Lithium-ion capacitor (LiC) is a newly developed hybrid energy storage technology and manufactured as a single energy storage device combining both the good characteristics of lithium-ion batteries and supercapacitors. As already indicated, lithium-ion battery has high energy density but low power density while supercapacitor has high power density but low energy density [144]. The idea behind LiC is to have a single unit energy storage device to combine these two characteristics (high energy and high power densities) [145], [146]. The chemical reaction in Lithium-ion capacitors occurs when adsorption and desorption processes take place at the positive electrode surface while at the same time at the negative electrode, the cations redox chemical reaction occurs. Therefore, a high energy density and high-power density are obtained in the LiC as a result of the ionic adsorption in the supercapacitor double layer and the redox chemical reaction due to the lithium-ion part [145], [147]. 27 Applications The selection of suitable energy storage technologies reviewed in the previous sub-section depends on the technical characteristics and the costs. Table 3 shows, in order to derive the comparisons and assessments, various criteria are needed such as the storage devices energy and power densities, efficiency, life-time for operation, life cycle, capital cost investment, self-discharge, depth of discharge, durability, size (the weight to volume ratio), time of discharge, and response rate (note that only the main characteristics are considered in Table 3) [39],[148]. The type of installation configuration is also one of the criteria for the comparisons and assessments. For instance, for onboard type of ESSs installation mode, criteria such as ESSs size in terms of weight and volume or space are to be considered so that an ESS is carefully selected for specific application [10], [148], [149]. Another criterion for comparisons and assessments is the application response rate categorise into short or long duration (during charging and discharging of the ESS). The first category being the urban rail transit systems which have high and fast response rate to charging and discharging of regenerative braking energy because of the frequent and numerous braking phases that occur, and hence are classified under short – duration applications. While applications such as renewable energy system where the response rate is not that high due to longer charging and discharging rate and load shifting, they are therefore classified under long duration applications. [10], [150]. To start with, the energy and power densities are among the main characteristics to be considered in selecting ESSs for electric railway system applications. For onboard ESS applications the size is one part of the main criteria as space and weight are of great concerns. From the Table 3, it could be observed that batteries indicate to have the highest energy density (Wh/kg) over supercapacitors and flywheels [151]. The lithium-ion batteries have a high energy capacity and they occupy less volume and this make them highly suitable for onboard applications. In the case of power densities, supercapacitors and flywheels have higher ratio power to weight values (W/kg) over batteries with flywheels having higher energy capacity. Efficiency is an important parameter to be considered and it indicates the amount of 28 energy to be discharged by the ESS during charging and discharging cycles as lower efficiency signifies lower energy delivered. Applications The efficiency of an ESS is a function of the energy storage cost. Supercapacitors, flywheels, and lithium are batteries that have the high efficiencies (≈ 95%). It should also be noted that self-discharge parameter has similar effect as the efficiency, which higher rates of self-discharge have adverse impacts on the ESS cost since the total stored energy is reduced, [39], [152]. Despite flywheels having a high self-discharge rate, they can be fitted in urban rail applications as the energy storage duration requirements is for a short duration. This is due to the frequent charging and discharging cycles. Another important characteristic is the ESSs durability where it could be seen that batteries (lead-acid, nickel metal hydride, and sodium – sulphur) have much lower charge – discharge life-cycles compared to supercapacitors, advanced lithium-ion and flywheels that have higher number of charge – discharge cycles. It should be noted that this parameter is very important to be considered in urban railway applications because of the high number of charging and discharging cycles [10], [153], [154]. Another critical and important criterion to be considered at selecting the ESSs is the cost of the system which is expressed in terms of the total cost per energy stored and cost per rated power of the ESS measured in $/kWh and $/kW respectively. The selection of the ESS based on total cost per energy and per rated power is done without compromising the efficiency. It could be observed that the supercapacitors and flywheels are cheaper than batteries (lead-acid, nickel metal hydride, and sodium – sulphur) when cost/kW is considered. While in terms of cost/kWh, batteries are the cheapest. Moreover, it should be noted that ESS costs also depend upon the types of application to be used e.g., for applications in power systems or railway or renewable energy systems. Reference [151] carried out a study on cost comparative analysis of ESSs for different types of applications. For instance, the cost of ESS for a particular (short or long duration) application depends on the rate of utilization of the stored energy in the operation. In urban railway systems where the stored energy in the ESSs is being discharged so frequently, then cost per/unit power output is to be considered. On a contrary, where the stored energy in the ESS stays for a longer duration then the cost/unit energy is to be considered. Applications Based Another critical and important criterion to be considered at selecting the ESSs is the cost of the system which is expressed in terms of the total cost per energy stored and cost per rated power of the ESS measured in $/kWh and $/kW respectively. The selection of the ESS based on total cost per energy and per rated power is done without compromising the efficiency. It could be observed that the supercapacitors and flywheels are cheaper than batteries (lead-acid, nickel metal hydride, and sodium 29 on this, batteries are the most cost effective for longer storage durations while supercapacitors and flywheels are for the shorter storage durations [10], [39], [151]. on this, batteries are the most cost effective for longer storage durations while supercapacitors and flywheels are for the shorter storage durations [10], [39], [151]. Table 3 presents the techno-economic comparisons of the energy storage technologies covered in this section. 3.2.6 Ragone Plot 3.2.6 Ragone Plot Another method in selecting a suitable type of an energy storage system for an application is by the use of Ragone plot. In the techno-economic comparisons done in the previous section, hybrid electrical energy storage system is not included as it is not a single entity energy storage device. Therefore, the Ragone plot includes the hybrid ESS to further shows its position with respect to other ESSs. Essentially, Ragone plot characterised energy storage devices on the basis of their specific energy and power densities. The plot shows the HESS abilities in meeting various applications demand of energy accumulation capacities and power transfer rates. As can be seen Fig. 13, the logarithmic axes of the plot indicates that the batteries have energy densities of up to 100kWhkg-1 but with low power densities while supercapacitors with high power densities of up to 1kWkg-1 but with low energy densities. Other energy storage devices are shown to fall within different energy – power densities regions which also signify for specific type of application based on energy and power requirements [101], [75],[155]. Fig. 13 below shows Ragone plot for various energy storage devices. 30 101 102 102 103 103 104 104 105 105 106 106 Specific Energy (Wh.kg -1 ) Specific Power (W.kg-1 ) 101 107 1 x10-4 S 1 x10-2 S 1 S 100 S 1 x104 S Superconducting Magnetic Energy Storage (SMES) Supercapacitors Batteries Li-ion NaS Ni-MH Ni-Cd Lead-acid Flywheels Electrolytic Capacitors Flim Capacitors Fig. 13: Ragone Plot showing features of various Energy Storage Devices [75], [101], [156]. 1 S Superconducting Magnetic Energy Storag (SMES) Specific Energy (Wh.kg -1 ) Flim Capacitors Fig. 13: Ragone Plot showing features of various Energy Storage Devices [75], [101], [156]. The Ragone plot for actual energy storage devices being used in electric railway systems including hybrid ESS is shown in Fig. 14. The Ragone plot for actual energy storage devices being used in electric railway systems including hybrid ESS is shown in Fig. 14. Specific Energy (Wh.kg-1 ) Specific Power (W.kg-1 ) 10 100 1000 0.01 1 10 100 36 S 3.6 S 6 min 60 min Supercapacitors Batteries Flywheels Supercapacitor (Met) Supercapacitor (Sta) HYBRID MITRAC ALSTOM SITSES FLYSta FLYMo NiMHSta NiMHMo Li-ionMo Li-ionSta KEY Fig. 14: Ragone plot of actual energy storage technologies used in urban railway system [101] [156]. 3.2.6 Ragone Plot Specific Energy (Wh.kg-1 ) Specific Power (W.kg-1 ) 10 100 1000 0.01 1 10 100 36 S 3.6 S 6 min 60 min Supercapacitors Batteries Flywheels Supercapacitor (Met) Supercapacitor (Sta) HYBRID MITRAC ALSTOM SITSES FLYSta FLYMo NiMHSta NiMHMo Li-ionMo Li-ionSta KEY Supercapacitor (Met) Supercapacitor (Sta) HYBRID MITRAC ALSTOM SITSES FLYSta FLYMo NiMHSta NiMHMo Li-ionMo Li-ionSta KEY Batteries Flywheels Specific Power (W.kg-1 ) Fig. 14: Ragone plot of actual energy storage technologies used in urban railway system [101] [156]. On the logarithmic plane, the time is represented by the slope lines and deduced from the ratio of the energy storage device’s energy and power densities which also signifies the device discharge time. It 31 could be observed that Li – ion batteries with discharging time of around 5 minutes could be used for long duration applications. Flywheels have discharging time of around 1 minute for onboard installation while between a range of around 1 – 10 minutes for the trackside installation. Supercapacitors have a fast discharging time of around 4 – 35 seconds. The hybrid energy storage system by Siemens called SITRAS consists of nickel metal hydride and supercapacitor. The Ni – MH batteries have a discharging time around 10 minutes while the supercapacitors with the value around 10 seconds [101]. Base on the plot, it is evident that supercapacitors have the fastest discharging time over batteries and flywheels in both installation modes (stationary and onboard) and therefore highly preferable for urban railway applications to be used for energy consumption reduction and autonomous driving (catenary – free driving) [101], [155]. The discharging time for flywheels is in between the supercapacitors and the batteries and this qualifies them to be used for urban railway applications except for mobile (onboard) configuration due to their large sizes, cost of maintenance and risk of shattering in the event of explosion. For the use of batteries, they are most preferable for wayside/trackside installations. However, their shorter life-cycle operation compared to supercapacitors and flywheels make them not suitable in electrified urban railway applications. The hybrid energy storage systems appear to be the most suitable ESSs nowadays in urban railway systems especially in mobile configuration due to their good performance over supercapacitors or batteries as separate ESS. However, the costs implications and complex control strategies are to be incurred [101], [155], [156]. 3.3.1 Onboard Energy Storage System Mode By onboard installation it means that the ESS is placed on the vehicle’s roof or under the floor. Fig. 3 in section 1 shows the onboard energy flow where the red dashed lines represent the regenerative braking energy flow from the vehicle to the ESS and release back from the ESS to the vehicle during acceleration process. The main objectives of this installation mode are, firstly, to store the vehicle’s regenerative braking energy and releases it later during acceleration phase. Secondly, this mode of installation is for power peak reduction during the vehicle’s acceleration hence reducing the total power 32 drained from the traction substation. It stabilises the catenary line voltage during acceleration phases. Finally, it allows the vehicle to have catenary – free driving [62], [157] Some research studies on onboard ESSs found that an energy saving about 14% to 34% could be achieved when onboard ESSs are deployed in urban railway applications [158], [159]. Advantages and Disadvantages: The onboard ESS advantages are, the peak power shaving during the vehicle’s acceleration resulting in total energy cost reduction. The onboard ESS regulates the catenary line voltage’s fluctuations which is caused during the vehicle’s acceleration and braking phases. Moreover, the onboard ESS enables the vehicle to have an autonomous driving in the event of power outage from substations, pantograph failure, or passing through city centres where no catenary lines are available. One of the major drawbacks of onboard ESS is that it increases the vehicle’s total weight by an average of 3 ton and this depends on the ESS’s total size. Due to this increase in weight, the vehicle’s energy consumption also increases. For this reason, it is actually the best to make provision for an approximate onboard ESS space and sizing during the vehicle design rather than retrofitting an existing one , [157], [160], [161]. 3.3.2 Wayside Energy Storage Mode Unlike onboard ESSs, in wayside ESSs (also known as trackside or stationery), the installation mode is stationary or at the ground level, and the ESSs capture and store the excess regenerated braking energy in the electric section (catenary line/third rail) provided by any braking vehicle in the same network. When any vehicle in the electric network brakes, the catenary line voltage increases and hence the ESSs stores the surplus braking energy (charging mode) whereas when the line voltage decreases due to any vehicle accelerating in the network, the ESSs discharges the stored braking energy (discharging mode) to aid the acceleration process as well as to maintain the line voltage threshold value. The installation of wayside is done either at a substation or at a trackside close to the train stations where the electric section experiences much fluctuations due to several braking and acceleration processes [160]–[162]. Fig. 3 in section 1 shows the trackside (wayside) energy flow where the green dashed lines represent regenerative braking energy flow from the vehicle to the catenary line and then to the ESS before it is released back to the catenary line for other vehicles absorb during their acceleration phases. 33 Advantages and Disadvantages: One of the advantages is, the wayside ESSs bring significant of cost savings due to power peaks shaving in the entire electrical network due to the vehicles’ accelerations. The wayside ESSs regulate the fluctuations of the catenary line voltage caused by the braking and accelerating vehicles [6], [163]. The wayside ESSs have more freedom of space and size allocation for the energy storage devices. Moreover, in case of a substation power supply failure, a vehicle can reach the nearest destination by getting the supply from the catenary line via the wayside ESSs. However, the wayside ESSs present lower efficiency because of the I2R losses along the catenary line (from the vehicle supplying the regenerative energy to the ESSs location) [157], [164], [165]. 4 Power Converter Topologies for ESSs in Regenerative Braking Energy Recovery The DC-DC power converters form a part of the ESSs in the recuperation process of the regenerative braking energy. In railway system networks, power converters are required to interface the energy storage devices to the traction network (DC-link) where all other power converters are connected to. One of the main functions of the power converters is to match the characteristics of the regenerated energy to the energy storage device working parameters including the voltage and current, and to provide an efficient energy flow in both directions (bi-directional) [10], [62], [166]. Depending on the energy storage technology and application, when making a topology selection for the bidirectional DC- DC power converter, some important working parameters for the converters need to be considered. Those parameters are high efficiency, fast response, ability to withstand frequent and high-power peaks, working over a wide temperature range, small in size and light weight (particularly in the case of onboard ESSs applications), providing autonomous driving capability when the need surges (also in onboard applications) and the power converters must able to provide the required voltage level (600,750,1500 or 3000V) to the DC-link [62], [167], [168]. The bidirectional DC-DC power converters are categorized into isolated and non-isolated. Generally, the non-isolated converters are used when the transfer voltage ratio is small (usually < 4 by 1) and the galvanic isolation is not necessary [62]. Due to these features, non-isolated DC-DC power converters do not have problems associated with magnetic interference, and their dimensions are small and lightweight. There are three main converter configurations namely buck (for stepping down voltage), 34 boost (for stepping up voltage) and buck-boost (for stepping up or down voltage) [168]. On the other hand, the isolated converters are used when galvanic/dielectric isolation is required and high voltage transfer ratio is required. In such converters, the DC voltage is first converted into AC, then passes a high frequency transformer, and finally converted back to DC. The high frequency transformer provides the magnetic isolation medium between the two linking voltages. For this reason, such converters are heavier and larger, [169], [170], [171]. In recuperating the regenerative braking energy using ESSs applications, the most commonly used bidirectional DC-DC converters are discussed in the following subsections.: 4.1 Non-Isolated Buck-Boost Bi-directional DC-DC Converter. 4.1 Non-Isolated Buck-Boost Bi-directional DC-DC Converter. As shown in Fig. 15, this converter topology (also known as half-bridge DC-DC converter) consists of two power semiconductor switches (IGBTs), one inductor and it is used when galvanic isolation is not needed. ESD DC-Link (Traction Network) L Cdc C S1 S2 Energy Storage Device Fig. 15: Non-Isolated Buck-Boost Bi-directional DC-DC Converter [168],[172]. Fig. 15: Non-Isolated Buck-Boost Bi-directional DC-DC Converter [168],[172]. It controls the power flow between two different voltage levels of DC buses (the high side and low side). The energy storage device is usually connected to the low DC bus of the converter whereas the energy source (in this case, regenerated braking energy from the traction DC-link) is connected to the high DC bus. During the dynamic braking, regenerated energy flows from the DC-link to the energy storage device, and in this mode, the converter acts as a buck converter, in which the transistor switch S1 conducts while S2 is off and the inductor works as a filter. In the reverse energy flow, the converter acts as a boost converter i.e. transistor switch S2 is on while S1 is off, and the ESS sends the stored 35 energy to the DC-link to facilitate vehicle’s acceleration. In each of the operation, the duty cycle for each transistor switch controls the amount of energy flow to the ESS or to the DC link [62], [173], [172]. [172]. One of the advantages of this topology is that, it has less effect in the energy storage device deterioration despite the high switching frequency ripples being produced by the current at the converter output as discussed in [174], Another advantage is that it is simple, has smaller size and lighter weight and his topology has high efficiency (≥90%). On the other hand, the use of this topology makes the ESS lacks of galvanic isolation from the DC link. This results in high switching losses and its inability to increase its voltage level during boost mode as required [175], [176]. 4.3 Cascaded Buck-Boost Bi-directional DC-DC converter Fig. 17 shows two non-isolated buck-boost bi-directional DC-DC converters connected in cascaded form by means of an inductor. This topology enables the converter to have a high voltage gain ratio and a low current stress. ESD S3 S4 S1 S2 Cdc DC-Link (Traction Network) L C Fig. 17: Cascaded Buck-Boost DC-DC converter [62], [172]. DC-Link (Traction Network) Fig. 17: Cascaded Buck-Boost DC-DC converter [62], [172]. The energy storage device is thus connected to the DC bus via S3 and S4 whereas S1 and S2 are connected to the DC-link traction network [172]. This type of converter is commonly used in electric vehicle (EV) applications. This topology has a high-power capability due to low filter current stresses and low current ripples when compared to the single non-isolated buck-boost converter. Moreover, the voltage transfer ratio is high for the same switching frequency of a single buck-boost converter. However, the drawback is that it is costlier because of the used of greater number of switches, [172], [180]–[182]. 4.2 Multiphase Buck-Boost Bi-directional DC-DC Converter In order to have a high-power capacity and to reduce the converter output current ripple without necessarily increasing the switching frequency and the inductor value, a multiphase converter is employed that is the interleaved buck-boost DC-DC converter which consists of two or more of the buck-boost converters. The buck-boost converters are arranged in parallel as shown in Fig.16. With such arrangement of the phases, the control signals for the semiconductor switches are displaced by a phase shift angle of 360o/N where N stands for the number of the phases. ESD C L1 L2 L3 S1 S2 S3 S4 S5 S6 Cdc DC-Link (Traction Network) Fig. 16: Multiphase Buck-Boost Bi-directional DC-DC Converter [168], [172]. Fig. 16: Multiphase Buck-Boost Bi-directional DC-DC Converter [168], [172]. The idea of this topology is to lower the output current ripple and to have a frequency N times of a single-phase arrangement. This topology increases the reliability of energy flow that if one of the phases fails, others can continue to conduct though the total power capability is lowered by one third of the 36 initial total power given that the number of the phases is three. Smaller number of filter components is required leading to reduce the weight. Moreover, switching losses are reduced compared to the single- phase buck-boost converter [62], [177]–[179]. However, due to increase in the number of switches, this topology requires a complex control strategy and more expensive compared to the single-phase buck-boost converter [168]. 4.4 Dual Active Full-Bridge Bi-directional DC-DC Converter This is an isolated bidirectional DC-DC converter which is formed by connecting two full-bridge converters with a high frequency transformer in between them as shown in Fig. 18. Other components 37 in the circuit are the capacitors which are connected at both sides of the DC buses, and an inductor connected to the primary side of the transformer. ESD S3 S1 S4 S2 Q3 Q1 Q4 Q2 L N1 N2 C Cdc DC-Link (Traction Network) Fig. 18: Dual Active Full-Bridge Bi-directional DC-DC Converter [62], [172]. Fig. 18: Dual Active Full-Bridge Bi-directional DC-DC Converter [62], [172]. The high frequency (HF) transformer provides the required galvanic isolation and hence enables the control of the amount of power to be transferred on both sides of the converter. When the energy flows from DC-link (DC bus 1) to the ESS (ESS charging mode), the converter connected to the primary side of the HF transformer acts as an inverter while the converter at the secondary side of the transformer becomes a full wave rectifier. In reverse energy flow direction (ESS discharging mode), the former becomes the rectifier while the latter becomes the inverter [172], [183], [184]. The advantage of this converter is that it has high efficiency of about 95% or more, therefore it is suitable for high power applications. Another advantage is that as it can operate at a high frequency and high power density is attained when a small size of the HF transformer is required. Moreover, the ESS is highly protected from the magnetic interference and from the DC link because of the galvanic isolation provided by the converter. However, the converter has the disadvantage of its dimensions which are heavier and larger in size due to the transformer and many components involved. The converter also has many switches and hence a complex control strategy is required compared to the other simple topologies. Therefore, the design and execution of such a converter is complicated [168], [185], [186]. 38 4.5 Dual Active Half-Bridge Bi-directional DC-DC Converter. In this type of topology, two transistor switches from the two converters of the dual active full bridge are replaced with capacitors and hence each converter becomes a half bridge as shown in Fig. 19. The idea of this topology is to reduce the number of the semiconductor switches used. 4.5 Dual Active Half-Bridge Bi-directional DC-DC Converter. 4.4 Dual Active Full-Bridge Bi-directional DC-DC Converter In this type of topology, two transistor switches from the two converters of the dual active full bridge are replaced with capacitors and hence each converter becomes a half bridge as shown in Fig. 19. The idea of this topology is to reduce the number of the semiconductor switches used. DC-Link (Traction Network) ESD Q1 Q2 C3 C4 C1 C2 S1 S2 N1 N2 L Cdc C Fig. 19: Dual Active Half-Bridge Bi-directional DC-DC Converter [62], [168]. DC-Link (Traction Network) Fig. 19: Dual Active Half-Bridge Bi-directional DC-DC Converter [62], [168]. This type of converter is commonly used in low power applications because of the less power transfer between the two sides of the converter when compared to the dual active full bridge. The converter operates in buck mode when energy flow from the DC-link (high voltage side) to the ESS DC bus (low voltage side) and in this case transistors S3 and S4 are conducting. When the energy flows in reverse direction, the converter operates in boost mode with the same transistors. The two capacitors on both sides of the HF transformer are called balancing capacitors which perform the functions of circulating the transformer winding currents during the operation, [62], [168], [187]. This converter topology has the advantage of providing fewer current ripples on the secondary side of the HF transformer (the ESS DC bus) and hence fewer current stresses in the switches. Moreover, it has a reduced number of transistors compared to the full bridge converter for the same power rating. However, this converter has a drawback of allowing load currents to circulate through the capacitors as mentioned previously. As for the transformer design and dimension, it has less power transfer ratio compared to the dual active full bridge [172], [188], [189]. 39 5 Energy Storage Systems Control and Energy Management As discussed earlier, the energy storage system consists of three components namely the energy storage device itself (ESD) which can be a supercapacitor, battery, flywheel or hybrid ESS, then a bidirectional dc-dc converter and a controller. The need for the control and energy management of the energy storage system applied to urban rail transit system is highly crucial because of the frequent and numerous vehicles acceleration and braking operations which result in a very high rate of charging and discharging cycles. The control and energy management of the ESS must provide an efficient, optimal recovery in re-using the regenerative braking energy while ensuring safe and reliable operations of the energy storage devices as well as the bidirectional DC-DC converter [10], [190]. Depending on what is required to be achieved in the traction network – energy saving or voltage stabilization, that is the indicator which control strategy should be applied to the ESS [62], [191]–[194]. In General, control of the ESS in recovering the braking energy is divided into two categories. The first category is the energy flow management among the energy storage devices, trains and the traction substations, and the second category is the control of the bidirectional DC-DC power converter that manages the energy flow to and from the energy storage device, [193]. The control strategies governing these two categories are applied onto a controller which provide the control actions and receive the required input parameters as shown in Fig. 4 in section 3 to ensure the overall control and energy management are achieved, [195]–[199]. In the following subsections, the controls for both energy storage devices and bidirectional DC-DC converters are presented. Some related research studies applying such control strategies are also presented. The aims of these energy storage and DC-DC converters controls are to achieve an efficient, optimal control and energy management for the ESS in urban railway systems. 5.1 Energy Storage Device Control The control and energy management for the energy storage device in electrified urban railway system is to regulate the amount of regenerative braking energy to be stored and to determine when to reuse it. These are governed by number of control strategies depending on the operating modes of the vehicles. 40 The control and energy management systems are also to ensure the energy storage devices are well protected against the full charge/discharge as well as preventing the entire system from oscillation [193]. For the normal operating mode, the energy storage devices are prepared to store the braking energy and also release the energy during the acceleration period and when the vehicle is passing through gaps (where no catenary line connection presence), [101], [200]. The manufacturers of energy storage products such as supercapacitors or batteries for energy savings and voltage regulation to be used in railway applications, provide only products’ specifications, drawings, functions and their advantages. However, they do not provide how to control the devices i.e., which control strategy or control technique is suitable for the optimal control and energy management. There are also no documentations on how to interface the storage devices to the traction network and which type of power converter to be used. The only solution provided to this is by the academic researchers who deal with the energy storage control strategies and the types of power converter topologies to be used [6], [201]. 5.2 Bidirectional DC-DC Converter Control The common control task in the bidirectional DC-DC converter as applied to the ESS for recuperating of regenerative braking energy is to control the power flow in both directions between the traction network and the energy storage device. The energy storage device cannot be connected directly to the traction DC-link for capturing the regenerative braking energy and releasing it back to enhance acceleration. This is because of their varying input and output conditions that are not suitable for railway applications [10]. However, if the energy storage device is connected directly to the DC-link, its utilization would be very minimal. Therefore, there is need for a power conversion system in the form of a bidirectional DC-DC converter to interface the energy storage device to the DC-link in order to manage the energy flow between the two (the DC-link and the storage device) [193]. Urban railway system is characterised as one of the high levels of power fluctuations resulting into intermittent operating conditions that require applications of fast response power converters to absorb the high spikes of the regenerative braking currents [193]. Moreover, during the operations, the DC- 41 link and the energy storage system both have varying voltage levels, and to overcome this a bidirectional power converter is required to serve as an interface between the two. These two main issues highly affect the steady and dynamic performance of the bidirectional DC-DC converter during system operation [193], [194]. Hence, there is a need for an efficient control strategy to ensure a stable operation of the bidirectional DC-DC converter in all working conditions and free from interferences. The converter must be controlled in order to supply the required voltages and currents to the energy storage device according to the designed energy management control strategy [195]. Fig. 23 shows the general structure of a feedback control system to control the bidirectional DC-DC converter. Control Strategy Bidirectional DC-DC Converter Controller Error Control Output Converter Output Desired Output (Input) (Output) Fig. 23: General Structure of Feedback Control System for the Bidirectional DC-DC Converter [172]. Control Strategy Bidirectional DC-DC Converter Controller Error Control Output Converter Output Desired Output (Input) (Output) Control Strategy Bidirectional DC-DC Converter Controller Error Control Output Converter Output Desired Output (Input) (Output) Controller Control Output Converter Output Desired Output Error Control Strategy (Input) Fig. 23: General Structure of Feedback Control System for the Bidirectional DC-DC Converter [172]. 5.2 Bidirectional DC-DC Converter Control The choice for the converter control method depends on the type of topology to be used and the nature of the control tasks required for a particular application. Therefore, for both isolated and non-isolated topologies and depending on an application’s control task, the commonly control strategies used for the converter control in the recovery of regenerative braking energy in urban railway systems are listed in the next subsections and some related applications of such control methods done in regenerative braking energy recovery as well as other areas. Generally, in regenerative braking applications, two main parameters become the target control variables for the DC-DC converter and these are the energy storage device current (IESD) and the traction DC-link voltage (also called network voltage, VDC-Link) [196], [197]. 42 5.2.1 Proportional Integral Derivative (PID) Control Method Due to its simplicity in design and implementation, PID control method is the most common, classic and traditional control strategy that is used to control various control problems for decades especially in the industry. In fact, over 90% of control loops found in the industries are controlled by using the PID or PI algorithms [202]. This control strategy is applied to either linear or nonlinear controllers in order to control and coordinate a closed loop control system to meet the required control targets. It plays a vital role in the control of processes, motor drive systems, power converters, and etc, [172]. Fig. 24 shows the PID feedback control system for the bidirectional DC-DC converter. Proportional, KP Bidirectional DC-DC Converter PID Controller Error Control Output Converter Output Desired Output (Input) (Output) Integral, KI Derivative, KD Fig. 24: PID Controller for the Converter Feedback Control System, [172], [203]. Control Output Converter Output Desired Output Error Fig. 24: PID Controller for the Converter Feedback Control System, [172], [203]. The basic principle of PID controller operation can be described by the following three-term control actions and with the controller transfer function: 𝐺𝑠𝐾𝐾𝐾𝑠 𝐺𝐺𝑐𝑐(𝑠𝑠) = 𝐾𝐾𝑃𝑃 + 𝐾𝐾𝐼𝐼 𝑠𝑠+ 𝐾𝐾𝐷𝐷𝑠𝑠 (1) (1) 𝐺𝐺𝑐𝑐(𝑠𝑠) = 𝐾𝐾𝑃𝑃 + 𝐾 𝐼𝐼 𝑠𝑠+ 𝐾𝐾𝐷𝐷𝑠𝑠 (1) 𝑢𝑢 (𝑡𝑡) = 𝐾𝐾𝑝𝑝𝑒𝑒(𝑡𝑡) + 𝐾𝐾𝐼𝐼 ∫𝑒𝑒(𝑡𝑡) 𝑑𝑑𝑑𝑑+ 𝐾𝐾𝐷𝐷 𝑑𝑑𝑑𝑑(𝑡𝑡) 𝑑𝑑𝑑𝑑 (2) 𝐾𝑃 𝑢𝑢 (𝑡𝑡) = 𝐾𝐾𝑝𝑝𝑒𝑒(𝑡𝑡) + 𝐾𝐾𝐼𝐼 ∫𝑒𝑒(𝑡𝑡) 𝑑𝑑𝑑𝑑+ 𝐾𝐾𝐷𝐷 𝑑𝑑𝑑𝑑(𝑡𝑡) 𝑑𝑑𝑑𝑑 (2) 𝐾𝑃 (2) 𝐾 (2) 𝐾 It could be seen from Eq. (1), the PID controller consists of three terms and these are the 𝐾𝐾𝑃𝑃 which is the proportional compensation, 𝐾𝐾𝐼𝐼 the integral compensation, and 𝐾𝐾𝐷𝐷 the derivative compensation. The controller output equation in time domain, 𝑢𝑢 (𝑡𝑡) in Eq. (2) shows the total sum for the control actions of the three-part on the error signal. With these three gains, a PID controller has been proved to guarantee an optimal control performance for a given closed – loop control system by ensuring high level of error reduction while maintaining a target level of stability and damping ratio, [204]. It could be seen from Eq. (1), the PID controller consists of three terms and these are the 𝐾𝐾𝑃𝑃 which is the proportional compensation, 𝐾𝐾𝐼𝐼 the integral compensation, and 𝐾𝐾𝐷𝐷 the derivative compensation. The controller output equation in time domain, 𝑢𝑢 (𝑡𝑡) in Eq. (2) shows the total sum for the control actions of the three-part on the error signal. 5.2.1 Proportional Integral Derivative (PID) Control Method With these three gains, a PID controller has been proved to guarantee an optimal control performance for a given closed – loop control system by ensuring high level of error reduction while maintaining a target level of stability and damping ratio, [204]. 43 Advantages and Disadvantages: One of the major advantages of the PID control method is, it can be used for various converter topologies as well as for most control problems. It has a simple functionality as well as easier to implement. Moreover, it can be used in combination with other control strategies to make the control scheme to be more robust and optimal control performances, [172]. On the other hand, one of the limitations of this control method is that it is a compulsory requirement to determine the dynamic model of the system to be controlled in order to design and implement the PID control strategy. Moreover, the models developed in most cases are based on assumptions which the parameters are not easy to measure and are in fact varying during working condition, [203], [204]. There are two main control issues with regard to PI control method for bidirectional DC-DC converter during charging and discharging of energy storage devices applications. During the discharging process (boost mode), the converter has nonminimum phase behaviour as its output voltage to duty transfer function has a Right- Hand Plane (RHP) zero and a pole P is in the Left-Hand Plane (LHP), [205]. During the charging process, (buck mode), a zero is in the Left-hand Plane while a pole P is in the RHP. The effect of RHP zero presents an undershoot in step response which limits the achievement of high dynamic performance of the system leading to instability while the LHP zero presents an unstable open-loop system [193], [205]. [205]. Applications: The most common control method usually applied to bidirectional DC-DC converter for recuperating of regenerative braking energy is the double closed – loop proportional – integral (PI) control strategy, in which the inner loop controls the current and the outer loop controls the voltage. This is because of the strategy has a simple functionality, parameter settlings, and easier to implement. 5.2.1 Proportional Integral Derivative (PID) Control Method 44 Applications: The most common control method usually applied to bidirectional DC-DC converter for recuperating of regenerative braking energy is the double closed – loop proportional – integral (PI) control strategy, in which the inner loop controls the current and the outer loop controls the voltage. This is because of the strategy has a simple functionality, parameter settlings, and easier to implement. In [193], hierarchical control strategies were proposed consisting of upper and lower levels. The upper- level control strategy was to control the supercapacitor energy management system while the lower- level control strategy was for the bidirectional DC-DC converter. The state machine control strategy was proposed for the energy management system whereas the double closed-loop PI control strategy was proposed for the bidirectional power converter. The outcome of the framework shows an outstanding performance and high disturbance rejection. In [195], the control of supercapacitor energy storage system was studied to provide an efficient energy management in traction network applications. 44 44 In the proposed control strategy, the researcher considered the effect of the inductor impedance on the stability of the system. In [197], a polynomial control strategy was proposed to control a bidirectional DC-DC converter between the DC-link and supercapacitor applied in a hybrid vehicle for power management applications. This polynomial strategy was compared to the conventional PI control strategy and the result exhibits that the system operated with a better performance and the strategy appears to be more precise and powerful compared to proportional integral control. In [206], the researchers outlined the classical linear control technique used to control the bidirectional DC-DC converters for energy storage systems which consists of two PI regulators (double closed-loop control) for controlling the voltage and current. This technique is easier to implement due to its simplicity but lacking in performance. 5.2.2 Sliding Mode Control Method Bidirectional DC-DC converters are inherently variable structure systems in their operations which constantly make their topologies to be changing from one form to another. As a result, they contain nonlinear elements in their dynamic equation which make the converters to be nonlinear systems. Moreover, the converters have an inherent chattering characteristic which is difficult to eliminate. The theory-based controllers, like the PID, require accurate mathematical models of the target control/plant and the controllers are also sensitive to parameters’ variations. In using such controllers for the design of nonlinear system, the system must first be linearized around its equilibrium point using method such as Taylor’s series, [207]. Hence, the models derived with such methods are approximate of the actual model due to the series of estimation within the process. The models derived from such methods do not account for perturbation and disturbances. Therefore, such controllers are not suitable for optimal control of nonlinear systems such as bidirectional dc-dc converters. In order to reliably control such nonlinear variable structure systems and by accounting for the perturbation and disturbance, there is a need to adopt nonlinear control strategies such as the sliding mode control, [172]. Sliding mode control is a type of nonlinear control strategies that can be used to control either linear or nonlinear systems because of its simplicity, fast and finite–time response, highly resistance to parameters’ variations, external perturbations and disturbances. The principle of sliding mode control 45 is to specify a virtual surface known as sliding surface where the system trajectory converges at an equilibrium point and a switching control input is applied to maintain the state trajectory on the sliding surface. The following equations show the principle of sliding mode control where the tracking error for a given system is calculated using the state vector 𝑥𝑥 and reference vector 𝑥𝑥𝑟𝑟, [208], [209]. 𝑥𝑥𝑥𝑟 𝑥𝑥̅ = 𝑥𝑥− 𝑥𝑥𝑟𝑟 (3) (3) (3) To design the sliding mode controller, the following equation is applied [208]: 𝑢𝑠𝑠𝑠𝑠𝑠 To design the sliding mode controller, the following equation is applied [208]: 𝑢𝑠𝑠𝑠𝑠𝑠 𝑢𝑢= 1 2 [1 + 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 (𝑠𝑠)] (4) (4) (4) rom Eq. (4), 𝑢𝑢 signifies the output controller action which is a function of s, the sliding surface. 𝑠𝑠= 𝑐𝑐𝑇𝑇 𝑥𝑥̅ (5) 𝑠𝑠= 𝑐𝑐𝑇𝑇 𝑥𝑥̅ (5) 𝑠𝑠= 𝑐𝑐𝑇𝑇 𝑥𝑥̅ Where c signifies the positive coefficient. The Lyapunov function is defined as shown in Eq. (6). 𝑉𝑠𝑠 Where c signifies the positive coefficient. 5.2.2 Sliding Mode Control Method The Lyapunov function is defined as shown in Eq. (6). 𝑉𝑠𝑠 𝑉𝑉(𝑠𝑠) = 0.5𝑠𝑠2 (6) 𝑉𝑉(𝑠𝑠) = 0.5𝑠𝑠2 (6) (6) The sliding mode controller is designed in such a way that the derivative of V(s) is always negative when the sliding surface, s is not zero, [208]. Fig. 25 shows a sliding mode feedback control system for the bidirectional DC-DC converter where the controller is seen to contain the sliding surface as the first block that receives the error signal and then the control law which in this case is the Lyapunov Stability Function that determines the switching functions for the converter. Bidirectional DC-DC Converter Sliding Mode Controller Error Control Output Converter Output Desired Output (Input) (Output) Control Law (Lyapunov Stability Function) Sliding Surface Fig. 25: Sliding Mode Controller for the Converter Feedback Control System, [172], [203]. Sliding Mode Controller Control Output Converter Output Desired Output Error Sliding Surface Fig. 25: Sliding Mode Controller for the Converter Feedback Control System, [172], [203]. 46 Advantages and Disadvantages: The advantages of the SMC include having fast response, easier to implement, highly robust against parameter changes and external perturbation and disturbances, [10], [11], capability of system order reduction, it does not require accurate mathematical models of the target control/plant, it can be designed to have first – order response irrespective of the system, suitable for systems with inherent variable structure like power converters. On the other hand, one main drawback of this control strategy is that it chatters along the sliding surface when trying to move to the sliding regime. It requires at least the knowledge of the range of system’s parameter variation for its effective implementation, [172], [204], [209]. Advantages and Disadvantages: The advantages of the SMC include having fast response, easier to implement, highly robust against parameter changes and external perturbation and disturbances, [10], [11], capability of system order reduction, it does not require accurate mathematical models of the target control/plant, it can be designed to have first – order response irrespective of the system, suitable for systems with inherent variable structure like power converters. On the other hand, one main drawback of this control strategy is that it chatters along the sliding surface when trying to move to the sliding regime. It requires at least the knowledge of the range of system’s parameter variation for its effective implementation, [172], [204], [209]. 5.2.2 Sliding Mode Control Method Applications: In [210], a sliding mode control strategy was applied to an electric drive in railway regenerative braking energy applications where the strategy was based on Lyapunov stability theorem designed and simulated in order to realize the system stability in all operating conditions. The strategy was applied to a bidirectional DC-DC converter (for charging/discharging the supercapacitor), and a unidirectional for charging the battery from the supercapacitor. The result revealed that the system stability was achieved together with the energy management system. In [211], a sliding mode control was used to control a coupled - inductor bidirectional DC-DC converter interfacing the supercapacitor energy storage system for the purpose of improving the converter’s performances. In this paper, a numerical application was used to a DC tramway system with stationary supercapacitor storage system (SESS). The results showed that the sliding mode control applied was highly resistive to external perturbations and disturbances. In [212], a sliding mode control was used to control a bidirectional DC- DC converter interfacing hybrid supercapacitor and battery energy storage system for recuperating of train kinetic energy based on kinetic energy estimation and supercapacitor internal voltage observation. The sliding control method improved the system stability and the DC-link voltage fluctuations/dynamics. The control method enabled the supercapacitor and battery currents to track their references effectively. In a related application, a single degree sliding mode control strategy was presented for controlling a bidirectional DC-DC converter in a standby renewable energy source which a solution based on adaptive equations was offered to overcome the problem of recalculation of the design equations in every cycle operation. However, the method presented, the converter losses were 47 47 not taken into account. Moreover, there was a chattering problem due to the first-degree level of the switching function used as against the second-degree level, [213]. 5.2.3 Model Predictive Control (MPC) Method MPC, also known as receding horizon optimization control (RHC), Dynamical Matrix Control (DMC) or Generalized Predictive Control (GPC) is one of the powerful advanced control methods that utilizes optimization control of tracking with constraints which is highly suitable for the control of power electronic converters and electric drives, [214]. It has been in industrial process applications since in the eighty’s (1980s) though the practical application of Dynamic Matrix Control started at early 1970’s by Shell Oil, [215]. It has a proven successful industrial application because of its stability proofs, excellent resource, and etc. Due to its excellent characteristics such as its simplicity, practicality, ease of implementation, and richness make it a successful method in control design. Moreover, it is highly suitable for systems or processes with problems that include time delays, variable control objectives, constraints contain in both manipulated and controlled variables, and with multiple number of controlled and manipulated variables, [216]. The principle of model predictive control strategy is that it uses a detailed nonlinear dynamic model of the system to be controlled and based on a predefined cost function of the system variables, a prediction of the system’s behaviour in the future is made for all possible input states. It has a fast-dynamic response and the state variables of the system follow a predefined value. It is also easier to execute once a cost function is established, [172], [204]. In terms of its application in power electronic converters, the design of the MPC controllers begins with the computation of future inputs sequence, and it is the first array of the future control inputs that is applied. The principles of MPC operation can be demonstrated by solving the optimization in a receding horizon as demonstrated in Eq. 7 below, [215], [216]: 𝐽𝑦𝜏𝑟𝑢𝜏𝑢𝜏 The principles of MPC operation can be demonstrated by solving the optimization in a receding horizon as demonstrated in Eq. 7 below, [215], [216]: 𝐽𝑦𝜏𝑟𝑢𝜏𝑢𝜏 min 𝐽𝐽= ∑ ൫𝑦𝑦 (𝜏𝜏)൯ 2 + 𝑟𝑟൫𝑢𝑢(𝜏𝜏) −𝑢𝑢(𝜏𝜏−1)൯ 2 ∞ 𝜏𝜏=𝑡𝑡+1 (7a) 𝑢𝜏𝑢 (7a) Subject to: |𝑢𝑢 (𝜏𝜏)| ≤ 𝑢𝑢𝑜𝑜 (7b) 𝑥𝑥 (𝑡𝑡+ 1) = 𝐴𝐴𝐴𝐴(𝑡𝑡) + 𝐵𝐵𝐵𝐵(𝑡𝑡) (7c) Subject to: |𝑢𝑢 (𝜏𝜏)| ≤ 𝑢𝑢𝑜𝑜 (7b) 𝑥𝑡𝐴𝐴𝑡𝐵𝐵𝑡 (7b) 𝑥𝑥 (𝑡𝑡+ 1) = 𝐴𝐴𝐴𝐴(𝑡𝑡) + 𝐵𝐵𝐵𝐵(𝑡𝑡) (7c) (7c) 48 𝑦𝑦(𝑡𝑡) = 𝐶𝐶𝐶𝐶(𝑡𝑡) (7d) 𝑦𝑦(𝑡𝑡) = 𝐶𝐶𝐶𝐶(𝑡𝑡) (7d) Eq. 7a represents the infinite horizon optimal control while equations Eq. 7c and Eq. 5.2.3 Model Predictive Control (MPC) Method 7d represent the system model with state vector x, input vector u, and output vector y. This set of equations is for the discrete MPC. The continuous MPC is similar in principle with the discrete MPC except that it is based on a continuous time model as described by equations Eq. 8,[215],[216]. 𝐽𝑦𝑡𝜏𝑦𝑡𝜏𝑢𝑡𝜏𝑑𝑑𝑇 min 𝐽𝐽= ∫൫|𝑦𝑦𝑟𝑟(𝑡𝑡+ 𝜏𝜏) −𝑦𝑦(𝑡𝑡+ 𝜏𝜏)|𝑄𝑄 2 + |𝑢𝑢(𝑡𝑡+ 𝜏𝜏)|𝑅𝑅 2൯𝑑𝑑𝑑𝑑 𝑇𝑇 0 (8a) 𝑔𝑢𝑡𝜏𝑥𝑡𝜏 (8a) Subject to: 𝑔𝑔൫𝑢𝑢(𝑡𝑡+ 𝜏𝜏), 𝑥𝑥(𝑡𝑡+ 𝜏𝜏)൯≤0 (8b) 𝑥𝐴𝐴𝐵𝐵 Subject to: 𝑔𝑔൫𝑢𝑢(𝑡𝑡+ 𝜏𝜏), 𝑥𝑥(𝑡𝑡+ 𝜏𝜏)൯≤0 (8b) 𝑥𝐴𝐴𝐵𝐵 Subject to: 𝑔𝑔൫𝑢𝑢(𝑡𝑡+ 𝜏𝜏), 𝑥𝑥(𝑡𝑡+ 𝜏𝜏)൯≤0 (8b) 𝑥𝐴𝐴𝐵𝐵 (8b) 𝑥𝑥̇ = 𝐴𝐴𝐴𝐴+ 𝐵𝐵𝐵𝐵 (8c) 𝑦𝑦= 𝐶𝐶𝐶𝐶 (8d) 𝑥𝑥̇ = 𝐴𝐴𝐴𝐴+ 𝐵𝐵𝐵𝐵 (8c) 𝑦𝑦= 𝐶𝐶𝐶𝐶 (8d) 𝑥𝑥̇ = 𝐴𝐴𝐴𝐴+ 𝐵𝐵𝐵𝐵 (8c) 𝑦𝐶𝐶 𝑥𝑥̇ = 𝐴𝐴𝐴𝐴+ 𝐵𝐵𝐵𝐵 (8c) 𝑦𝐶𝐶 (8c) 𝑦𝑦= 𝐶𝐶𝐶𝐶 (8d) 𝑦𝑦= 𝐶𝐶𝐶𝐶 (8d) (8d) Fig. 26 shows a model predictive feedback control system for the bidirectional DC-DC converter in which the model predictive controller’s optimization function and other elements to process the error as well as to send the control command to the converter. After beginning with the discrete time model of the Bidirectional DC-DC Converter MPC Error Control Output Converter Output Desired Output (Input) (Output) Model Optimizer Constraint Future I/P Cost Function Future I/P Predicted O/P Fig. 26: MPC Controller for the Converter Feedback Control System [172], [204]. Cost Function Constraint Control Output Converter Output Desired Output Error Future I/P Optimizer Fig. 26: MPC Controller for the Converter Feedback Control System [172], [204]. system, To begin with, the model of the system is first discretized and then the optimization and prediction systems are designed. At the prediction stage, measurements are obtained from the previous defined model while the future predicted value is being provided by a function of current values of the control variables for each mode of the converter operation. At the end of the MPC cycle operation, all predictions are sent to the optimization block where the solution of the optimization problem is done 49 online using the predicted values and the predefined cost function. Finally, the MPC provides the required optimal control actions with continuous cycle operations, [172]. Advantages and Disadvantages: MPC is a simple control strategy that is highly robust and is capable of handling multiple objectives. It is easy to maintain and modify the models or the specifications without necessarily redesigning the complete system. It explicitly accommodates constraints and use explicit model. 5.2.3 Model Predictive Control (MPC) Method It has clear tunning parameters which include the prediction horizon and optimization problem setup MPC compared to other control strategies, depends on the accuracy of the discrete-time model of the system to execute the MPC strategy. It also depends on the tuning of the MPC feedback control performance which is also a challenging task as it requires infinite prediction horizon or terminal constraints. Moreover, during its design process, all states may not be available, therefore, there is a need for an integrator feedback in order to handle the steady state error, and finally there is large angle deviation as a result of soft constraints and linearized model deficiencies, [172], [215],[216]. Applications: In [217], an explicit model predictive control was used to control the bidirectional DC- DC converter for supercapacitor energy storage system in Light Rail Vehicle in order to achieve real time control of the system and to reduce the amount of online computation. The optimal control problem with the system parameters and the constraints of the duty cycle are derived from the model that is based on v-resolution method which capable of capturing the hybrid nature of the supercapacitor in the form of piece wise affine. The model predictive control law was computed using multi-parameter programming. In [218], a model predictive control was used to control three-level bidirectional DC-DC converter for supercapacitor energy storage system in order to address the converter’s high voltage stress and large output current ripple. In this strategy, the DC-bus voltage and flying capacitor voltage are the control objectives, and the MPC optimizes the objectives using prediction modelling and reducing the objective function. The results showed that the high voltage stress has been reduced and the low output current ripple for the supercapacitor energy storage system have been achieved. In another application, a model predictive control strategy was used to control a bidirectional DC-DC converter for a battery energy storage system (BESS) in renewable energy system. The purpose of the strategy, was to overcome the fluctuation problem of the energy source output, and resulting a 50 smoothed output as well as a stable DC-bus voltage, [219]. In [220], MPC strategy was applied to control a bidirectional DC-DC converter that links a battery bank to the DC bus for the purpose of maintaining the DC bus voltage in a specified range. 5.2.3 Model Predictive Control (MPC) Method The results showed that the control strategy proved to reduce to minimize the DC bus voltage fluctuations by keeping the DC bus voltage within the predefined rated range. In [221], an accurate MPC was used to control a bidirectional DC-DC converter connected to DC distributed power systems. The outcome of the control strategy showed that the converter has achieved the desired transient response as well the required stability. 5.2.4 Fuzzy Logic Control (FLC) Method Fuzzy logic control (FLC), does not require an accurate mathematical model, and it is capable of working with both linear and nonlinear systems with imprecise inputs, and it is highly resistant to disturbance as compared to most nonlinear control strategies, [204],[209]. The fuzzy logic concept was first developed by Prof. L.A. Zadeh in the year 1965 from the University of California, USA. However, it was not practically implemented until in the year 1974 by Dr. E. H. Mamdani, a Prof. in London University, UK who used the concept into practice in controlling an automatic steam engine. Later in the year of 1976, the industrial implementation took place by the Blue Circle in England and SIRA in Denmark in controlling cement kiln. From 1980 onwards, several applications of FLC continued to diverge in other sectors which include but not limited to automobile manufacturing, automatic controls, manufacturing/process industries, academic institutions, hospitals, and etc, [203]. The principle of fuzzy logic control is based on fuzzy sets where a smooth transition of such sets take place from membership to non-membership not in a sudden manner. The first step in designing FLC is, to specify the input and output variables which are the state variables, their errors, and error variations with respect to their accumulation, [209]. In the operation of DC-DC power converters, the FLC input variables consist of output voltage/current error and/or accumulation/variation of the error. The FLC technique consists of the following consecutive steps which need to be implemented one after the other: 51 51 a. Fuzzification: is a process of converting crisp also called classic data (data understandable by computer machines) into the fuzzy data called membership functions (MFs) b. Fuzzy Inference Process: provides the fuzzy output by working on the MFs and the control rules. c. Defuzzification: is a step calculating each output which a different approach is used and the results are placed in table called the lookup table. For each application process, an output is picked up from the table based on the current input. c. Defuzzification: is a step calculating each output which a different approach is used and the results are placed in table called the lookup table. For each application process, an output is picked up from the table based on the current input. 5.2.4 Fuzzy Logic Control (FLC) Method The result of this control strategy enables the bidirectional DC-DC to have 90% efficiency and could regulate the battery charging and discharging current, while maintaining the output voltage at a constant value. In another application of electric vehicle systems, a comparison of FLC strategy was done against the Proportional Integral (PI). The fuzzy control proved to have better control performances over the PI control strategy, [224]. In [225], a fuzzy logic based control strategy was applied to a proposed bidirectional DC-DC in electric vehicle for the purpose of efficient energy management. 5.2.4 Fuzzy Logic Control (FLC) Method As a DC-DC converter is a nonlinear and time varying system, and in order to use the fuzzy control method for the converter control, the dynamic model, parameters or working conditions are not required but rather a well-defined qualitative knowledge of the system dynamics is required. As shown in Fig. 27, fuzzy control technique is explained [172], [203]. Bidirectional DC-DC Converter Fuzzy Logic Controller Error Control Output Converter Output Desired Output (Input) (Output) Inference System Fuzzifier Defuzzifier Knowledge Base Fig. 27: Fuzzy Controller for the Converter Feedback Control System [172], [204]. Control Output Converter Desired Output Error Bidirectional DC-DC Converter Output Inference System Defuzzifier Fuzzifier (Input) (Output) Fig. 27: Fuzzy Controller for the Converter Feedback Control System [172], [204]. Advantages and Disadvantages: The main advantage of the fuzzy controllers is that no prior knowledge is needed about the system parameters which applicable for those systems whose dynamic models are difficult or impossible to be obtained. They can be used for both linear and nonlinear control systems which makes them become robust controllers. The FLCs are almost resistant to system parameter fluctuations since they do not take into account their values. However, one of the major drawbacks of FLCs is that they are wholly dependent on human knowledge and expertise as there is a need for regular updating of the fuzzy rules. Moreover, the controllers require manual tuning and time-consuming retuning processes. There is a trade-off in the case of the controller performance robustness since in most cases it is not taken into consideration in the fuzzy logic tuning process, [ 202], [203], [209]. 52 52 Applications: In one of the fuzzy control applications, an energy consumption reduction from the substation for an electric elevator was achieved while ensuring the charging and discharging of the electric double layer capacitor (EDLC) are regulated. In that system, the EDLC stores the braking energy of the elevator, and then releases it back when the elevator accelerates during its operation, hence reduces the energy being drawn from the supply. With the help of the fuzzy control strategy, the EDLC stabilizes the DC-link voltage due to fluctuations caused by the power supply voltage variations, [222]. In [223], a fuzzy logic control strategy was used to control a bidirectional DC-DC converter for the purpose of optimizing battery performance in electric vehicle applications. 5.2.5 Artificial Neural Network (ANN) Method Unlike the traditional linear control systems, in which the key requirement of the controller design and implementation is that the plant/process’s mathematical model is mandatory and it must be in the form of algebraic and differential equations. These controllers suffer several drawbacks because of the assumptions made, parameter variations, nonlinear systems approximated to linear and time – invariant systems. Thus, these control challenges could be solved effectively by an artificial intelligence-based control strategies called the Artificial Neural Network (ANN) control. The ANN control is another intelligent control strategy that could deal with nonlinear systems or whose models cannot be obtained, and are highly insensitive to parameter variations. The strategy is based on learning the system/process by studying previous data provided by the system then it develops an appropriate control action to provide an optimal control in the system, [172], [204]. Fig. 28 shows ANN feedback control system for a bidirectional DC-DC converter. 53 ANN Controller Desired Model ANN Converter Model Bidirectional DC-DC Converter Control Input Model Error Converter Output Control Error Command Input Fig. 28: ANN Controller for the Converter Feedback Control System [204]. Desired Model ANN Converter Model Model Error Command Input Fig. 28: ANN Controller for the Converter Feedback Control System [204]. Advantages and Disadvantages: The main advantage of using artificial neural control methods is that does not require detailed information about the system or its analytical model (mathematical model) to be known. Instead, the control methods are based on learning by studying the previous data in controlling the system. Other advantages are, they are capable of handling large and complicated systems with complex control problems. Their design is based on linguistic information provided by the expert knowledge or real data of the system/plant. Controllers based on ANN do not require much tuning effort compared to the classical controllers. In addition to the linguistic information process design, a response-based information can be used in conjunction to carry out the design. An improved performance is achieved when ANN controllers are tuned appropriately. On the other hand, some of their disadvantages are similar to those of FLC strategies as both of them are intelligent-based control techniques. Other disadvantage is their designs are wholly dependent on linguistic information obtained from human knowledge and expertise or by using clustering method, and such information need to be updated from time to time, [172], [204]. 5.2.5 Artificial Neural Network (ANN) Method Applications: In [226], an artificial neural network control method was applied to a boost converter in order to improve its transient response performance by regulating the output voltage. The result of the simulation showed a fast-dynamic response which reduced the percent overshoot. In [227], ANN control was combined with an FLC strategy was applied to control the temperature of the water. The ANN control was combined in order to overcome the control problem which could not be fully solved by only using FLC control method. An improved performance was achieved by combining the two control strategies. In [228], a new ANN control strategy called Neural Network Predictor was applied 54 to control a multiple outputs DC-DC converter and the dynamic characteristics of this new method were compared to a traditional PID feedback control. The results of the new control method showed improved performances in terms of the convergence time of the output voltage, the undershoot voltage, and the overshoot voltage. In [229], an ANN control was applied to control a static DC-DC converter for an onboard electrical energy management in electric vehicle. In this method, the parameters update has been done by Levenberg-Marquardt method while the network validation by the model assumption Nonlinear Autoregressive with Exogenous input (NARX). The comparison between the ANN method and the classical linear control method showed that the ANN control method has a better control system stability. 5.2.6 Other Potential Control Methods Some other potential control methods worth to be mentioned, and which are used in the control of bidirectional DC-DC converter are Dynamic Evolution Control, Backstepping Control and Boundary Control. In dynamic evolution control, the aim is to minimize the error state by ensuring that the error is brought down zero level with respect to the time increase. One main advantage of this method is that no expert knowledge of the system model parameters is required [172]. In reference [230], it was used to control a bidirectional DC-DC converter that interfaced a supercapacitor energy storage system in a fuel cell electric vehicle system. The supercapacitor served as a secondary power supply to enhance the efficiency of the system by improving the fuel cell dynamic response. The bidirectional converter managed the power flows between the supercapacitor and the fuel cell DC-link. The backstepping control method is a nonlinear control strategy which employs the use of Lyapunov second method that enables it to provide robust control and stability of the control system. One main advantage of this method is that, it tracks the error to zero level irrespective of the external disturbances and variations in the parameters [204]. A boundary control strategy is also used in the control of bidirectional DC-DC converters because it has large signal trajectories. Boundary control is based on a technique applied to inverse problems with respect to control theory [172]. Table 4 presents the summary of the control methods discussed in this section able 4 presents the summary of the control methods discussed in this section 55 6 Discussion and Recommendation 56 Energy storage systems prove to be a viable solution among other methods for the utilization of regenerative braking energy in electrified urban railway system, which they are used in either onboard or stationary applications to maximise the use of such energy. The major features that make ESSs to have special priority over other methods are, the recovering of the train’s kinetic energy during dynamic braking (regenerative braking), they assist the vehicle with the stored regenerated energy during its acceleration process hence reducing of the energy consumption from the traction substation, a vehicle with onboard ESSs can freely move without having to be connected to the catenary line supply or in 56 the event of power outage or failure. The ESSs allow for peak power shaving hence power and size of the substations are reduced. As a system, it consists of three main components and those are the energy storage device itself, the bidirectional electronic DC-DC power converter, and a controller. The detail discussion and recommendation of energy storage technologies, topologies of DC-DC converter and control methods are presented below. 6.2 Power Electronic Converter Topologies For the bidirectional DC-DC power converter topologies, the selection of a particular topology depends on the energy storage technology and installation configuration to be used. For instance, in onboard applications, the size and weight of the converter need to be considered as smaller size and less heavier converters are required. However, these considerations do not matter in the case of stationary/wayside installation. The choice of the topology also depends on whether a galvanic isolation is required or not. The parameters that are considered in choosing a particular converter topology for an energy storage technology and for a given application are the efficiency, transfer voltage ratio, operating temperature range, galvanic isolation, number of transistor switches, magnitude of converter output ripple, and etc. For a non-isolated application, three converters are presented, the buck-boost DC-DC converters, interleaved buck-boost and cascaded buck-boost. Among these converters, cascaded buck-boost DC- DC converter can be considered to be the most suitable for recuperating of regenerative braking energy because of the advantages they have over the buck-boost and interleaved DC-DC converters. Although the cascaded buck-boost converter has twice number of power semiconductor switched over the conventional bidirectional buck-boost converter, it has the best performances in terms of high voltage gain ratio, very fewer current ripples and inductor current stresses. This also result in less current stresses in the semiconductor switches as well as other passive components. The interleaved buck-boos converter also has good performances but it has high number of switches and inductors, all of which make it heavier, expensive and requires a complex control algorithm over the cascaded. For the bidirectional DC-DC power converter topologies, the selection of a particular topology depends on the energy storage technology and installation configuration to be used. For instance, in onboard applications, the size and weight of the converter need to be considered as smaller size and less heavier converters are required. However, these considerations do not matter in the case of stationary/wayside installation. The choice of the topology also depends on whether a galvanic isolation is required or not. The parameters that are considered in choosing a particular converter topology for an energy storage technology and for a given application are the efficiency, transfer voltage ratio, operating temperature range, galvanic isolation, number of transistor switches, magnitude of converter output ripple, and etc. For a non-isolated application, three converters are presented, the buck-boost DC-DC converters, interleaved buck-boost and cascaded buck-boost. 6.1 Energy Storage Technologies In general, there are three storage devices available for the energy storage system in urban railway applications. They are supercapacitors, batteries and flywheels. The important key performance indicators used in describing and comparing the performances of the energy storage technologies are energy and power densities, efficiency, lifecycle, response rate, durability, and self-discharge rate. As discussed, and presented in the advantages, disadvantages, techno-economic comparisons, and Ragone plot, the energy and power densities are the most critical indicators to consider in making a selection for an energy storage device in regenerative braking applications especially in onboard ESS applications. Among the three storage devices, batteries possess the highest energy densities (especially the lithium-ion then followed by sodium sulphur), followed by supercapacitors and then flywheels. The compactness of Li-ion batteries makes them highly suitable for onboard applications over supercapacitors and flywheels. However, batteries present a very low power densities compared to supercapacitors and flywheels, and they have a long charging time. On the other hand, supercapacitors have a high-power density, long life cycle, and able to withstand high rate of charging and discharging cycles. Flywheels are known to have both high energy density as well as high power density compared to batteries which can also be applicable for regenerative braking energy recovery. In urban rail transit systems, there are frequent and numerous trains acceleration and braking operations especially during the peak periods, and resulting into frequent and numerous charging and discharging cycles. The characteristics of regenerative braking energy is for a short duration and high peak currents. With these urban rail features, it can be concluded that among the three energy storage devices, only supercapacitor seems to be a more suitable option for such applications because of its high-power 57 density, being able to absorb high and frequent charging and discharging cycles for such short durations, and also having a long-life cycle without failure. 6.2 Power Electronic Converter Topologies Among these converters, cascaded buck-boost DC- DC converter can be considered to be the most suitable for recuperating of regenerative braking energy because of the advantages they have over the buck-boost and interleaved DC-DC converters. Although the cascaded buck-boost converter has twice number of power semiconductor switched over the conventional bidirectional buck-boost converter, it has the best performances in terms of high voltage gain ratio, very fewer current ripples and inductor current stresses. This also result in less current stresses in the semiconductor switches as well as other passive components. The interleaved buck-boos converter also has good performances but it has high number of switches and inductors, all of which make it heavier, expensive and requires a complex control algorithm over the cascaded. For the isolated applications, the bidirectional dual active half-bridge DC-DC converter should be selected over the full-bridge because the former has a half of the switches’ quantity for the same power rating, having low transistor current stresses and low current ripples at the low voltage side. 58 6.3 Control for the Bi-directional DC-DC Converter The classical linear PID control method is limited in providing the optimal control required especially for systems like power converters which are nonlinear in their dynamic state and with high parameter variations during their operations. The sliding mode control method provides a better control over the classical PID method because it can deal with the nonlinearity issues but still the inherent chattering problem exists. The MPC also provides a better control but requires a well informative nonlinear dynamic of the converter in order to predict the future state for every switching vector. However, both FLC and ANN employ the learning methods from the knowledge of the converter dynamics in their control strategies which enable them to provide a highly robust control without the need to have the model of the converter. Therefore, among these control methods outlined for the DC-DC power converter, both FLC and ANN control methods can provide the best control compared to other methods due to the stated features. The recommendations derived from this review paper are as follows: The control methods of bidirectional power converter The FLC and ANN methods are hereby recommended to be used separately or combined over the classical linear PID control, sliding control, and MPC because of their excellent features. The FLC and ANN are suitable for systems like electronic power converter whose dynamic model are highly nonlinear, and for their ability to provide optimal control with no model of the converter required. Moreover, they are highly resistant to system parameter fluctuations compared to the other control methods. The storage technologies Generally, the supercapacitors are the best for onboard applications over batteries and flywheels because of their fast response to high rate of charging and discharging cycles, having high power density, and long-life cycles. However, Li-ion battery are able to compete in the future if its power density can be increased to the required level. The flywheels also have high energy and power densities but are limited to a number of applications because of the risk of explosion and shattering and they are heavier. On the other hand, a hybrid combination of supercapacitor and Li-ion energy technologies can give the best performance for onboard applications especially when catenary-free driving is required but by compromising the cost, complex control strategies, and weight of the system. As for the wayside applications, supercapacitor storage is recommended over batteries and flywheels due to its outstanding features such as fast response rate to charging and discharging, high power density, and etc. The second suitable storage device are batteries since there is no restriction for space 59 in wayside application, however, their lifetime limitation will be highly affected due to the characteristics of regenerative braking operations which have fast and high cycling frequency. For flywheels, they can also be used like the supercapacitors but for safety reasons, their use in such wayside application is also limited. The bidirectional DC-DC converter topologies The bidirectional DC-DC converter topologies The recommended topology according to this review is the cascaded configuration for non-isolated onboard and wayside ESSs applications. When isolation is required for wayside ESSs application, then a dual-active half-bridge DC-DC converter is recommended over the full-bridge configuration. The control methods of bidirectional power converter 7. Challenges and Future Research Direction 60 The most widely used ESSs in railway regenerative braking energy recovery are batteries and supercapacitors due to their simple implementation, accessibility, and weight, compared to flywheels and the rest of the energy storage technologies. However, they too have their associated issues which include but not limited to overcharging, over-discharging, internal self-discharge, short circuit, temperature, and etc [39], [83]. The bidirectional DC-DC converter, being a variable structure nonlinear dynamical system, is highly affected by the urban railway intermittent operating nature due to the shocking regenerative braking current, wide range variation of train load current, energy storage device voltage and the traction network voltage. All of these, highly influence both the steady and dynamic performances of the converter , [193]. Furthermore, the converter has some control challenges during the charging and discharging of the ESS. During the discharging operation, the converter has a 60 nonminimum phase behaviour as its output voltage to duty transfer function has a right-hand plane zero while pole in the left-hand plane. While in the charging mode, the behaviour reverses. The overall effect leads to system’s instability and limitation to achieving high dynamic system performance, [193], [205]. It could be observed that the majority of the literature in ESSs applications for the regenerative braking energy recovery in urban rail transit are concerned mostly with the energy management issues for the storage devices in terms overcharging and over discharging (SOCmin, SOCmax, Iesdmax, and Vesdmax) rather than optimal control of the bidirectional DC-DC converter that manages the power flow. In such studies, the converter operations were assumed to be ideal whereas those are not the case in real practice. nonminimum phase behaviour as its output voltage to duty transfer function has a right-hand plane zero while pole in the left-hand plane. While in the charging mode, the behaviour reverses. The overall effect leads to system’s instability and limitation to achieving high dynamic system performance, [193], [205]. It could be observed that the majority of the literature in ESSs applications for the regenerative braking energy recovery in urban rail transit are concerned mostly with the energy management issues for the storage devices in terms overcharging and over discharging (SOCmin, SOCmax, Iesdmax, and Vesdmax) rather than optimal control of the bidirectional DC-DC converter that manages the power flow. In such studies, the converter operations were assumed to be ideal whereas those are not the case in real practice. 7. Challenges and Future Research Direction Therefore, a deeper research work and analyses are needed for the bidirectional DC-DC power converter that link the traction network and the energy storage device in terms of suitable topology selection and the most efficient control strategy in recuperating the regenerative braking energy for ESSs application in urban railway system. These research work and analyses are important so that the braking energy is harvested maximally while ensuring the best energy management condition in the entire system. The future direction of these studies is to take into account the analyses and designs of both classical and intelligent control strategies for the bidirectional DC-DC converter in regenerative braking energy recovery applications, simulations and hardware experiments, and then comparisons of their performances. It is hoped that an optimal and robust control strategy in terms of the improved converter dynamic performances as well highly efficient energy management system can be recommended. 8 Conclusion In this paper, comprehensive reviews of the ESS technologies, relevant topologies of bidirectional DC- DC converters and control schemes being used in railway systems for regenerative braking energy recovery and management have been presented. Unlike most studies and reviews which are limited to only the ESS characteristics, configurations, advantages and disadvantages, this paper also reviewed in detail the applications of the storage technologies, power converters and control strategies in both academic research studies and commercial implementations. For each of these components, the most suitable technology has been recommended although, in most cases, this depends on the type of 61 application and ESS installation configuration. The energy storage technologies (supercapacitors, batteries, and flywheels) have been analysed and discussed in terms of their working principle, advantages, disadvantages, and applications. Their comparisons in terms of energy and power densities have been graphically represented and analysed by means of a Ragone plot. Moreover, the techno – economic comparisons of the storage devices have been presented and analysed in terms of key performance parameters like efficiency, durability, energy density, power density, self-discharge, cost per kW, cost per kWh. For the energy storage technologies, their applications in terms of installation configurations which could be either stationary (wayside/trackside) ESS installation or onboard installation also have been discussed. This study concludes that among the storage technologies, supercapacitor appears to be the most suitable choice in terms of regenerative braking energy recovery and management for all types of applications, followed by Lithium-ion batteries and then flywheels. For the bidirectional DC-DC converter topology selection, cascaded BDC is recommended for non-isolated type while dual active bridge (DAB) BDC is selected in the case of isolated type. It has been proven that in terms of the control methods for the bidirectional DC-DC converter, FLC and ANN have been recommended over the other control strategies for robust and stable control in regenerative braking energy recovery and management. Thus, the key contribution of this paper is the comprehensive reviews and analyses given for the energy storage system components in the recovery of regenerative braking in urban railway applications. The review also highlighted the key performance indicators, important parameters, possible solutions and recommendations for the three ESS components which may assist the researchers, academicians, manufacturers and other stakeholders in improving the energy efficiency and economic benefits in urban railway applications to the next level. onflicts of Interest: The authors declare no conflict of interest Conflicts of Interest: The authors declare no conflict of interest DATA AVAILABILITY STATEMENT DATA AVAILABILITY STATEMENT Data sharing not applicable to this article as no datasets were generated or analysed during the current study. ORCID Danlami Sadiq https://orcid.org/0000-0002-2948-9108 Azri H. Hasani https://orcid.org/0000-0002- 0285-2747 Acknowledgements The authors acknowledge the Ministry of Education, Malaysia for supporting this work under Universiti Tenaga Nasional with grant code LRGS/1/2018/UNITEN/01/1/2. 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Closed Gastroschisis

Journal of neonatal surgery

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Journal of Neonatal Surgery 2017; 6:61 Doi:10.21699/jns.v6i3.599 Journal of Neonatal Surgery 2017; 6:61 Doi:10.21699/jns.v6i3.599 Journal of Neonatal Surgery 2017; 6:61 Doi:10.21699/jns.v6i3.599 INTRODUCTION within normal limits. Echocardiography showed small patent foramen ovale. within normal limits. Echocardiography showed small patent foramen ovale. Gastroschisis is a congenital abdominal wall defect that is usually located on the right side of the umbili- cal cord, rarely; it is located in a mirror image position on its left side. Intestine, stomach, and occasionally other abdominal organs e.g. ovaries eviscerate through that defect [1]. In very rare cases, the defect itself closes around the eviscerated organs causing exit and/or entry level ischemia; a state which has been termed closed or closing gas- troschisis and is representing around 6% of all cases of gastroschisis [2]. Sometimes the whole midgut is lost, a condition termed vanishing midgut [3]. Figure 1: Extra-abdominal bowel remnant and x-ray showing small bowel obstruction. Figure 1: Extra-abdominal bowel remnant and x-ray showing small bowel obstruction. ABSTRACT Closed gastroschisis is a rare entity usually associated with intestinal atresia and short bowel syndrome. We report two cases of closed gastroschisis presenting with neonatal intestinal obstruction and para- umbilical evisceration without an abdominal defect. Key words: Closed gastroschisis; Closing gastroschisis; Short bowel; Intestinal atresia Closed Gastroschisis Closed Gastroschisis Mohammed Abdel-Latif1, Mohamed H. Soliman2, Khaled M. El-Asmar2,* Mohamed Abdel-Sattar2, Ibrahim M. Abdelraheem3, Ehab El-shafei2 1 Pediatric surgery unit, Helwan University 2 Pediatric surgery department, Ain Shams University 3 Pediatric surgery unit, Al Galaa Teaching Hospital How to cite: Abdel-Latif M, Soliman MH, El-Asmar KM, Abdel-Sattar M, Abdelraheem IM, El-shafei E. Closed gastroschisis. J Neonatal Surg. 2017;6:61. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence*: Dr. Khaled Mohamed EL-Asmar, 14 mostafa sadek el-rafeay, Heliopolis, Cairo, Egypt E mail: [email protected] © 2017, Abdel-Latif et al Submitted: 14-04-2017 Accepted: 30-06-2017 Conflict of interest: Nil Source of Support: Nil CASE SERIES The baby was explored on day 2 via circum-umbilical incision. There was small bowel atresia. Remaining small bowel was 30 cm of blind-ended dilated proximal jejunum and 40 cm of small caliber distal ilium. Two fibrous strings were connecting the mesentery of both jejunum and ilium to an area in the inner aspect of the abdominal wall corresponding to the extra-abdominal bowel remnant. There was no abdominal wall defect. There was associated colonic Case 1: A full term 3 kg male presented with extra- abdominal bowel remnant just to the left side of a normally appearing umbilicus (Fig 1). Antenatal scan prior to delivery revealed intra-abdominal dilated bowel loops. Clinically, there was upper abdominal distension and bilious vomiting. Initial resuscitation was initiated. Abdominal X-ray showed suspicion of jejunal obstruction. Laboratory investigations were The baby was explored on day 2 via circum-umbilical incision. There was small bowel atresia. Remaining small bowel was 30 cm of blind-ended dilated proximal jejunum and 40 cm of small caliber distal ilium. Two fibrous strings were connecting the mesentery of both jejunum and ilium to an area in the inner aspect of the abdominal wall corresponding to the extra-abdominal bowel remnant. There was no abdominal wall defect. There was associated colonic Correspondence*: Dr. Khaled Mohamed EL-Asmar, 14 mostafa sadek el-rafeay, Heliopolis, Cairo, Egypt E mail: [email protected] © 2017, Abdel-Latif et al Submitted: 14-04-2017 Accepted: 30-06-2017 Conflict of interest: Nil Source of Support: Nil Correspondence*: Dr. Khaled Mohamed EL-Asmar, 14 mostafa sadek el-rafeay, Heliopolis, Cairo, Egypt E mail: [email protected] © 2017, Abdel-Latif et al Submitted: 14-04-2017 Accepted: 30-06-2017 Conflict of interest: Nil Source of Support: Nil Closed Gastroschisis atresia type IIIa at the level of transverse colon. The extra-abdominal bowel remnant and the connecting fibrous strings were excised. Short tapering of the proximal dilated jejunum, then primary end to end anastomosis was fashioned. Double barrel colostomy was done at the site of the colonic atresia leaving a length of 70 cm viable small bowel in addition to an intact ileocecal valve. Oral feeding was started on 10th postoperative day which was increased gradually to full feed on day 28. He was discharged on day 30. early during pregnancy may close spontaneously around the herniated bowel and the supplying superior mesenteric artery resulting in strangulation and necrosis of the midgut. CASE SERIES Recently, marked improvement in the outcome has occurred due to advances in neonatal care and nutritional support and the introduction of home TPN regimens for pediatric age group [7]. Our survived case had ade- quate bowel length; 30 cm jejunum, 40 cm ilium, in addition to the viable part of the colon and ileocaecal valve. However, our second case succumbed to complications of management of short bowel syn- drome. Infants suffering gastroschisis and vanishing midgut have nearly 70% mortality. Recently, marked improvement in the outcome has occurred due to advances in neonatal care and nutritional support and the introduction of home TPN regimens for pediatric age group [7]. Our survived case had ade- quate bowel length; 30 cm jejunum, 40 cm ilium, in addition to the viable part of the colon and ileocaecal valve. However, our second case succumbed to complications of management of short bowel syn- drome. DISCUSSION Spontaneous prenatal closure of the anterior ab- dominal wall defect in gastroschisis is very rare. Closure of the defect can be complete or incomplete with variable sequelae affecting bowel loops [4]. In cases of mild affection, there is a closing abdominal defect with viable bowel. Next in severity are cases with complete closure of the defect with a tiny stalk connecting the intra-abdominal bowel to an extracorporeal bowel remnant. These forms are termed closing gastroschisis and closed gas- troschisis [2]. CASE SERIES Second, the herniated fetal bowel may incarcerate within the defect leading to its ischemia and atrophy with subsequent closure of the defect. Third scenario starts by volvulus of the midgut with subsequent infarction, resorption, and closure of the defect. Houben et al in 2009 clarified the criteria for successful antenatal ultrasonographic follow up in patients with gastroschisis [6]. They stated that after diagnosis of gastroschisis, fetal progress is monitored at 4 to 6 weeks intervals. More frequent scans were required in cases of intraabdominal bowel dilatation (>10 mm) or growth restriction of the fetus. The key for diagnosing closing gastroschisis is increased or persisted intraabdominal bowel dilatation with hyperperistalsis. At the same time, there is shrinkage or no increase in the size of extracorporeal bowel. After birth, they defined clos- ing gastroschisis as circumferential or partial (>50%) closure of the ring around the protruding bowel associated with intestinal atresia, bowel ischemia, bowel necrosis, or viable intestine. No defect could be detected in our cases that could be explained by the delay in the initial ultrasound; however, progressive intraabdominal bowel dilatation was detected. Case 2: A preterm (33weeks) male neonate weighing 1.9kg presented with extra-abdominal bowel rem- nant, 6 cm long, located to the right margin of an intact umbilical ring. Antenatal scan at 30 weeks of gestation showed dilated bowel loops. Follow-up scan performed 2 weeks later showed progressive dilatation of bowel loops without any abdominal wall defect. The baby developed neonatal intestinal obstruction. X-ray abdomen was suggestive of jeju- nal obstruction. On exploration, the jejunum was ending blindly 20 cm after the DJ junction. The eviscerated bowel remnant was connected from in- side with the colon by a short fibrous stalk. There was 30 cm of non-atretic colon until the peritoneal reflection. Primary end-to end jejuno-colic anastomosis was performed. The patient was put on management for short bowel. One month later, the patient was re-explored due to partial intestinal obstruction. Second exploration revealed an ectatic poorly functioning jejunal loop. Side to side jejuno- colic anastomosis was done for better bowel drain- age. The baby died at the age of 4 months due to cholestatic liver failure and sepsis a complication of TPN. Infants suffering gastroschisis and vanishing midgut have nearly 70% mortality. 7. Vogler SA, Fenton SJ, Scaife ER, Book LS, Jackson D, Nichol PF, et al. Closed gastroschisis: total parenteral nutrition-free survival with aggressive attempts at bowel preservation and intestinal adaptation. J Ped Surg. 2008; 43:1006-10. Journal of Neonatal Surgery Vol. 6; 2017 REFERENCES 1. Chabara S, Gleason CA. Gastroschisis: embryology, pathogenesis, epidemiology. NeoReviews. 2005; 6:493-9. 2. Davenport M, Haugen S, Greenough A, Nicolaides K. Closed gastroschisis: Antenatal and postnatal features. J Pediatr Surg. 2001; 36:1834-7. 3. Johnson N, Lilford R, Irving H, Crabbe D, Cartmill R. The vanishing bowel. Case report of bowel atresia following gastroschisis. Br J Obstet Gynaecol. 1991; 98:214-5. The sequence of events is not well known. It is not clear whether the closure of the defect is the primary event or occurs secondary to loss of the eviscerated bowel. Bhatia and colleagues in1996 proposed 3 scenarios [5]. All proposals attributed bowel atresia to vascular insult. First, abdominal wall defect seen 4. Basaran UN, Inan M, Gücer F, Yardim T, Pul M. Prenatally closed gastroschisis with midgut atresia. Pediatr Surg Int. 2002; 18:550-2. 4. Basaran UN, Inan M, Gücer F, Yardim T, Pul M. Prenatally closed gastroschisis with midgut atresia. Pediatr Surg Int. 2002; 18:550-2. 5. Bhatia AM, Musemeche CA, and Crino JP. Gastroschisis complicated by midgut atresia and closure of the defect in utero. J Ped Surg. 1996;31:1288-9. 5. Bhatia AM, Musemeche CA, and Crino JP. Gastroschisis complicated by midgut atresia and closure of the defect in utero. J Ped Surg. 1996;31:1288-9. Journal of Neonatal Surgery Vol. 6; 2017 6. Houben C, Davenport M, Ade-Ajayl N, Flack N, Patel S. Closing gastroschisis. diagnosis, management, and outcomes. J Ped Surg. 2009 ; 44:343-7.

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Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.)

GenomeBiology.com

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UCLA UCLA Previously Published Works Title Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) Permalink https://escholarship.org/uc/item/8569w8zd Journal Genome Biology, 14(5) ISSN 1465-6906 Authors Ming, Ray VanBuren, Robert Liu, Yanling et al. Publication Date 2013-05-10 DOI http://dx.doi.org/10.1186/gb-2013-14-5-r41 Peer reviewed UCLA UCLA Previously Published Works Title Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) Permalink https://escholarship.org/uc/item/8569w8zd Journal Genome Biology, 14(5) ISSN 1465-6906 Authors Ming, Ray VanBuren, Robert Liu, Yanling et al. Publication Date 2013-05-10 DOI http://dx.doi.org/10.1186/gb-2013-14-5-r41 Peer reviewed Open Access Open Access RESEARCH Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) Ray Ming1,2*†, Robert VanBuren2†, Yanling Liu1†, Mei Yang1†, Yuepeng Han1, Lei-Ting Li2,3, Qiong Zhang1,2, Min-Jeong Kim4, Michael C Schatz5, Michael Campbell6, Jingping Li7, John E Bowers8, Haibao Tang9, Eric Lyons10, Ann A Ferguson11, Giuseppe Narzisi5, David R Nelson12, Crysten E Blaby-Haas13, Andrea R Gschwend2, Yuannian Jiao7,14, Joshua P Der14, Fanchang Zeng2, Jennifer Han2, Xiang Jia Min15, Karen A Hudson16, Ratnesh Singh17, Aleel K Grennan2, Steven J Karpowicz18, Jennifer R Watling19, Kikukatsu Ito20, Sharon A Robinson21, Matthew E Hudson22, Qingyi Yu17, Todd C Mockler23, Andrew Carroll24, Yun Zheng25, Ramanjulu Sunkar26, Ruizong Jia27, Nancy Chen28, Jie Arro2, Ching Man Wai2, Eric Wafula14, Ashley Spence2, Yanni Han1, Liming Xu1, Jisen Zhang29, Rhiannon Peery2, Miranda J Haus2, Wenwei Xiong30, James A Walsh2, Jun Wu3, Ming-Li Wang27, Yun J Zhu27,31, Robert E Paull28, Anne B Britt32, Chunguang Du30, Stephen R Downie2, Mary A Schuler2,33, Todd P Michael34, Steve P Long2, Donald R Ort2,35, J William Schopf36, David R Gang4, Ning Jiang11, Mark Yandell6, Claude W dePamphilis14, Sabeeha S Merchant13, Andrew H Paterson7, Bob B Buchanan37, Shaohua Li1* and Jane Shen-Miller36* Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Abstract Background: Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan. Results: The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment. Conclusions: The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots. © 2013 Ming et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 2 of 11 Page 2 of 11 Additional file 1). The nine anchored megascaffolds have a combined size of 543.4 Mb, accounting for 67.6% of the genome assembly, and they are mostly proportional to the karyotype of the lotus chromosomes (Figure S2 and S3 in Additional file 1). The high quality of the lotus gen- ome assembly is largely due to the unexpected homozyg- osity of the ‘China Antique’ variety. Repeat content of the sacred lotus genome p g Repetitive sequences account for 57% of the assembled genome, including 47.7% recognizable transposable ele- ments (Table S4 in Additional file 1). Unlike most plants, which exhibit relatively inconsequential non-long terminal repeat retrotransposons (approximately 1% of the genome) [7-9], such non-long terminal repeat retro- transposons contribute 6.4% to the lotus genome. Differ- ing from other plants that usually have more Gypsy-like elements [9,10], Copia and Gypsy-like elements are comparable in copy number and genomic fraction in lotus. Most major DNA transposon families are detected in sacred lotus (occupying 16% of the lotus genome), albeit with more than 10-fold variation in relative abun- dance. An exception, the Tc1/Mariner super-family, is absent from both the lotus and grape genomes [7], sug- gesting the frequent loss of this family of elements. Surprisingly, hAT (Ac/Ds-like) elements contribute to nearly 7% of the lotus genome, represented by more than 100,000 copies, more than in any other sequenced plant genome. Of these, CACTA elements are least abundant (0.4%) while MULE, PIF and Helitron ele- ments have amplified to a moderate degree (2.5%, 2.7% and 3.6%, respectively). The lotus genome further includes 1,447 Pack-mutator-like elements that carry genes or gene fragments [11]. Analysis using expressed sequence tags (ESTs) indicated that at least 10 Pack- mutator-like elements are expressed, suggesting that they may play functional roles. Here, we report the sequencing and analysis of the sacred lotus genome, which descends from the most ancient lineage of angiosperms. We have studied the evolutionary history of the genome and genes involved in relevant processes governing the unique features of this ancient land plant, including its adaptation to aqua- tic environments. Abstract Although lotus is an out-crossing plant, its cultivation and vegetative propaga- tion via rhizomes over the past 7,000 years may have imposed a narrow genetic bottleneck. This could be partly the consequence of its unique feature, seed longev- ity, which might have further reduced the number of generations in its evolutionary history in addition to vegetative propagation. The estimated heterozygosity in ‘China Antique’ is 0.03%, lower than the 0.06% of the sequenced papaya cultivar ‘SunUp’ after 25 generations of inbreeding [6]. The estimated heterozygosity in the American lotus N. lutea ’AL1’ variety is 0.37%, also low. Background Sacred lotus, so named because of its religious signifi- cance in both Buddhism and Hinduism, belongs to the small plant family Nelumbonaceae, with only one genus, Nelumbo, and two species: N. nucifera (Asia, Australia, Russia) and N. lutea (eastern and southern North Amer- ica) [1]. Lotus is in the eudicot order Proteales, which lies outside of the core eudicots (Figure S1 in Additional file 1); its closest relatives are shrubs or trees belonging to the families Proteaceae and Platanaceae. Lotus was a land plant that has adapted to aquatic environments. Used as a food for over 7,000 years in Asia, lotus is cultivated for its edible rhizomes, seeds and leaves. Its buds, flowers, anthers, stamens, fruits, leaves, stalks, rhizomes and roots have been used as herbal medicines for treatment of cancer, depression, diarrhea, heart pro- blems, hypertension and insomnia [2,3]. Its seeds have exceptional longevity, remaining viable for as long as 1,300 years, and its vegetative rhizomes remain healthy for more than 50 years [1,2]. The nanoscopic closely packed protuberances of its self-cleaning leaf surface have been adapted in Europe for the manufacture of a ‘self-cleaning’ industrial paint, Lotusan. The use of this paint results in the so-called lotus effect that is now widely advertised for self-cleaning automobiles, buildings and fabrics. Genome sequencing and assembly We sequenced the genome of the sacred lotus variety ‘China Antique’ with 94.2 Gb (101×) Illumina and 4.8 Gb (5.2×) 454 sequences. The final assembly includes 804 Mb, 86.5% of the estimated 929 Mb lotus genome [4]. The contig N50 is 38.8 kbp and the scaffold N50 is 3.4 Mbp (Table S1 in Additional file 1). The largest 429 scaffolds account for 94.8% of the assembled genome and 98.0% of the annotated genes. Among the 39 plant gen- omes published to date, the median N50 scaffold length is about 1.3 Mb, making lotus the eighth best assembled genome (Table S2 in Additional file 1). We constructed a high-density genetic map using 3,895 sequence-based restriction-associated DNA sequencing markers and 156 simple sequence repeat markers [5]. The former were sorted into 562 co-segregating bins and a total of 698 informative markers were mapped into nine linkage groups for the eight lotus chromosomes, with one gap remaining between two linkage groups (Table S3 in Genome annotation and gene expression Following repeat-masking and annotation, we inferred 26,685 protein-coding genes in lotus, including all 458 core eukaryotic proteins [12]; 82% of the genes have similarity to proteins in SwissProt as identified by Basic Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 3 of 11 Local Alignment Search Tool (E < 0.0001). The average gene length is 6,561 bp with median exon and intron lengths of 153 bp and 283 bp, respectively (Table S1 in Additional file 1). The average gene density is one gene per 30 kb, with genes spread more evenly over the assembled genome than in many other plant genomes (Figure S2 in Additional file 1), which are characterized by gene-rich regions often found at the distal regions of chromosomes arms. A total of 12,344 ESTs were aligned to 11,741 gene models, and 174 alternative splicing events were identified from 164 genes involving 380 EST contigs (Table S5 in Additional file 1). Of the anno- tated genes in lotus, 22,803 (85.5%) show expression in rhizomes, roots, leaves or petioles based on RNAseq data (Figure S4 in Additional file 1). Expression of the remaining genes is likely confined to seeds, flowers and other unsurveyed tissues. Expression of 3,094 protein- coding genes was tissue-specific, including 1,910 genes showing expression only in rhizomes and 841 only in roots; 14,477 genes are expressed across all tissues sur- veyed. Of the 1,910 rhizome-specific genes, we found several AP2-like ethylene-responsive transcription fac- tors, BTB/POZ domain-containing proteins, heat shock proteins, homeobox transcription factors, kinesins and pentatricopeptide repeat-containing proteins (PPRs) (Table S6 in Additional file 1). In lotus, 544 genes were annotated as PPRs, with 201 of these expressed in the four tissues tested, and 199 only expressed in the rhizome. PPRs have been identified as a group of RNA-binding pro- teins involved in RNA processing, stability, editing, maturation and translation in plants. Although the mole- cular mechanism of their function has not yet been eluci- dated, their broad expression in lotus rhizome is notable. We reconstructed the ancestral gene content at key nodes of the evolutionary series, as well as the adapta- tional changes occurring along the branches leading to these nodes: the greatest changes observed in orthogroup presence and absence are specific to terminal lineages (Tables S8 and S9 in Additional file 1 and Figure 1). Synteny and genome evolution l f h A major evolutionary force shaping genome architecture in angiosperms is whole genome duplication (WGD) [14,15]. This process is followed by the ‘diploidization’ of genome organization through rearrangement, and of gene content through ‘fractionation,’ or homeologous gene loss. Intragenomic analysis of lotus indicates that it has experienced at least one WGD (paleotetraploidy, see Figure S6 in Additional file 1), named l, but implies that the Nelumbo lineage did not experience g, the paleo- hexaploidy (triplication) event around 125 million years ago detected in all other sequenced eudicot genomes [6,16-20]. Using lotus as a reference, as many as three post-g grape subgenomic copies are equally evident, the syntenic regions of which show extensive collinearity of homologous genes (Figure 2). Among the 87.1% of the lotus genic regions retained from this duplication, 5,279 (33.3%) are singletons, 8,578 (54.1%) are duplicated, and 2,007 (12.6%) have more than three homeologs, imply- ing there may have been additional paleo-duplications (Table S10 in Additional file 1). Based on three lines of evidence, the lineage nucleotide substitution rate in lotus is about 30% slower than that of grape, widely used in angiosperm comparative genomics due to its basal phylogenetic position in rosids, slow mutation rate, and lack of reduplication. First, while phy- logenetic evidence firmly dates the lotus-grape diver- gence before the pan-eudicot g triplication affecting only grape, synonymous substitution rates (Ks) between gen- ome-wide lotus-grape syntelog pairs (Figure S7 in Addi- tional file 1) are smaller than those among triplicated grape genes. Second, the lotus lineage mutation rate also appears slower (about 29.26% slower) than that of Vitis based on a maximum-likelihood tree of 83 plastid genes [21] and expert dating of the respective speciation events [22] using the r8s program [23] with penalized likelihood. Third, the lotus genome has retained more ancestral loci following its lineage-specific WGD. Lotus is a basal eudi- cot, and its genome is the one from the most ancient lineage of angiosperm sequenced to date (Figure S1 in Additional file 1). Lotus represents an even better model than grape for inferences about the common ancestor of eudicots. Genome annotation and gene expression More than three times as many orthogroup gains occur in the lineage leading to all eudicots, as compared to core eudicots (Figure S5 in Additional file 1), an increase second only to that of the grasses. Ortholog classification and ancestral gene content in eudicots The protein-coding gene sets from lotus and 16 other sequenced angiosperm species were used to identify putative orthologous gene clusters with Proteinortho v4.20 [13]. A total of 529,816 non-redundant genes were classified into 39,649 orthologous gene clusters (orthogroups) containing at least two genes (Table S7 in Additional file 1). Of the 26,685 protein-coding genes in lotus, 21,427 (80.3%) were classified into 10,360 orthogroups, of which 317 contained only lotus genes. From this gene classification, we estimate a minimum gene set of 7,165 genes in 4,585 orthogroups for eudi- cots (Table S7 in Additional file 1). The minimum gene set for core eudicots (7,559 genes in 4,798 orthogroups) is only slightly larger than the eudicot-wide set, suggest- ing that the minimal gene set of the eudicot-monocot ancestor (6,423 genes in 4,095 orthogroups) would add at least 490 orthogroups associated with the eudicots as a whole. Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 4 of 11 Figure 1 Orthogroup dynamics in lotus and other angiosperm genomes. Ancestral gene content and gene family (orthogroup) dynamics in lotus and other eudicot and monocot genomes identify expansion of the number of gene families and gene content associated with the ancestral eudicot. Figure 1 Orthogroup dynamics in lotus and other angiosperm genomes. Ancestral gene content and gene family (orthogroup) dynamics in lotus and other eudicot and monocot genomes identify expansion of the number of gene families and gene content associated with the ancestral eudicot. The remarkably slow mutation rate in lotus compli- cates the dating of the l duplication. l-duplicated lotus genes have a median synonymous substitution rate (Ks) of 0.5428, corresponding to an age of 27 million years ago (MYA) on the basis of average rates in plants [24] or 54 MYA on the basis of the grape lineage rate (Figure S7 in Additional file 1). Because lotus diverged from its clo- sest sister lineage approximately 135 to 125 MYA [21], before the g triplication, this suggests that the mutation rate in lotus is much lower than that in grape, and that the lotus-specific WGD event occurred about 65 MYA with a range between 76 and 54 MYA. This date coin- cides with the Cretaceous-Tertiary mass extinction that led to the loss of roughly 60% of plant species [25]. Ortholog classification and ancestral gene content in eudicots Polyploidization has been associated with increased adap- tation and survivability, and the numerous plant species inferred to have undergone polyploidy within this time- frame suggests a possible advantage to polyploid lineages during the Cretaceous-Paleogene transition, an interpre- tation supported by the l duplication in lotus. By tracing the phylogenetic histories of 688 pairs of grape genes in 528 orthogroups from each of the g dupli- cation blocks [26], we tested the timing of the g paleohexa- ploid event that has been observed in the genomes of Vitis [7], papaya [6], Populus [20] and other core eudicots [14,17]. About 50% of the resolved trees support the Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 5 of 11 Figure 2 High resolution analysis of syntenic regions of Nelumbo nucifera (Nn1/Nm2) and Vitis vinifera (Vv1/Vv2/Vv3). Synteny regions were identified from Figure S5 in Additional file 1. Gene models are arrays in middle of each panel; Colored boxes and lines connect regions of sequence similarity (LastZ) for protein-coding sequences between pair-wise comparisons. Figure 2 High resolution analysis of syntenic regions of Nelumbo nucifera (Nn1/Nm2) and Vitis vinifera (Vv1/Vv2/Vv3). Synteny regions were identified from Figure S5 in Additional file 1. Gene models are arrays in middle of each panel; Colored boxes and lines connect regions of sequence similarity (LastZ) for protein-coding sequences between pair-wise comparisons. Figure 2 High resolution analysis of syntenic regions of Nelumbo nucifera (Nn1/Nm2) and Vitis vinifera (Vv1/Vv2/Vv3). Synteny regions were identified from Figure S5 in Additional file 1. Gene models are arrays in middle of each panel; Colored boxes and lines connect regions of sequence similarity (LastZ) for protein-coding sequences between pair-wise comparisons. of g block duplications were eudicot-wide [26], even though the signal is primarily observed in core eudicots (Figure 3). timing of the g event to have occurred ‘core-eudicot-wide’ after the divergence of lotus, consistent with synteny ana- lysis. By contrast, gene family phylogenies for about half of the g block duplications include lotus genes (Table S11 in Additional file 1), although, in rare cases, duplicated monophyletic groups contain both lotus and eudicot-wide genes. Ortholog classification and ancestral gene content in eudicots (A) Summary of polyploidy events in the history of angiosperm evolution, with a focus on the possible phylogenetic origins of the three subgenomes comprising the gamma paleohexaploidy event in core eudicots. Synteny analysis of the Nelumbo genome indicates that gamma is shared only within the core eudicots; however, phylogenomic analysis suggests a more complex history since around half of the gamma pairs were duplicated core-eudicot-wide and the other half eudicot- wide (See Table S10 in Additional file 1). AA, BB, and CC are three subgenomes of the ancestral hexaploidy. Three possible phylogenetic origins of the ancestral AA genome involved in gamma are denoted by 1, 2 and 3. Lamda is defined as the most recent polyploidy event in the evolutionary history of Nelumbo. All the other Greek symbols are well-known polyploidy events in the evolutionary history of angiosperms. Gamma: genome-triplication (hexaploid) event in core eudicot genomes [7,23]; Sigma and rho: genome duplications detected in grass genomes [8]; Epsilon: angiosperm-wide duplication detected in large-scale gene family phylogenies. Based on gene tree phylogenomics, we hypothesize that the triplication event involved a tetraploid event (BBCC red star) first, then subgenome AA combined with BBCC to form hexaploidy AABBCC (blue dashed line). (B) Predicted gene tree topologies of hypothetical origins of the AA subgenome of the gamma paleohexaploidy. A, B, C indicate surviving genes inherited from AA, BB, CC subgenomes of the AABBCC ancestral hexaploidy. N indicates genes of Nelumbo. comprised of grasses (Poales) and palms (Arecales), was associated with relatively large gains in gene family num- ber and size. proteins in lotus compared to other plants is attributed to expansions in COG2132, a family of multi-copper oxidases. Most plant genomes encode one or two mem- bers of COG2132, whereas lotus has at least 16 members due to WGD and repeated tandem duplications (Figure 4, and see Figure S8 in Additional file 1). The only COG2132 members in Arabidopsis, LPR1 and LPR2, are involved in phosphate starvation signaling in root meristems. Simi- larly, in lotus, expression of COG2132 family members is confined largely to the roots (Figure 4). The lotus-specific expansion appears to form a separate phylogenetic clade from the LPR1 and 2-like proteins, suggesting a novel Ortholog classification and ancestral gene content in eudicots This is consistent with an earlier phylogenomic analysis using data from numerous plant genomes and basal eudicot transcriptomes, suggesting that 18% to 28% Such data suggest that a relatively large amount of genetic novelty is specifically associated with eudicots as a whole, even though the core eudicots shared a genome- triplication after divergence from the basal eudicots. By contrast, in monocots it appears that the evolution of the grass family specifically, rather than the earlier node Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 6 of 11 Figure 3 Polyploidy events in the history of angiosperm evolution. (A) Summary of polyploidy events in the history of angiosperm evolution, with a focus on the possible phylogenetic origins of the three subgenomes comprising the gamma paleohexaploidy event in core eudicots. Synteny analysis of the Nelumbo genome indicates that gamma is shared only within the core eudicots; however, phylogenomic analysis suggests a more complex history since around half of the gamma pairs were duplicated core-eudicot-wide and the other half eudicot- wide (See Table S10 in Additional file 1). AA, BB, and CC are three subgenomes of the ancestral hexaploidy. Three possible phylogenetic origins of the ancestral AA genome involved in gamma are denoted by 1, 2 and 3. Lamda is defined as the most recent polyploidy event in the evolutionary history of Nelumbo. All the other Greek symbols are well-known polyploidy events in the evolutionary history of angiosperms. Gamma: genome-triplication (hexaploid) event in core eudicot genomes [7,23]; Sigma and rho: genome duplications detected in grass genomes [8]; Epsilon: angiosperm-wide duplication detected in large-scale gene family phylogenies. Based on gene tree phylogenomics, we hypothesize that the triplication event involved a tetraploid event (BBCC red star) first, then subgenome AA combined with BBCC to form hexaploidy AABBCC (blue dashed line). (B) Predicted gene tree topologies of hypothetical origins of the AA subgenome of the gamma paleohexaploidy. A, B, C indicate surviving genes inherited from AA, BB, CC subgenomes of the AABBCC ancestral hexaploidy. N indicates genes of Nelumbo. Fi 3 P l l id t i th hi t f i l ti (A) S f l l id t i th hi t f i Figure 3 Polyploidy events in the history of angiosperm evolution. Adaptation to an aquatic environment Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. function not found in Arabidopsis (Figure 4, and see Figure S8 in Additional file 1). Several other gene families also show unusual compo- sitions that may reflect adaptation to aquatic lifestyles. Adaptation to an aquatic environment Submersed plant growth presents unique physiological challenges. Lotus has had to evolve novel features to cope with its aquatic lifestyle. Possible adaptations include an astonishing number of putative copper-dependent pro- teins, of which 63 proteins contain at least one COX2 domain, 55 contain a ‘copper-binding-like’ domain, and 4 contain polyphenol oxidases. The abundance of copper Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 7 of 11 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. Figure 4 Lotus-specific expansion in LPR1/LPR2 proteins. (A) The number of LPR1/LPR2 homologs in land plants. Homologs detected by Basic Local Alignment Search Tool against the genomes of land plants are represented by a box. A protein similarity network of those proteins is also shown; lotus proteins are represented as purple nodes, Arabidopsis proteins (LPR1 and LPR2) are represented as green nodes and other land plant proteins are represented as grey nodes. (B) Heatmap of COG2132 gene family member expression in lotus. Reads per kilo base per million (RPKM) values were log2 transformed, where blue correlates to high expression, and yellow to low expression. (C) A maximum-likelihood tree of LPR1/LPR2-like lotus proteins. Branch support was calculated using an Approximate Likelihood-Ratio Test. Lotus homologs are connected with a dashed bracket, whereas proteins whose genes are found in tandem on the genome are connected with a solid bracket. A detailed phylogeny of COG2132 members can be found in Figure S8 in Additional file 1. Adaptation to an aquatic environment The basic helix loop helix (bHLH) family, implicated in light responses including germination, control of flower- ing and de-etiolation, and root and flower development, lacks three of its 20 subfamilies in lotus: Va, implicated in brassinosteroid signaling; VIIIc2, implicated in root hair development; and XIII, implicated in root meristem development [28]. The largest families of bHLH factors in lotus are XII, involved in developmental processes including control of petal size, brassinosteroid signaling Adaptation to phosphate starvation in lotus is also evi- denced by expansion of the UBC24 family and the miR399 family that regulates it (Table S12 in Additional file 1). The miR169 family, implicated in adaptation to drought stress in Arabidopsis [27], also shows expansion in lotus, totaling 22 members. The fact that lotus grows aquatically and may rarely be subjected to drought sug- gests that the miR169 family is involved in other physio- logical processes. Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 8 of 11 and floral initiation, and Ia, implicated in stomatal development and patterning. were detected only rarely in another phylogenomic analy- sis that introduced transcriptome data from the basal eudicots Gunnera and Pachysandra [29]. The 34 uni- genes available from that study were used to populate five MADS box orthogroups with larger taxon sampling in this study. Phylogenies of these orthogroups identify (at boostrap >50%) one eudicot-wide and three core- eudicot-wide duplications (Table S11 in Additional file 1), consistent with the rest of the findings in the pre- sent study. The PRR1/TOC1 circadian clock family, which coordi- nates internal biology with daily light/dark cycles and is highly conserved across many plant species, includes three predicted members in lotus compared to the one or two present in other plant genomes. The fact that PRR proteins have key roles in modulating light and tempera- ture input into the circadian clock suggests that lotus may require more sensitive adjustments to its environ- ment than other plants. Consistent with this, the crypto- chrome (CRY) family of blue light photoreceptors is also increased with five (two CRY1, two CRY2, one CRY3) compared to three in Arabidopsis and four in poplar (Additional file 1, Table S13). Similar expansion in the CRY family was also noted in another aquatic organism, Ostreococcus, a micro green algae. Adaptation to an aquatic environment Lotus is adapted to both temperate and tropical climates and day lengths with a wide range of flowering times, perhaps associated with increased numbers of flowering time and circadian clock-associated genes. In contrast to the phylogenomic results, syntenic com- parison showed one lotus region matched with up to three Vitis homologous regions, indicating that the lotus genome did not share the g event. We propose that the g event occurred after the separation of the lotus lineage (Proteales), and involved hybridization with a now extinct species that branched off around the same time (Figure 3A, AA at position #2), or even earlier than lotus (Figure 3A, AA at position #3). This model explains why the phylogenomic analyses could identify some g duplications occurring before the divergence of lotus, but not observable as a triplication in the lotus genome structure. A similar two-step model was sug- gested by Lyons et al. [30] on the basis of fractionation patterns seen in Vitis, and evidence for a two-step hexa- ploid process is clearly observed in the much more recent paleohexaploid Brassica rapa [31]. Additional whole plant genome sequences from lineages close to the g event, especially ones without the confounding effects of lineage-specific genome duplications, may also help to clarify genome-wide patterns of fractionation among the three g subgenomes, which could provide further evidence bearing on the timing and event(s) associated with the g paleohexaploidy event that is asso- ciated with what is arguably one of the most important radiations in angiosperm history. Discussion Paleopolyploids are widespread among eukaryotes and particularly common in angiosperms [14,15]. Lotus diverged from other eudicots early in eudicot history, prior to the g genome-triplication characteristic of most members of the group [14,15,17,26], and provides insight into the timing and nature of this event asso- ciated with a rapid radiation of the large eudicot lineages. When plant genomes of high paleopolyploidy levels are compared, differentiated gene loss (fractiona- tion) among several homologous subgenomes tends to diminish the signals of synteny. In such cases, genomes with few paleopolyploidy events (such as those of grape or papaya) can be used to take advantage of the smaller evolutionary distances between orthologous segments. Extensive collinearity within itself, as well as with other plant genomes such as those of Arabidopsis, grape, rice and sorghum, makes the lotus genome not only a eudi- cot evo-genomic reference (Figure S9 in Additional file 1), but also a better resource for reconstructing the pan- eudicot genome and facilitating comparative analysis between eudicots and monocots. The higher homeolog retention rate in lotus compared with most other genomes studied provided an opportu- nity to study subfunctionalization [32], a major driving force affecting fates of duplicated genes following paleo- polyploidy. Most pairs of lotus homeologs have no dif- ference in PFAM domain families, whereas 453 pairs (11.6%) differ by up to five domains. The unshared domains have mean length 17 amino acids with a range of 0 to 890 amino acids. Between homeologous lotus gene pairs, mRNA length (excluding 5′ and 3′ untrans- lated regions), coding sequence length, and intron length differences all follow geometric-like distributions (Figure S10 in Additional file 1), consistent with inde- pendent accumulation of small insertions and deletions. The changes of length in exonic and intronic regions seem uncorrelated, implying that subfunctionalization affects gene regulation at multiple transcriptional and post-transcriptional levels. Surprisingly, the phylogenomic analysis of gene families associated with the g include a substantial fraction of eudicot-wide duplications, suggesting the possibility of a two-step model that involved genetic material from a lineage that branched off earlier than the core eudicots (Figure 3A). A substantial fraction of eudicot-wide gene duplications was also observed in phylogenomic analyses that contained large collections of transcriptome data from early branching basal eudicots such as Platanus, Aquilegia and poppies [26]. Eudicot-wide duplications Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 Page 9 of 11 Ming et al. Discussion Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 When divergence of lineages is followed by WGD, one predicts similar divergence of the paralogs in one spe- cies’ genome from a shared ortholog in the other spe- cies, confirmed in previous studies [16,33]. Comparison of paired l paralogs and their grape ortholog generally fit this prediction (Figure S11 in Additional file 1); how- ever, comparisons to cereal (sorghum) orthologs show consistent differentiation in branch lengths. This discre- pancy in the lotus-cereal comparison could be explained by fast evolutionary rates in cereal genomes and/or l being older than it appears, due to the slow Nelumbo evolutionary rate. Alternatively, this is also consistent with structural compartmentalization, with genes within the same genome undergoing different evolutionary tra- jectories [33]. Wider taxa sampling at neighboring branches will help better distinguish the possibilities. as rhizome development and flowering time. The assembly of the lotus genome is surprisingly high quality, largely due to the high level of homozygosity resulting from domesti- cation and vegetative propagation. The lotus genome has a lineage-specific WGD event that occurred about 65 MYA, but shows no structural evidence for the g hexaploid event shared among core eudicot species. The lotus genome has a 30% slower nucleotide mutation rate than that of grape, contributing in part to the outstanding genome assembly using next-generation sequencing technologies. Analysis of sequenced plant genomes yielded a minimum gene set for vascular plants of 4,223 genes. Strikingly, lotus has 16 COG2132 multi-copper oxidase family proteins with root- specific expression. COG2132 members are involved in root meristem phosphate starvation, reflecting lotus’ adap- tation to limited nutrient availability in an aquatic environ- ment. The slow nucleotide substitution rate and the lack of the triplication event make lotus genome an excellent reference for reconstructing the pan-eudicot genome and for accelerating comparative analysis between eudicots and monocots. The lotus genome will accelerate the iden- tification of genes controlling rhizome yield and quality, seed size and nutritional profile, flower morphology, and flowering time for crop improvement. The extraordinary seed longevity and vegetative propa- gation via rhizomes are likely the causes of the slow evolu- tionary rate in lotus. The ‘China Antique’ has a highly homozygous genome, yielding arguably the best assembled genome using next-generation sequencing technologies with pseudo-molecules proportional to its karyotype. Conclusions Sacred lotus has many unique biological features, most noticeable seed longevity and the lotus effect, in addition to its agricultural and medicinal importance. The purpose of sequencing the lotus genome is to facilitate research in these areas and on agronomic and horticultural traits such Materials and methods Illumina (Illumina HiSeq 2000) libraries were generated from purified N. nucifera ’China Antique’ nuclear DNA with inserts of 180 bp, 500 bp, 3.8 kb and 8 kb and assembled using ALLPATHS-LG. 454/Roche (GSFLX pyrosequencing platform) 20 kb mate pair reads were used for scaffolding. RNAseq data generated from various lotus tissues were used for annotation and RNAseq dif- ferential gene expression analysis using CLC Genomics Workbench 5.0 (CLC Bio, Aarhus, Denmark). MAKER version 2.22 was used in combination with the assembled RNAseq data to annotate 26,685 genes in the lotus gen- ome. Detailed methods for genome assembly, annotation and analyses are provided in Additional file 1. Sacred lotus is the first true aquatic plant to be sequenced and comparative genomics reveal unique gene family expansions that may have contributed to its adap- tations to an aquatic environment. Submersed soils are largely hypoxic and have a decreased reduction-oxidation potential, causing heavy metal precipitation and reduced nutrient availability. Lotus has a dramatic expansion of the COG2132 family, a group of multi-copper oxidases involved in phosphate starvation in root meristems. A role in root-specific processes is supported by the expression of these unique genes in root tissue. Adapta- tion to phosphate starvation can also be seen in an expansion of the UBC24 family and the miR399 family that regulates it. Lotus lacks four bHLH subfamilies involved in iron uptake and root hair and root meristem development, suggesting novel root growth and iron reg- ulation. These gene family expansions and preferential retention of duplicated genes reflect the challenges of aquatic growth. Data access The assembled N. nucifera genome was submitted to Gen- Bank (AQOG00000000; PID PRJNA168000, http://www. ncbi.nlm.nih.gov/Traces/wgs/?val=AQOG01). Whole gen- ome shotgun raw reads are deposited under SRA study: SRP021228 (http://trace.ncbi.nlm.nih.gov/Traces/sra/? study=SRP021228). The raw RNAseq data are deposited under BioProject 196884 (http://www.ncbi.nlm.nih.gov/ bioproject/196884). Discussion The lotus genome provides the foundation for revealing the molecular basis of its many distinguishing biological prop- erties, including seed longevity, adaptation to aquatic environment, the distinctive superhydrophobicity and self- cleaning property of its leaves, and the thermogenesis that is thought to enhance its pollination success. Authors’ details 1 Yang M, Han Y, VanBuren R, Ming R, Xu L, Han Y, Liu Y: Genetic linkage maps for Asian and American lotus constructed using novel SSR markers derived from the genome of sequenced cultivar. BMC Genomics 2012, 13:653. 6. 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Authors’ details 1 13Department of Chemistry and Biochemistry and Institute for Genomics and Proteomics, University of California, Los Angeles, 607 Charles E Young Drive East, CA 90095, USA. 14Department of Biology and Intercollege Graduate Program in Plant Biology, The Pennsylvania State University, 201 Life Sciences Building, University Park, PA 16802, USA. 15Center for Applied Chemical Biology, Department of Biological Sciences, Youngstown State University, 1 University Plaza, Youngstown, OH, 44555, USA. 16USDA-ARS, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA. 17Texas A&M AgriLife Research, Department of Plant Pathology & Microbiology, Texas A&M University System, 17360 Coit Road, Dallas, TX 75252, USA. 18Department of Biology, University of Central Oklahoma, 100 North University Drive, Edmond, OK 73034, USA. 19School of Earth and Environmental Sciences, University of Adelaide, North Terrace, Adelaide, 5005, Australia. 20Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japan. 21Institute for Conservation Biology, The University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia. 22Department of Crop Sciences, University of Illinois at Urbana-Champaign, 1101 West Peabody Drive, Urbana, IL 61801, USA. 23Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO 63132, USA. 24Lawrence Berkeley National Laboratory, 1 Cyclotron Road Berkeley, Emeryville, CA 94720, USA. 25Institute of Developmental Biology and Molecular Medicine & School of Life Sciences, Fudan University, 220 Handan Road, Shanghai, 200433, China. 26Department of Biochemistry and Molecular Biology, 246 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA. 27Hawaii Agriculture Research Center, 94-340 Kunia Road, Waipahu, HI 96797, USA. 28Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI 96822, USA. 29Fujian Normal University, Qishan Campus, Minhou, Fuzhou, 350117, China. 30Department of Biology and Molecular Biology, Montclair State University, 1 Normal Avenue, Montclair, NJ 3. Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke AK: Handbook of Medicinal Herbs 2002, Boca Raton: CRC Press. 3. Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke AK: Handbook of Medicinal Herbs 2002, Boca Raton: CRC Press. 4. Diao Y, Chen L, Yang G, Zhou M, Song Y, Hu Z, Lin JY: Nuclear DNA C-values in 12 species in Nymphaeales. Caryologia 2006, 59:25-30. 4. Diao Y, Chen L, Yang G, Zhou M, Song Y, Hu Z, Lin JY: Nuclear DNA C-values in 12 species in Nymphaeales. Caryologia 2006, 59:25-30. 4. Diao Y, Chen L, Yang G, Zhou M, Song Y, Hu Z, Lin JY: Nuclea 5. Abbreviations bHLH: basic helix loop helix; bp: base pair; CRY: cryptochrome; EST: expressed sequence tags; MYA: million years ago; PPR: pentatricopeptide repeat-containing proteins; WGD: whole genome duplication. Authors’ contributions RM, RV, YL, MY, YH and S L designed research; RM, RV, YL, MY, YH, LTL, QZ, JEB, HT, EL, AAF, GN, DRN, CEBH, ARG, YJ, JPD, FZ, JH, XM, KAH, KI, SAR, MEH, QY, TCM, AC, YZ, RS, RJ, NC, JA, CMW, EW, AS, YH, LX, JZ, RP, MJH, WX, JAW, JW, MLW, YJZ, REP, ABB, CD, SRD, MAS, TPM, SPL, DRO, JWS, DRG, NJ, MY, CWD, SSM, AHP, BBB, SL and JSM performed research and analyzed data; RM, RV, JL, AHP, CEBH, JRW, KI, SAR, CWD, SSM and BBB wrote the paper. All authors read and approved the final manuscript. RM, RV, YL, MY, YH and S L designed research; RM, RV, YL, MY, YH, LTL, QZ, JEB, HT, EL, AAF, GN, DRN, CEBH, ARG, YJ, JPD, FZ, JH, XM, KAH, KI, SAR, MEH, QY, TCM, AC, YZ, RS, RJ, NC, JA, CMW, EW, AS, YH, LX, JZ, RP, MJH, WX, JAW, JW, MLW, YJZ, REP, ABB, CD, SRD, MAS, TPM, SPL, DRO, JWS, DRG, NJ, MY, CWD, SSM, AHP, BBB, SL and JSM performed research and analyzed data; RM, RV, JL, AHP, CEBH, JRW, KI, SAR, CWD, SSM and BBB wrote the paper. All authors read and approved the final manuscript. Received: 4 January 2013 Revised: 19 April 2013 Received: 4 January 2013 Revised: 19 April 2013 Acknowledgements Received: 4 January 2013 Revised: 19 April 2013 Accepted: 10 May 2013 Published: 10 May 2013 Accepted: 10 May 2013 Published: 10 May 2013 We thank K. Hasenstein for collection of the fruits of Nelumbo lutea. This project was supported by the University of California, Los Angeles (JSM); Wuhan Botanical Garden, Chinese Academy of Sciences, P.R. China (SL); and the University of Illinois at Urbana-Champaign (RM). Wuhan Botanical Garden, Chinese Academy of Sciences, P.R. China (SL); and the University of Illinois at Urbana-Champaign (RM). Additional material Additional file 1: Supplementary data, including detailed materials and methods, and supplementary tables S1-S13, and figures S1-S14. Page 10 of 11 Page 10 of 11 Page 10 of 11 Ming et al. Genome Biology 2013, 14:R41 http://genomebiology.com/2013/14/5/R41 07043, USA. 31Institute of Tropical Biosciences and Biotechnology, China Academy of Tropical Agricultural Sciences, 4 Xueyuan Road, Haikou, Hainan 571101, China. 32Department of Plant and Microbial Biology, University of California, 1 Shields Avenue, Davis CA, 95161, USA. 33Department of Cell and Developmental Biology, University of Illinois, 1201 West Gregory Drive, Urbana IL, 61801, USA. 34The Genome Analysis Center, Monsanto, St Louis, MO 63167, USA. 35Global Change and Photosynthesis Research Unit, Agricultural Research Service, United States Department of Agriculture, 1206 West Gregory Drive, Urbana, IL, USA. 36IGPP Center for the Study of Evolution and Origin of Life, Geology Building, Room 5676, University of California, Los Angeles, 595 Charles E Young Drive East, Los Angeles, CA 90095-1567, USA. 37Department of Plant and Microbial Biology, University of California, 411 Koshland Hall, Berkeley, CA 94720, USA. 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Intake and Dietary Food Sources of Fibre in Spain: Differences with Regard to the Prevalence of Excess Body Weight and Abdominal Obesity in Adults of the ANIBES Study

Nutrients

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nutrients Article Intake and Dietary Food Sources of Fibre in Spain: Differences with Regard to the Prevalence of Excess Body Weight and Abdominal Obesity in Adults of the ANIBES Study Liliana G. González-Rodríguez 1,2 , José Miguel Perea Sánchez 1,2 , Javier Aranceta-Bartrina 3,4 , Ángel Gil 4,5 , Marcela González-Gross 4,6 , Lluis Serra-Majem 4,7 , Gregorio Varela-Moreiras 8,9 and Rosa M. Ortega 2,10, * 1 2 3 4 5 6 7 8 9 10 * Department of Nutrition and Dietetics, Faculty of Health Sciences, University Alfonso X El Sabio, Madrid 28691, Spain; [email protected] (L.G.G.-R.); [email protected] (J.M.P.S.) VALORNUT Research Group, Department of Nutrition, Faculty of Pharmacy, Complutense University, Madrid 28040, Spain Department of Preventive Medicine and Public Health, University of Navarra, Pamplona, Navarra 31008, Spain; [email protected] Biomedical Research Networking Center for Physiopathology of Obesity and Nutrition (CIBEROBN), Carlos III Health Institute, Madrid 28029, Spain; [email protected] (A.G.); [email protected] (M.G.-G.); [email protected] (L.S.-M.) Department of Biochemistry and Molecular Biology II and Institute of Nutrition and Food Sciences, University of Granada, Granada 18100, Spain ImFINE Research Group, Department of Health and Human Performance, Technical University of Madrid, Madrid 28040, Spain Research Institute of Biomedical and Health Sciences, University of Las Palmas de Gran Canaria, Faculty of Health Sciences, c/Doctor Pasteur s/n Trasera del Hospital, Las Palmas de Gran Canaria, 35016 Las Palmas, Spain Department of Pharmaceutical and Health Sciences, Faculty of Pharmacy, CEU San Pablo University, Madrid 28668, Spain; [email protected] Spanish Nutrition Foundation (FEN), Madrid 28010, Spain Department of Nutrition, Faculty of Pharmacy, Madrid Complutense University, Madrid 28040, Spain Correspondence: [email protected]; Tel.: +34-913-941-837; Fax: +34-913-941-810 Received: 8 December 2016; Accepted: 21 March 2017; Published: 25 March 2017 Abstract: The aim was to study the intake and food sources of fibre in a representative sample of Spanish adults and to analyse its association with excess body weight and abdominal obesity. A sample of 1655 adults (18–64 years) from the ANIBES (“Anthropometric data, macronutrients and micronutrients intake, practice of physical activity, socioeconomic data and lifestyles”) cross-sectional study was analysed. Fibre intake and dietary food sources were determined by using a three-day dietary record. Misreporters were identified using the protocol of the European Food Safety Authority. Mean (standard deviation) fibre intake was 12.59 (5.66) g/day in the whole sample and 15.88 (6.29) g/day in the plausible reporters. Mean fibre intake, both in the whole sample and the plausible reporters, was below the adequate intake established by European Food Safety Authority (EFSA) and the Institute of Medicine of the United States (IOM). Main fibre dietary food sources were grains, followed by vegetables, fruits, and pulses. In the whole sample, considering sex, and after adjusting for age and physical activity, mean (standard error) fibre intake (adjusted by energy intake) was higher in subjects who had normal weight (NW) 13.40 (0.184) g/day, without abdominal obesity 13.56 (0.192) g/day or without excess body weight and/or abdominal obesity 13.56 (0.207) g/day compared to those who were overweight (OW) 12.31 (0.195) g/day, p < 0.001 or obese (OB) 11.83 (0.266) g/day, p < 0.001, with abdominal obesity 12.09 (0.157) g/day, p < 0.001 or with excess body weight and/or abdominal obesity 12.22 (0.148) g/day, p < 0.001. There were no significant differences in relation with the fibre Nutrients 2017, 9, 326; doi:10.3390/nu9040326 www.mdpi.com/journal/nutrients Nutrients 2017, 9, 326 2 of 22 intake according to the body mass index (BMI), presence or absence of abdominal obesity or excess body weight and/or abdominal obesity in the plausible reporters. Fibre from afternoon snacks was higher in subjects with NW (6.92%) and without abdominal obesity (6.97%) or without excess body weight and/or abdominal obesity (7.20%), than those with OW (5.30%), p < 0.05 or OB (4.79%), p < 0.05, with abdominal obesity (5.18%), p < 0.01, or with excess body weight and/or abdominal obesity (5.21%), p < 0.01, in the whole sample. Conversely, these differences were not observed in the plausible reporters. The present study demonstrates an insufficient fibre intake both in the whole sample and in the plausible reporters and confirms its association with excess body weight and abdominal obesity only when the whole sample was considered. Keywords: fibre; food sources; obesity; abdominal obesity; misreporting; adults; Spain; ANIBES 1. Introduction In recent decades, there has been a significant increase in the prevalence of overweight (OW) and obesity (OB) in children and adults, in both developed and developing countries [1]. Excess body weight increases the risk of developing various diseases, such as cardiovascular disease, type 2 diabetes, some types of cancers, musculoskeletal disorders, and neurodegenerative diseases, which have an important health and social impact [1–3]. Diet and physical activity are the main factors that influence the development of OW and OB [1,4]. In this respect, several studies have shown that the intake of dietary fibre may have a positive effect on body weight control; however, the available results are inconsistent [5–8]. There are several possible mechanisms to explain the anti-obesogenic effect of dietary fibre. Among the most documented mechanisms is the one that refers to the benefits of the fibre capacity (mainly soluble fibre) to form viscous gels that delay the gastric emptying, which helps, on the one hand, to increase the satiety sensation and consequently reduces energy intake [9] and, secondly, to control the postprandial glycaemia by delaying the intestinal absorption [10]. Another possible mechanism is associated with short-chain fatty acid produced during fermentation of fibre in the gastrointestinal tract [11] since it has been shown that this can contribute to regulating the secretion of some gastrointestinal hormones, such as glucagon-like peptide-1 (GLP-1), involved in satiety, and ghrelin, involved in appetite control [12,13], and to increase the oxidation of fatty acids and energy expenditure and to regulate glucose metabolism [11]. In addition, there are few recent studies regarding the intake of fibre in a representative Spanish adults sample and none of these studies have analysed the relation between the fibre intake, fibre from different meals of the day and food sources and the problematic of excess body weight and abdominal obesity [14,15]. Additionally, it is the first Spanish study that takes into account the dietary misreporting of the participants. Therefore, the aim of the present work was to study the intake and dietary food sources of fibre in a representative sample of the Spanish adults from the ANIBES (“Anthropometric data, macronutrients and micronutrients intake, practice of physical activity, socioeconomic data and lifestyles”) study and to analyse the differences in fibre intake between people with different body weight and with or without abdominal obesity. The present work shows the analysed data for the total sample and plausible reporters of the study. 2. Materials and Methods 2.1. Study Design and Sampling Procedure The complete design, protocol, and methodology of the ANIBES study have been already described in detail elsewhere [16,17]. In summary, the ANIBES study was carried out to analyse Nutrients 2017, 9, 326 3 of 22 anthropometric data, physical activity, intake of food and beverages, and dietary habits in the Spanish population (9–75 years old, n = 2009). The participants were randomly selected from the Northeast, East, Southwest, North-Central, Barcelona, Madrid, Balearic, and Canary Islands areas of Spain, including rural, semi-urban and urban populations [16,17]. The present study is focused on 1655 adults (779 men and 858 women) with ages ranging 18–64. The following exclusion criteria were applied: • • • • Those individuals living in an institutional setting (e.g., colleges, nursing homes, hospitals, and others); Individuals following a therapeutic diet owing to recent surgery or taking any medical prescriptions; Potential participants with a transitory illness (i.e., flu, gastroenteritis) at the time of the fieldwork; and Individuals employed in areas related to consumer science, marketing, or the media [16,17]. The fieldwork for the ANIBES study was carried out from mid-September 2013 to mid-November 2013. The final protocol was approved by the Ethical Committee for Clinical Research of the Region of Madrid (Spain) (code FEN 2013/31, May 2013). All participants were informed of the protocol and risks and benefits of their participation in the study and a written informed consent was obtained from all the study’s participants. 2.2. Anthropometric Data Weight, height, and waist circumference (WC) were measured by trained interviewers following standardized procedures [18]. Weight was measured once with a Seca® model 804 weighing scale (Medizinische Messsysteme und Waagen seit 1840, Hamburg, Germany; range: 70–205 cm, precision: 1 mm). Height was assessed in triplicate using a Seca® model 206 stadiometer (Medizinische Messsysteme und Waagen seit 1840, Hamburg, Germany; range: 0.1–150 kg, precision: 100 g). WC was measured in triplicate using a Seca® 201 tape measure (Seca, Hamburg, Germany; range: 0–150 cm, precision: 1 mm). Excess body weight was assessed using the body mass index (BMI) and abdominal obesity by the waist to height ratio (WHtR). BMI was calculated as weight (kg)/height (m)2 and WHtR as WC (cm)/height (cm). Participants were classified into the following categories using the BMI: underweight (UW) (BMI <18.5 kg/m2 ), normal weight (NW) (BMI 18.5–24.9 kg/m2 ), OW (BMI 25–29.9 kg/m2 ), and OB (BMI ≥30 kg/m2 ), based on the World Health Organization classification [19], and using the WHtR respondents were classified into two categories: “non-abdominal obesity” (WHtR <0.5) and those with “abdominal obesity” (WHtR ≥0.5) [20–23]. BMI and WHtR were combined into one category called “excess body weight and/or abdominal obesity” (BMI ≥25 kg/m2 and/or WHtR ≥0.5) for subsequent analysis [19,21–23]. 2.3. Physical Activity To obtain physical activity data of the studied participants, a detailed interview using the International Physical Activity Questionnaire (IPAQ) for adults was performed [24]. In addition, different physical activity measurements were obtained with an accelerometer ActiGraph (model GT3x and GT3x+; ActiGraph, Pensacola, FL, USA) in a subsample of 167 adults. Individuals were asked to wear the ActiGraph on a belt above the right hip, for three consecutive full days. This previous information was used to validate the physical activity questionnaire administered to the whole sample. There were calculated the following indicators of physical activity in the participants of the present study: • • Total physical activity expressed as minutes of activity per week; Time spent in vigorous physical activity expressed as minutes of activity per week. Nutrients 2017, 9, 326 4 of 22 Subjects were classified into two levels of physical activity based on two questions of the IPAQ [24]: “Sedentary activity level” as those subjects who have performed less than 30 min per day of physical activity of daily life and less than 2 h per week of structured physical exercise. “Active activity level” as those subjects who have performed at least 30 min per day of physical activity of daily life or have performed at least 2 h per week of structured physical exercise. 2.4. Food and Beverage Record Study participants were provided with a tablet device (Samsung Galaxy Tab 2 7.0, Samsung Electronics, Suwon, South Korea) and trained in how to record information by taking photos of all food and beverages consumed during the three days of the study, both at home and outside the home. Photos had to be taken before beginning to eat and drink, and again after finishing, so as to record the actual intake. Additionally, a brief description of meals (including the type of food), recipes, brands, and the use of dietary supplements were recorded using the device. Energy and fibre intake were calculated from food consumption records using VD-FEN 2.1 software (Dietary Evaluation Programme, Spanish Nutrition Foundation, Madrid, Spain) which was newly developed for the ANIBES study by the Spanish Nutrition Foundation and is based mainly on Spanish food composition tables [25], with several expansions and updates. Data obtained from food manufacturers and nutritional information provided on food labels were also included. A food photographic atlas was also used to assist in assigning gram weights to serving sizes [26]. The present study was focused on the intake of fibre expressed in grams per day (g/day) raw (before the energy intake adjustment) and after the energy intake adjustment by the residual methods [27,28] and the intake of fibre expressed in grams per 1000 kcal per day. The mean intake of fibre of the studied participants was compared with the adequate intake set out by European Food Safety Authority (EFSA): 25 g/day in adults [29], and by the Institute of Medicine of the United States (IOM): 38 g/day for men and 25 g/day for women of 19–50 years and 30 g/day for men and 21 g/day for women of 51–70 years (14 g/1000 kcal/day) [30]. Once fibre intake has been established, the percentage of fibre from each one of the meal times (breakfast, mid-morning snack, lunch, afternoon snack, and dinner) was calculated. The contribution of each food to total fibre intake has been calculated by adding the amount of fibre provided by each specific food and dividing by the total intake of fibre, all multiplied by 100 [31]. 2.5. Evaluation of Misreporting The assessment of dietary intake based on self-reported or self-recorded data by the study subjects is often prone to bias. One of the main sources of error is misreporting, including both under- and over-reporting, which are often influenced by age, sex, and other individual characteristics, including BMI [32]. The magnitude of misreporting of the intake of food may lead to biased interpretation of the results. In the present study the protocol of the EFSA was used to identify misreporters [33,34]. This method is based mainly on the Goldberg [35] and Black [36,37] work. This method evaluates the reported energy intake (EIrep) against the presumed energy requirements. EIrep is expressed as a multiple of the mean basal metabolic rate estimated (BMRest) (from formulas), and it is compared with the presumed energy expenditure of the studied population. Then the ratio EIrep:BMRest is referred to as the physical activity levels (PAL) [33,34]. The PAL is established for adults (≥18 years) in three levels: low, 1.4; moderate, 1.6; and vigorous, 1.8. Additionally, the protocol indicates that analyses should be performed at two levels, group and individual. The group level determines the overall bias to the reported energy intake, and the individual level shows the rate of under and over reporters. The BMRest was calculated using the Schöfield equations [38]. Misreporting cut-offs at group and individual levels for the ANIBES study has been described in a previous paper [39]. Subjects were classified into two categories: Nutrients 2017, 9, 326 5 of 22 “Plausible reporters” as those subjects with estimated values of the ratio EIrep:BMRest ranging between calculated lower cut-off and upper cut-off values for the studied population allocated to the different category of physical activity [33,34]. “Misreporters” as those subjects with estimated values of EIrep:BMRest below the calculated lower cut-off value (under-reporters) and those subjects with estimated values of EIrep:BMRest above the upper calculated cut -off value (over-reporters) [33,34]. In the present study, the results are showed in the total sample (before the exclusion of the non-plausible reporters) and in the plausible reporters. 2.6. Statistical Analysis Descriptive data were presented as mean and standard deviation (SD), median and interquartile range (IQR) (both without any adjustment), and frequencies (%) stratified by sex, BMI, presence of abdominal obesity, and excess body weight and/or abdominal obesity. The normality of the data and equality of the variances were tested using the Kolmogorov–Smirnov and Levene’s test, respectively. The statistical differences of the studied quantitative variables by sex were assessed using Student’s t-test when data were normally distributed, and using the Mann–Whitney test, otherwise. A two-way ANOVA test was used to assess the differences within the studied variables using sex and the variables of BMI, abdominal obesity or excess body weight and/or abdominal obesity. Two-way ANOVA main effects for BMI, abdominal obesity, and excess body weight and/or abdominal obesity were obtained. The Bonferroni post hoc test for comparison for more than two groups was used. A two-way ANCOVA test was used to assess the differences within the studied variables using the previous ANOVA model taking into account the age and the physical activity (PA) of the participants as covariates to control their influence on the dependent variable. Two-way ANCOVA main effects and interactions were analysed and they are only reported in the text as estimated marginal means and the standard error (SE) when a significant inverse trend, significant changes, or an interaction with sex were observed. The level of significance was set at p < 0.05. All calculations were performed using IBM SPSS version 22.0 (IBM Corp., Armonk, NY, USA). 3. Results 3.1. Study Population A sample of 1655 adults (48.2% men and 51.8% women) with ages ranging from 18 to 64 years was studied. In total, 26.16% (n = 433) (36.4% men and 63.5% women) of the studied adults were classified as plausible reporters. Table 1 shows the anthropometric and physical activity data of the whole sample and of the plausible reporters. The combined prevalence of OW and OB in the whole sample was 55.70% and 32.80% in the plausible reporters. The prevalence of abdominal obesity was 58.40% and 39.3% and the presence of excess body weight and/or abdominal obesity was 63.93% and 42.7% in the whole sample and in the plausible reporters, respectively. A 44.40% of the whole sample and 44.3% of the plausible reporters were sedentary. 3.2. Fibre Intake and Dietary Food Sources in the Whole Sample and in the Plausible Reporters and by Sex Mean dietary fibre intake (raw) was 12.59 (SD 5.66) g/day, and adjusted by energy was 12.59 (SD 4.91) g/day, while the intake of fibre expressed as grams per 1000 kcal per day was 7.05 (SD 2.81) in the whole sample (Table 2). Fibre intake (raw) was significantly lower in women than in men, but when it was adjusted by energy intake or when was expressed as grams per 1000 kcal per day, it was significantly lower in men than in women in the whole sample (Table 2). Meanwhile, in the group of plausible reporters, the mean dietary fibre (raw) was 15.88 (SD 6.29) g/day, and it was also significantly lower in women than in men, however, there were no statistically significant differences in relation with the fibre intake adjusted by the energy intake or expressed as g/1000 kcal/day (Table 2). Lunch and dinner contributed the highest proportions of fibre to the total daily intake in the whole Nutrients 2017, 9, 326 6 of 22 sample and in the plausible reporters (75.85% and 70.59%, respectively). It is noteworthy that the fibre from breakfast was quite low in the whole sample (13.04%) as well as in the plausible reporters (14.27%). The proportion of fibre from breakfast and afternoon snacks was higher in women and from dinner in men in the whole sample. No significant differences were observed regarding the fibre from the different meals in the plausible reporters (Table 2). The main food sources of fibre in the whole studied population and in the plausible reporters were grains (39.13% and 39.14%), vegetables (24.17% and 20.99%), fruits (16.60% and 18.51%), pulses (9.28% and 9.14%), ready-to-eat-meals (4.50% and 4.99%), sauces and condiments (2.18% and 1.97%), appetizers (1.53% and 2.30%), sugars and sweets (0.67% and 1.15%), non-alcoholic beverages (0.49% and 0.53%), dairy products (0.39% and 0.28%), and supplements and meal replacers (0.14% and 0.09%, respectively). 3.3. Fibre Intake and Dietary Food Sources in the Whole Sample and in the Plausible Reporters by Body Mass Index Classification Considering the whole sample, the fibre intake (raw) was higher in subjects who had NW compared to those who had OW or OB, taking into account sex and after adjusting for the age and PA (Table 3). When the interaction of sex by BMI was analysed, it was noted that the intake of fibre (raw) was slightly different according to sex. Men who presented NW 14.83 (SE 0.327) g/day had a higher intake compared to those who had OW 12.45 (SE 0.304) g/day (p < 0.001) or OB 11.67 (SE 0.413) g/day (p < 0.001). While women who were UW 14.52 (SE 1.077) g/day had a higher intake than those with OW 11.48 (SE 0.335) g/day (p < 0.05) or OB 11.33 (SE 0.455) g/day (p < 0.05). Fibre intake adjusted by energy intake (considering sex) was similar for the different groups of BMI in the whole sample (Table 3). However, after adjusting for the age and the PA, it was observed that subjects with NW 13.29 (SE 0.183) g/day had a higher intake than subjects with OW 12.26 (SE 0.194) g/day (p < 0.001) or OB 11.79 (SE 0.265) g/day (p < 0.001). Analysing the interaction of sex at different levels of BMI, these differences were observed only in men. Thus, men who presented NW 13.46 (SE 0.279) g/day had a higher intake in comparison with those who had OW 11.81 (SE 0.260) g/day (p < 0.001) or OB 11.33 (SE 0.353) g/day (p < 0.001). The intake of fibre per 1000 kcal per day (considering sex) was similar for the different groups of BMI in the whole sample (Table 3). However, after adjusting for the age and the PA, it was observed that subjects with NW 7.35 (SE 0.104) g/1000 kcal/day had a higher intake than subjects with OW 6.92 (SE 0.110) g/1000 kcal/day (p < 0.05) or OB 6.68 (SE 0.151) g/1000 kcal/day (p < 0.01). No significant differences were observed regarding the fibre intake (raw, adjusted by the energy intake and expressed as grams per 1000 kcal/day) according with the BMI in the plausible reporters, considering sex and after adjusting for the age and the PA (Table 3). The proportion of fibre from the lunch was higher in individuals with OW than those with NW taking into account sex, but this difference disappeared when an adjustment for age and PA was performed. Fibre from the afternoon snack was higher in individuals with NW than those with OW or OB (taking into account sex and even after adjusting for age and PA) (Table 3). While fibre from dinner was lower (after adjusting for the age and PA) in individuals who had OW 27.50% (SE 0.581) compared to those with OB 30.27% (SE 0.795) (p < 0.05). However, the proportion of fibre from the different meals was similar according the BMI in the plausible reporters (Table 3). Regarding the fibre food sources according to the BMI in the whole sample, we observed that fibre from breakfast cereals and cereal bars was higher in individuals with NW than those with OW and fibre from vegetables was higher in individuals with OB than those with NW, but these differences disappeared when an adjustment for age and PA was performed (Table S1). Fibre from fruits (taking into account sex and after adjusting for the age and PA) was significantly higher in individuals with NW 18.36% (SE 0.574%) compared to subjects with OW 15.16% (SE 0.605%) (p < 0.01). In analysing the interaction of sex, it was observed that only in men, the fibre from fruits was higher in those with NW 18.50% (SE 0.881%) than those with OW 14.01% (SE 0.819%) (p < 0.001) or OB 12.92% (SE 1.094%) (p < 0.001). Nutrients 2017, 9, 326 7 of 22 After excluding misreporters, we observed that fibre from grains and flours was higher in subjects with NW than those subjects with OW, but this difference disappeared when an adjustment for age and PA was performed (Table S2). Fibre from pasta (taking into account sex and after adjusting for age and PA) was significantly higher individuals with UW 10.83 (SE 2.231%) compared to subjects with NW 4.72% (0.383%) (p < 0.05) or OW 4.59% (0.580%) (p < 0.05). In analysing the interaction of sex, it was observed that only in men, the fibre from pasta was higher in those with UW 17.98% (SE 4.207%) than in those with NW 4.71% (SE 0.616%) (p < 0.05) or OW 4.62% (SE 0.835%) (p < 0.05). 3.4. Fibre Intake and Dietary Food Sources in the Whole Sample and in the Plausible Reporters by the Presence of Abdominal Obesity Determined by the Waist to Height Ratio Having into account the whole sample, fibre intake (raw) was higher in subjects without abdominal obesity (taking into account sex and even after adjusting for the age and PA) than those with abdominal obesity (Table 4). In analysing the interaction of sex was found that fibre intake in both men and women without abdominal obesity was higher 15.14 (SE 0.337) g/day and 12.76 (SE 0.276) g/day than those subjects with abdominal obesity 12.04 (SE 0.244) g/day (p < 0.001) and 11.47 (SE 0.264) g/day (p < 0.01), respectively. Fibre intake (adjusted by energy intake) considering the sex of the participants was similar independently of the presence or absence of abdominal obesity in the whole sample (Table 4). However, after adjusting for the age and PA, subjects without abdominal obesity 13.43 (SE 0.191) g/day had significantly higher intake than subjects with abdominal obesity 12.05 (SE 0.157) g/day (p < 0.001). In analysing the interaction of sex was found that fibre intake in both men and women without abdominal obesity was higher 13.63 (SE 0.288) g/day and 13.24 (SE 0.236) g/day that those with abdominal obesity 11.57 (SE 0.208) g/day (p < 0.001) and 12.52 (SE 0.226) g/day (p < 0.05), respectively. The intake of fibre per 1000 kcal per day (considering sex) was similar regardless the presence or absence of abdominal obesity in the whole sample (Table 4). However, after adjusting for the age and the PA, it was observed that the subjects without abdominal obesity 7.44 (SE 0.109) g/1000 kcal/day had a higher intake than subjects with abdominal obesity 6.79 (SE 0.089) g/1000 kcal/day (p < 0.001). An interaction of sex was found and it was observed that fibre intake only in men without abdominal obesity was higher 7.36 (SE 0.164) g/1000 kcal/day than in those men with abdominal obesity 6.40 (SE 0.119) g/1000 kcal/day (p < 0.001). Nevertheless, no significant differences were observed regarding the fibre intake (raw, adjusted by the energy intake and expressed as grams per 1000 kcal/day) according with the presence or absence of abdominal obesity, considering sex and after adjusting for the age and the PA in the plausible reporters (Table 4). Fibre from the mid-morning snack was higher in subjects without abdominal obesity than in subjects with abdominal obesity in the whole sample, but this difference disappeared when an adjustment for age and PA was performed (Table 4). Considering sex and after adjusting for age and PA, the proportion of fibre from lunch was higher in subjects with abdominal obesity than those without abdominal obesity. In contrast, fibre from the afternoon snack was higher in individuals without abdominal obesity unlike those with abdominal obesity (Table 4). The proportion of fibre from the different meals was similar according the presence or absence of abdominal obesity in the plausible reporters (Table 4). Concerning the fibre food sources according to the presence or absence of abdominal obesity in the whole sample, we observed that fibre from grains, breakfast cereals, and cereal bars, ready-to-eat meals, and sauces and condiments was higher in individuals without abdominal obesity than those with abdominal obesity and fibre from vegetables was higher in individuals with abdominal obesity than those subjects without abdominal obesity, but all of these differences disappeared when an adjustment for age and PA was performed (Table S3). We only found that fibre from bread (taking into account sex and after adjusting for the age and PA) was higher in subjects with abdominal obesity than those who did not have this problem (Table S3). Similarly, after adjusting for the age and the PA, it was observed Nutrients 2017, 9, 326 8 of 22 that the fibre from pasta was higher in individuals with abdominal obesity 6.73% (SE 0.272%) unlike those participants without abdominal obesity 5.68% (SE 0.332%) (p < 0.05). In contrast, the fibre from fruits (taking into account sex and after adjusting for the age and PA) was higher in subjects without abdominal obesity 18.46% (SE 0.600%) than in subjects with abdominal obesity 15.46% (SE 0.491%) (p < 0.001). However, when the interaction with sex was analysed (Table S3), the previous result only was observed in men without abdominal obesity 19.15% (SE 0.902%) compared to those with abdominal obesity 13.78% (SE 0.654%) (p < 0.001). In relation to the group of sugars and sweets and specifically chocolates, individuals without abdominal obesity had a higher intake than subjects with abdominal obesity (Table S3). In the plausible reporters, we observed that fibre from pasta was higher in subjects without abdominal obesity than those with abdominal obesity, but this difference disappeared when an adjustment for age and PA was performed (Table S4). Fibre from ready-to-eat-meals (taking into account sex and after adjusting for age and PA) was significantly higher in individuals with abdominal obesity 6.39% (SE 0.585%) compared to subjects without abdominal obesity 4.02% (0.484%) (p < 0.01). 3.5. Fibre Intake and Dietary Food Sources in the Whole Sample and in the Plausible Reporters by the Presence of Excess Body Weight and/or Abdominal Obesity Using the Body Mass Index and Waist to Height Ratio Fibre intake (raw) was higher in individuals without excess body weight and/or abdominal obesity in the whole sample (taking into account sex and after adjusting for the age and PA) (Table 5). An interaction of sex was found and it was observed that fibre intake in both men and women without excess body weight and/or abdominal obesity was higher 15.22 (SE 0.371) g/day and 12.81 (SE 0.291) g/day than in those who had excess body weight and/or abdominal obesity 12.28 (SE 0.232) g/day (p < 0.001) and 11.55 (SE 0.252) g/day (p < 0.01), respectively. Fibre intake (adjusted by energy intake) considering sex was similar according to the presence or absence of excess body weight and/or abdominal obesity in the whole sample (Table 5). However, after adjusting for the age and PA, it was found that subjects without excess body weight and/or abdominal obesity 13.43 (SE 0.206) g/day had significantly higher intake than subjects with excess body weight and/or abdominal obesity 12.18 (SE 0.148) g/day (p < 0.001). An interaction of sex was found and it was observed that fibre intake only in men without excess body weight and/or abdominal obesity was higher 13.68 (SE 0.317) g/day than in those men with excess body weight and/or abdominal obesity 11.74 (SE 0.198) g/day (p < 0.001). The intake of fibre per 1000 kcal per day (considering sex) was similar regardless the presence or absence of excess body weight and/or abdominal obesity in the whole sample (Table 5). However, after adjusting for the age and the PA, it was observed that the subjects without excess body weight and/or abdominal obesity 7.43 (SE 0.117) g/1000 kcal/day had a higher intake than subjects with excess body weight and/or abdominal obesity 6.86 (SE 0.084) g/1000 kcal/day (p < 0.001). An interaction of sex was found and it was observed that fibre intake only in men without excess body weight and/or abdominal obesity was higher 7.36 (SE 0.180) g/1000 kcal/day than in those men with excess body weight and/or abdominal obesity 6.48 (SE 0.113) g/1000 kcal/day (p < 0.001). No significant differences were observed regarding the fibre intake (raw, adjusted by the energy intake and expressed as grams per 1000 kcal/day) according with the presence or absence of excess body weight and/or abdominal obesity in the plausible reporters, considering sex and after adjusting for the age and the PA (Table 5). Considering the sex and after adjusting for the age and the PA, the proportion of fibre from lunch was higher in subjects with excess body weight and/or abdominal obesity than those who did not have excess body weight and/or abdominal obesity. In contrast, the fibre from the afternoon snack was higher in individuals who had no excess body weight and/or abdominal obesity, unlike those with excess body weight and/or abdominal obesity (Table 5). The proportion of fibre from the different meals was similar according the presence or absence of excessive body weight and/or abdominal obesity in the plausible reporters (Table 5). Nutrients 2017, 9, 326 9 of 22 Table 1. Anthropometric and physical activity data in the whole sample and in plausible reporters of the ANIBES study of the Spanish adult population (18–64 years). Personal, Anthropometric and Physical Activity Data Statistical Data Whole Sample Plausible Reporters Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) % % % % Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) % % % % Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) % % 1655 39.97 (12.19) 39.00 (20.00) 74.18 (16.47) 72.10 (21.80) 167.67 (9.35) 167.00 (14.00) 26.31 (5.14) 25.60 (6.20) 1.80 42.50 35.80 19.90 88.06 (14.50) 86.83 (19.74) 0.52 (0.08) 0.51 (0.11) 41.60 58.40 36.07 63.93 856.60 (637.72) 735.00 (920.00) 149.20 (264.15) 0 (180.00) 44.40 55.60 433 38.53 (11.67) 37.00 (17.00) 65.71 (13.06) 63.80 (17.2) 165.98 (9.56) 165.00 (14.00) 23.78 (3.88) 23.10 (5.00) 4.60 62.60 25.20 7.60 81.42 (11.70) 79.73 (16.17) 0.49 (0.07) 0.48 (0.09) 60.70 39.30 57.30 42.70 889.22 (640.23) 800.00 (960.00) 168.68 (275.13) 0 (260) 44.30 55.70 n Age (years) Weight (kg) Height (cm) Body mass index (kg/m2 ) Underweight Normal Overweight Obesity Waist circumference (cm) Waist to height ratio Non-abdominal obesity Abdominal obesity Non-excess body weight and/or abdominal obesity Excess body weight and abdominal obesity Physical activity (minutes/week) Vigorous activity (minutes/week) Sedentary activity level Active activity level SD: standard deviation; P50: 50th percentile; IQR: interquartile range; Abdominal obesity: waist to height ratio ≥ 0.5; Excess body weight and/or abdominal obesity: body mass index ≥25 kg/m2 and/or waist to height ratio ≥ 0.5. Nutrients 2017, 9, 326 10 of 22 Table 2. Fibre intake in the whole sample and in plausible reporters of the ANIBES study of the Spanish adult population (18–64 years) by sex. Whole Sample Energy (kcal/day) Total fibre (g/day) (raw) Total fibre (g/day) (adjusted by energy intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Plausible Reporters Energy (kcal/day) Total fibre (g/day) (raw) Total fibre (g/day) (adjusted by energy intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Statistical Data Total (n = 1655) Men (n = 798) Women (n = 857) p Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 1815.58 (511.96) 1758.00 (683) 12.59 (5.66) 11.63 (6.98) 12.59 (4.91) 11.75 (6.01) 7.05 (2.81) 6.55 (3.53) 13.04 (11.59) 10.84 (15.02) 5.16 (8.19) 7.51 (11.69) 47.46 (16.60) 47.10 (22.74) 5.92 (8.67) 1.52 (9.66) 28.39 (14.05) 27.03 (18.67) 1966.22 (543.22) 1919.00 (770) 13.09 (6.09) 12.15 (7.27) 12.26 (5.14) 11.32 (5.79) 6.71 (2.66) 6.25 (3.24) 12.15 (11.68) 9.73 (15.68) 4.99 (8.21) 0.00 (8.01) 48.00 (16.83) 47.61 (23.11) 5.48 (8.94) 0 (8.55) 29.35 (14.43) 27.82 (19.40) 1675.31 (436.85) 1648.00 (598) 12.13 (5.20) 11.27 (6.68) 12.91 (4.67) 12.25 (6.00) 7.37 (2.91) 6.99 (3.78) 13.88 (11.44) 11.78 (14.48) 5.31 (8.18) 0.87 (8.20) 46.96 (16.37) 46.17 (22.50) 6.34 (8.39) 3.13 (10.62) 27.49 (13.64) 26.53 (18.49) *** Statistical data Total (n = 433) Men (n = 158) Women (n = 275) p Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 2352 (425) 2297 (625) 15.88 (6.29) 14.89 (7.38) 15.88 (5.81) 14.78 (7.69) 6.79 (2.44) 6.31 (3.36) 14.27 (11.39) 12.04 (13.54) 6.32 (8.35) 3.38 (9.32) 42.98 (14.84) 42.22 (20.90) 8.81 (9.66) 6.15 (13.72) 27.61 (12.52) 26.01 (17.25) 2712 (358) 2640 (490) 17.97 (7.19) 16.63 (8.26) 15.93 (6.87) 14.26 (8.38) 6.64 (2.52) 6.07 (3.03) 13.68 (10.89) 11.75 (13.88) 5.99 (7.91) 2.53 (9.44) 44.05 (15.06) 43.32 (21.27) 8.16 (9.76) 5.05 (12.22) 28.12 (12.11) 27.04 (16.31) 2145 (306) 2095 (403) 14.68 (5.36) 14.10 (7.40) 15.85 (5.12) 14.94 (7.46) 6.87 (2.38) 6.47 (3.46) 14.61 (11.66) 12.45 (12.90) 6.50 (8.60) 3.78 (9.28) 42.37 (14.70) 41.05 (22.17) 9.19 (9.60) 6.46 (13.25) 27.32 (12.76) 25.79 (18.58) *** ** *** *** *** NS NS *** * *** NS NS NS NS NS NS NS SD: standard deviation; P50: 50th percentile; IQR: interquartile range; p: denotes statistical mean differences by sex. * p < 0.05. ** p < 0.01. *** p < 0.001. NS: non-significant. Nutrients 2017, 9, 326 11 of 22 Table 3. Fibre intake in the whole sample and in the plausible reporters of the ANIBES study of the Spanish adult population (18–64 years) by body mass index. Whole Sample Energy intake (kcal/day) Total fibre intake (g/day) (raw) Total fibre (g/day) (adjusted by energy) intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Plausible Reporters Energy intake (kcal/day) Total fibre intake (g/day) (raw) Total fibre (g/day) (adjusted by energy) intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Statistical Data Underweight (n = 30) Normal Weight (n = 704) Overweight (n = 592) Obesity (n = 329) Two-Way ANOVA Two-Way ANCOVA Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 1930.90 (592.45) 1857.00 (2114.00) 12.86 (6.35) 11.20 (22.98) 12.22 (4.56) 11.19 (7.68) 6.58 (2.39) 6.15 (9.10) 10.51 (11.89) 7.34 (41.49) 6.29 (9.91) 0.29 (35.40) 47.19 (15.69) 46.20 (57.35) 7.43 (5.93) 6.80 (19.78) 28.58 (12.39) 29.00 (66.69) 1872.51 (526.97) 1827.00 (4018.00) 13.05 (5.98) 11.82 (40.66) 12.73 (5.13) 11.86 (6.19) 7.06 (2.78) 6.56 (18.82) 13.25 (11.56) 11.01 (69.25) 5.32 (8.00) 1.08 (58.11) 45.83 (16.24 44.81 (93.52) 6.92 (9.11) 3.25 (52.26) 28.66 (13.96) 27.95 (79.03) 1773.45 (502.33) b 1712.50 (2843.00) 12.27 (5.40) b 11.46 (34.16) 12.50 (4.71) 11.90 (6.07) 7.05 (2.79) 6.64 (18.35) 13.16 (11.79) 10.94 (61.66) 5.19 (8.48) 0 (58.29) 48.99 (16.87) b 48.96 (88.61) 5.30 (8.49) b 0.0 (66.44) 27.35 (14.06) 25.80 (91.30) 1759.05 (475.65) b 1676.00 (3109.00) 12.19 (5.33) b 11.46 (35.71) 12.50 (4.83) 11.57 (5.81) 7.08 (2.97) 6.42 (23.84) 12.65 (11.30) 10.37 (64.58) 4.64 (7.93) 0 (63.99) 48.25 (16.71) 48.18 (97.88) 4.79 (7.99) b 0.0 (60.28) 29.65 (14.32) 27.64 (76.37) S ** BMI *** S ** BMI *** BMI ** I * BMI *** I * NS BMI *** I * S* S * BMI ** NS NS NS NS BMI ** NS BMI ** BMI * NS BMI * Statistical Data Underweight (n = 20) Normal Weight (n = 271 ) Overweight (n = 109) Obesity (n = 33) Two-Way ANOVA Two-Way ANCOVA Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 2153 (466) 2070 (799) 14.12 (6.35) 11.96 (10.08) 15.25 (4.93) 14.24 (8.93) 6.44 (2.19) 6.15 (4.26) 11.81 (13.33) 8.03 (19.27) 9.07 (11.13) 6.05 (16.17) 42.40 (15.06) 42.29 (29.63) 8.12 (6.06) 6.66 (10.27) 28.61 (12.83) 26.64 (16.05) 2307 (425) 2244 (619) 15.58 (6.35) 14.62 (7.13) 15.83 (5.81) 14.89 (7.09) 6.78 (2.44) 6.38 (3.12) 14.47 (11.63) 12.23 (14.34) 6.47 (8.57) 3.78 (9.31) 42.61 (15.11) 40.16 (20.22) 8.60 (9.28) 6.15 (13.48) 27.85 (12.69) 26.68 (17.34) 2443 (394) a,b 2405 (616) 16.69 (5.86) 16.45 (8.34) 16.17 (5.84) 15.48 (8.71) 6.93 (2.46) 6.53 (3.96) 14.12 (10.64) 13.08 (12.56) 6.10 (7.86) 2.64 (9.42) 43.72 (14.52) 45.93 (21.66) 9.58 (11.37) 5.27 (14.07) 26.48 (11.89) 25.48 (16.86) 2539 (395) a,b 2413 (459) 16.75 (6.97) 15.82 (6.45) 15.69 (6.41) 14.31 (7.62) 6.59 (2.48) 6.08 (3.26) 14.69 (10.81) 13.45 (14.12) 4.14 (5.30) 1.71 (7.15) 43.97 (14.00) 43.65 (22.68) 8.43 (8.50) 7.79 (12.94) 28.77 (13.26) 26.58 (18.43) BMI ** S *** BMI *** NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS SD: standard deviation; P50: 50th percentile; IQR: interquartile range; Two-way ANOVA was performed taking into account sex (S) and body mass index (BMI); Two-way ANCOVA was performed taking into account S and BMI and age and physical activity as covariates; Two-way ANOVA significant differences between BMI classification: a: regarding underweight, b: regarding normal weight, c: regarding overweight; I: Interaction; * p < 0.05, ** p < 0.01, *** p < 0.001; NS: non-significant. Nutrients 2017, 9, 326 12 of 22 Table 4. Fibre intake in the whole sample and in plausible reporters of the ANIBES Study Spanish adult population (18–64 years) by the presence or absence of abdominal obesity using the waist to height ratio. Whole Sample Energy intake (kcal/day) Total fibre intake (g/day) (raw) Total fibre (g/day) (adjusted by energy intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Plausible Reporters Energy intake (kcal/day) Total fibre intake (g/day) (raw) Total fibre (g/day) (adjusted by energy intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Statistical Data Non-Abdominal Obesity (n = 689) Abdominal Obesity (n = 966) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 1886.03 (543.28) 1832.00 (701.00) 12.96 (5.97) 11.94 (7.34) 12.58 (5.06) 11.71 (6.06) 6.97 (2.80) 6.46 (3.54) 13.15 (11.81) 10.75 (15.44) 5.70 (8.59) 1.44 (8.23) 45.35 (16.26) 44.29 (23.29) 6.97 (8.96) 3.31 (11.75) 28.81 (14.26) 27.96 (18.71) 1765.33 (482.43) 1705.00 (653.00) 12.33 (5.43) 11.46(6.52) 12.61 (4.81) 11.81 (5.83) 7.11 (2.82) 6.59 (3.49) 12.97 (11.43) 10.87 (15.04) 4.77 (7.88) 0 (7.80) 48.97 (16.67) 48.81(22.57) 5.18 (8.38) 0 (8.03) 28.09 (13.91) 26.64 (18.59) Statistical Data Non-Abdominal Obesity (n = 263) Abdominal Obesity (n = 170) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 2319.06 (446.79) 2244.00 (641.00) 15.36 (6.28) 14.47 (7.36) 15.54 (5.57) 14.65 (7.01) 6.63 (2.31) 6.22 (3.21) 14.36 (11.94) 11.89 (14.42) 6.88 (9.00) 4.11 (10.44) 41.97 (14.99) 39.91 (19.86) 8.57 (9.10) 6.28 (13.48) 28.19 (12.64) 26.87 (17.32) 2401.78 (384.418) 2366.50 (558) 16.67 (6.23) 16.32 (7.67) 16.39 (6.13) 15.27 (8.23) 7.03 (2.59) 6.52 (3.59) 14.12 (10.49) 13.16 (12.53) 5.43 (7.14) 2.60 (8.64) 44.54 (14.50) 45.79 (23.04) 9.18 (10.48) 6.03 (13.92) 26.70 (12.31) 25.50 (16.84) Two-Way ANOVA Two-Way ANCOVA S *** WHtR *** S *** WHtR *** S *** WHtR ** I ** S *** WHtR *** I ** I* WHtR *** I ** S *** S *** WHtR *** I * S ** S* WHtR * NS WHtR *** WHtR ** WHtR *** WHtR ** S ** S* Two-Way ANOVA Two-Way ANCOVA S *** S *** S *** S *** NS NS NS NS NS NS NS NS NS NS NS NS NS NS SD: standard deviation; P50: 50th percentile; IQR: interquartile range; Two-way ANOVA was performed taking into account sex (S) and the waist to height ratio (WHtR); Two-way ANCOVA was performed taking into account S and the WHtR and the age and physical activity as covariates. I: interaction. * p < 0.05. ** p < 0.01. *** p < 0.001. NS: non-significant. Nutrients 2017, 9, 326 13 of 22 Table 5. Fibre intake in the whole sample and in plausible reporters of the ANIBES study of the Spanish adult population (18–64 years) by the presence or absence of excess body weight and/or abdominal obesity using the body mass index and the waist to height ratio. Whole Sample Energy intake (kcal/day) Total fibre intake (g/day) (raw) Total fibre (g/day) (adjusted by energy intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Plausible Reporters Energy intake (kcal/day) Total fibre intake (g/day) (raw) Total fibre (g/day) (adjusted by energy intake) Fibre per 1000 kcal/day Fibre from breakfast (%) Fibre from mid-morning snack (%) Fibre from lunch (%) Fibre from afternoon snack (%) Fibre from dinner (%) Statistical Data Non-Excess Body Weight and/or Abdominal Obesity (n = 597) Excess Body Weight and/or Abdominal Obesity (n = 1058) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 1892.54 (539.47) 1848.00 (686.00) 13.00 (5.96) 11.94 (7.25) 12.58 (5.09) 11.71 (6.14) 6.97 (2.78) 6.47 (3.56) 13.21 (11.85) 10.76 (15.27) 5.66 (8.31) 1.46 (8.31) 45.17 (16.17) 44.13 (22.98) 7.20 (9.09) 3.61 (12.03) 28.76 (13.84) 27.91 (18.71) 1772.15 (490.72) 1707.00 (669.00) 12.36 (5.49) 11.47 (6.54) 12.60 (4.81) 11.79 (5.86) 7.1 (2.84) 6.59 (3.5) 12.96 (11.45) 10.85 (15.07) 4.88 (8.13) 0 (7.76) 48.76 (16.71) 48.64 (22.37) 5.21 (8.35) 0 (8.03) 28.19 (14.18) 26.79 (18.62) Statistical Data Non-Excess Body Weight and/or Abdominal Obesity (n = 248) Excess Body Weight and/or Abdominal Obesity (n = 185) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) Mean (SD) P50 (IQR) 2296 (434) 2223 (623) 15.16 (6.09) 14.42 (7.27) 15.47 (5.47) 14.62 (6.84) 6.62 (2.31) 6.22 (3.10) 14.34 (12.12) 11.47 (14.64) 6.98 (9.08) 4.22 (10.74) 41.93 (14.99) 39.75 (20.10) 8.71 (9.22) 6.37 (13.86) 28.05 (12.81) 26.71 (17.24) 2426 (402) 2385 (601) 16.85 (6.43) 16.36 (7.87) 16.43 (6.20) 15.33 (8.33) 7.02 (2.58) 6.53 (3.71) 14.19 (10.35) 13.31 (12.24) 5.43 (7.17) 2.63 (8.70) 44.40 (14.56) 45.69 (22.85) 8.96 (10.25) 5.98 (13.44) 27.02 (12.13) 25.64 (17.04) Two-Way ANOVA Two-Way ANCOVA S *** BMI_WHtR *** S *** BMI_WHtR *** S *** BMI_WHtR** I** S *** BMI_WHtR *** I** I* BMI_WHtR *** I ** S *** S *** BMI_WHtR ** I * S*I* S*I* NS NS BMI_WHtR *** BMI_WHtR ** BMI_WHtR *** BMI_WHtR ** S ** S* Two-Way ANOVA Two-Way ANCOVA S *** S *** BMI_WHtR ** S *** S *** NS NS NS NS NS NS NS NS NS NS NS NS NS NS SD: standard deviation; P50: 50th percentile; IQR: interquartile range; Two-way ANOVA was performed taking into account sex (S) and the body mass index and/or waist to height ratio (BMI-WHtR); Two-way ANCOVA was performed taking into account S and the BMI-WHtR and the age and physical activity as covariates. I: interaction. * p < 0.05. ** p < 0.01. *** p < 0.001. NS: non-significant. Nutrients 2017, 9, 326 14 of 22 Relating to the fibre food sources, according to the presence or absence of excess body weight and/or abdominal obesity in the whole sample, we found that fibre from grains, breakfast cereals, and cereal bars, chocolates, ready-to-eat meals, sauces, and condiments, was higher in individuals without excess body weight and/or abdominal obesity than those with excess body weight and/or abdominal obesity, and fibre from vegetables was higher in individuals with excess body weight and/or abdominal obesity than those subjects without excess body weight and/or abdominal obesity, but all these differences disappeared when an adjustment for age and PA was performed (Table S5). Fibre from bread (taking into account sex and after adjusting for the age and PA) was higher in subjects who had excess body weight and/or abdominal obesity than in those who did not have this problematic (Table S5). Similarly, after adjusting for the age and the PA, it was observed that the fibre from the pasta was higher in individuals with excess body weight and/or abdominal obesity 6.65% (SE 0.256%) as opposed to those without it 5.63% (SE 0.358%) (p < 0.05). In contrast, fibre from fruits (taking into account sex and after adjusting for the age and PA) was higher in subjects without excess body weight and/or abdominal obesity 19.06% (SE 0.644%) than in subjects with excess body weight and/or abdominal obesity 15.45% (SE 0.462%) (p < 0.001). However, when analysing the interaction with sex (Table S5), this result was only observed in men without excess body weight and/or abdominal obesity 20.12% (SE 0.990%) compared to those with excess body weight and/or abdominal obesity 13.87% (SE 0.619%) (p < 0.001). In the plausible reporters, we observed that fibre from the groups of grains and flours, pasta and juices and nectars was higher in subjects without excess body weight and/or abdominal obesity than those with excess body weight and/or abdominal obesity, but these differences disappeared when an adjustment for age and PA was performed (Table S6). Taking into account sex and after adjusting for age and PA, fibre from fruits was significantly higher in individuals without excess body weight and/or abdominal obesity 20.2% (SE 1.042%) compared to subjects with excess body weight and/or abdominal obesity 16.9% (1.144%) (p < 0.05). Conversely, the fibre from ready-to-eat-meals was higher in those subjects with excess body weight and/or abdominal obesity 6.41 (SE 0.558%) than those without excess body weight and/or abdominal obesity 3.81% (SE 0.508%) (p < 0.01). 4. Discussion The present study provides updated information on fibre intake and dietary sources and their association with the condition of excess body weight and abdominal obesity in a representative sample of the Spanish adult population. It is highlighted the ANIBES is the first national diet and nutrition survey in Spain that has taken into account the plausible reporters in the analysis of the data, based on well-harmonised procedures [33,34]. A great proportion of participants of the whole sample and of the plausible reporters had OW or OB, which is in concordance with other studies performed in the Spanish population [40,41]. Nonetheless, it is highlighted the prevalence of OW or OB was lower in the plausible reporters in comparison with the whole sample. When comparing our results with the FANPE study (carried out in 2009 on a representative sample of the Spanish population), we found that the combined prevalence of OW and OB of the study (47.80%) was lower than that observed in our study in the whole sample (55.70%) and higher than that observed in the plausible reporters (32.80%) [41], while the combined prevalence observed in the ENPE study, conducted in 2014–2015, also in Spain, was higher (60.9%) than that observed in the present study both in the whole sample and in the plausible reporters [40]. Some studies have indicated that the WHtR is a better predictor of metabolic syndrome or cardiovascular disease and mortality than the WC or BMI [21,23]. Possibly, the most important advantage of using the WHtR resides in the fact that this ratio takes into account the height of the subject, which avoids the overestimation or underestimation of individuals who have a high or low height [21,23,42]. In our study, the mean WHtR in the whole sample was 0.52 (SD 0.08) and 0.49 (SD 0.07) in the plausible reporters. These values are in line to those indicated by the FANPE and ENPE studies [40,43]. Using this parameter, 58.4% of the whole sample and 39.30% of the plausible Nutrients 2017, 9, 326 15 of 22 reporters had abdominal obesity, lower than those indicated in the DARIOS Study (conducted in 2013 in Spanish population), in which 89% of men and 77% of women had abdominal obesity [44]. Moreover, when the presence of excess body weight and/or the presence of abdominal obesity were examined, it was found that 63.93% of the whole sample and 42.70% of the plausible reporters had one or both of these problems. Furthermore, we found that a high proportion of the studied population was sedentary, which has been already discussed in detail in a previous paper [45]. The mean dietary fibre intakes (raw, adjusted by energy intake and expressed in grams per 1000 kcal per day) both in the whole sample and in the plausible reporters were very similar and were lower in comparison with the observed in other studies performed in population with similar ages and characteristics [14,15,46,47]. When comparing the results of our study with those observed by the ENIDE study (carried out in a representative sample of the Spanish population aged 18 to 64 years in 2011), the fibre intake of the participants of our study was lower than the results of such study (men: 20.94 (SD 11.38) g/day and women: 18.85 (SD 10.06) g/day) [14]. Likewise, the mean fibre intakes shown in other study also performed in Spain 20.2 (SD 7.8) g/day and in a study carried out in Irish population 25.7 (SD 8.1) g/day were higher than that observed in our study [15,46]. However, our results were more similar to those observed in the National Health and Nutrition Examination Survey (NHANES 2009–2010) performed in United States adults aged 19 years and older where the fibre intake was 17.0 g/day [47]. The mean intake of fibre (raw) both in men and women was below the adequate intake established by the EFSA and IOM [29,30] in the whole sample and in the plausible reporters. This situation was close to the results shown in various studies performing in similar population [14,46,48,49]. We observed only in the whole sample that the fibre intake after adjusting for energy intake and the fibre per 1000 kcal per day were significantly higher in women than in men. This may be explained because women are usually more concerned about following healthy eating habits and tend to include more healthy foods in their diet compared to men [50]. The analysis of the source of daily fibre intake depending on the different meals throughout the day, revealed that nearly half came from lunch (47.46% and 42.98%) and almost a third from dinner (28.39% and 27.61%) in the whole sample and plausible reporters, respectively. It is noteworthy that the fibre from breakfast was too low in the whole sample (13.04%) and in the plausible reporters (14.27%). The number of frequency of meals per day, including snacks, has been positively related to the intake of various nutrients including the fibre intake [51]. In our study, the mid-morning and afternoon snacks provided the 11.08% and the 15.13% of the daily fibre in the whole sample and in the plausible reporters, respectively. In contrast to our results, the 2001–2010 NHANES study observed that most of the daily fibre came from dinner (37% for adults 19–50 years) [52]. Furthermore, the pattern of fibre intake from the different meals of the day differs according to sex. The proportion of fibre from breakfast and afternoon snacks was higher in women and from dinner in men only in the whole sample. These differences in the pattern of fibre intake are probably due to the differences in the food choices made by the subjects at each meal of the day [52]. A better contribution of fibre from breakfast or afternoon snack, with respect to other meals, could help to reduce appetite and food intake at subsequent meals [6,9]. This could be related to the better situation observed in women compared to men in connection with the presence of excess body weight and abdominal obesity described in a previous paper in more detail for the ANIBES Spanish adult population [53]. Regarding dietary sources of fibre, the main sources in the studied sample were grains, followed by vegetables, fruits, and pulses and were very similar in both studied samples. Similar food groups were identified as the major contributors in the Belgian population [49]. Unlike our study, the main sources of fibre in the ENIDE study were fruits (30%), followed by legumes and nuts (26%), cereals (22%), and vegetables (14%) [14]. Differences were also observed when compared to the results of the 2001–2010 NHANES study, where it was observed that the main sources were the vegetables, followed by cereals and fruits [52]. Nutrients 2017, 9, 326 16 of 22 Some studies performed on populations from the United States [5], the Netherlands [4], and Spain [54] have observed an inverse association between fibre intake and BMI. In accordance with this, in our study, taking into account sex, and after adjusting for the age and physical activity of the participants, fibre intake (raw, adjusted by energy intake and expressed as grams per 1000 kcal per day) was different according to the BMI only in the whole sample. Participants with NW had a significantly higher intake of fibre (raw and expressed as grams per 1000 kcal per day) than those subjects with OW or OB. However, the differences on fibre intake adjusted by the energy intake only were observed in the male sex, where men with NW had a greater intake than those who had OW or OB, which is consistent with what is stated in the study conducted in the Dutch population, where they have found an inverse association between fibre intake and BMI only in men [4]. Moreover, the pattern of fibre intake in the different meals during the day varied depending on the body weight situation only in the whole sample. Specifically, we found that the percentage of fibre that comes from the afternoon snack was higher in individuals with NW than those with OW or OB, while the fibre from dinner was higher in individuals who had OB than those who had OW. The difference found regarding the contribution of fibre from the afternoon snack according to BMI, as previously mentioned, could be related, on the one hand, to the fact that a higher fibre content may favour a reduced appetite which, in turn, can help to take in less food at subsequent meals, in this case during dinner, thus balancing the daily energy intake [6,9]. On the other hand, this also could be explained due to an afternoon snack that contains a higher amount of fibre, which could also include healthier foods with a lower content of energy or fat. An inverse relation between dietary fibre from cereals and fruits and body weight gain has been described in a study performed in male adults (40–75 years old) from the United States [55]. Although the fibre from grains was the most important fibre food source in the present study, there were no significant differences according to BMI in the whole sample. Conversely, we found that the intake of fibre coming from fruits was higher in men with NW compared to those who had OW or OB in the whole sample. These differences may be explained due to fruits that are rich in soluble fibre, which may help control appetite, or because people who consume fruits and vegetables regularly also tend to have a healthier lifestyle [9,49]. We only observed in the men of the group of plausible reporters that fibre from pasta was higher in those with UW than in those with NW or OW. This may be due to subjects who have an excessive body weight try to control their weight by the reduction of carbohydrates of the diet reducing the intake of this type of food. When the data were analysed according to the presence or absence of abdominal obesity using the WHtR, we found that the intake of fibre (raw, adjusted by energy intake and express per 1000 kcal per day) only in the whole sample was higher in those subjects without abdominal obesity. In this manner, it becomes clear that the fibre intake may help to avoid the appearance of abdominal obesity, which has also been described in a study performed in Chinese adults that observed subjects with a lower WHtR had a higher fibre intake [56]. On the other hand, the pattern of fibre intake in the different meals during the day varied depending on the presence or absence of abdominal obesity only in the whole sample. The proportion of fibre from lunch was higher in the participants with abdominal obesity compared to their normal counterparts, which could be due to the style of eating in Spain, since in general, lunch is characterized by the presence of an abundant amount of foods like cereals, pulses and vegetables that provide a great amount of fibre, respect to other meals of the day. This type of lunch is largely respected by the general population regardless of individual food habits. However, in relation to the snack and dinner, in Spain is observed that there is a much more marked difference in the composition depending on the food habits of each person. On the other hand, the proportion of fibre from afternoon snacks was higher in individuals without abdominal obesity than in those who had this problem. This finding suggests that a higher intake of fibre in the afternoon could have a beneficial effect in relation to the abdominal accumulation of body fat and not only with respect to the body weight situation described previously. Nevertheless, further studies are needed to clarify this aspect. Nutrients 2017, 9, 326 17 of 22 Some studies have indicated that the consumption of whole grain foods seems to have benefits regarding weight control and abdominal adiposity, emphasizing that the consumption of whole grain products seems to have not promote weight gain, while refined-grain products are directly associated with the excess of weight and abdominal fat [7,57]. In our study, the contribution to the intake of fibre from bread and pasta was higher only in subjects with abdominal obesity of the whole sample. However, the information about the type of bread or pasta consumed by the population was not available in our study. Moreover, various studies have found an inverse association between dietary fibre from fruits and the WC, insulin resistance, and metabolic syndrome [58–60]. In line with this, in our study, the fibre from fruits was higher only in men of the whole sample without abdominal obesity than in those with this problem. This difference, as previously noted, could be explained because of the beneficial effect of soluble fibre on the reduction of the appetite or due to a healthier lifestyle [9,49]. A similar trend was also observed in relation to the group of sugars and sweets and, in particular, with the subgroup of chocolates. However, because the food groups disaggregated by food items have not been analysed, it was not possible to give an explanation. However, it is assumed that this difference may be due to participants without abdominal obesity selecting and consuming some type of chocolate or similar healthier product with a higher content of fibre, unlike the group who has abdominal obesity. We only observed in the group of plausible reporters that fibre from ready-to-eat-meals was higher in individuals with abdominal obesity compared to subjects without abdominal obesity, which is according with following an unhealthy diet rich in ready-to-eat-meals, which is common in individuals with obesity. Likewise, when analysing the excess body weight and abdominal obesity individually, it was confirmed that there was a greater fibre intake in subjects without excess body weight and abdominal obesity compared with those who had one or two of these problems only in the whole sample. It is noted that the fibre from the afternoon snack seems to play a major role in the body weight situation and abdominal obesity. Even when considering the presence or absence of both problems in the same individual, we also found that those subjects in the whole sample with excess body weight or abdominal obesity had a lower proportion of fibre from the afternoon snack and higher from lunch than their counterparts. This suggests that, probably, the meal of the day in which the fibre is consumed is of relevance to obtaining the benefits of the fibre in relation to excess body weight or abdominal obesity. In relation to the dietary fibre sources according to the excess body weight and/or abdominal obesity, we found a consistent tendency when the sources were analysed according to the excess body weight and abdominal obesity separately. A higher proportion of fibre from bread and pasta (in men and woman), and less from fruit (only in men), is associated with excess body weight and/or abdominal obesity in comparison with subjects with NW and without abdominal obesity. As in the whole sample, in the plausible reporters we also observed that fibre from fruits was significantly higher in individuals without excess body weight and/or abdominal obesity compared to subjects with excess body weight and/or abdominal obesity. However, only in the plausible reporters, the fibre from ready-to-eat-meals was higher in those subjects with excess body weight and/or abdominal obesity than those without excess body weight and/or abdominal obesity, which is consistent with the results when the fibre dietary sources were analysed according to the presence or absence of abdominal obesity individually. A limitation of our study was the inability to analyse the types of fibre (soluble and insoluble) and the food groups disaggregated by food items, since such information was not available. Nonetheless, this did not represent an impediment to achieve the aim of our work that was to study the intake and dietary food sources of fibre and analyse the differences in the fibre intake between people with different body weight situations, and with or without abdominal obesity. In contrast, the main strengths of our study include the methodological design used in the ANIBES study, such as the fact that all anthropometric data were measured and they were not self-reported by the participants, Nutrients 2017, 9, 326 18 of 22 which improves the validity of the study, and the possibility of extrapolating our results to the Spanish population because it was conducted in a representative sample. It is important to highlight that this is the first Spanish study at national level that analyses the data for the whole population and the plausible reporters. The findings regarding the association between diet and the health outcomes analysed in the present study should be interpreted with caution given the discrepancy observed between both samples. Further studies considering different methods to address misreporting are needed to confirm the association between the fibre intake and the excess body weight and/or abdominal obesity. At the same time, the information derived from our study can be useful in designing nutrition intervention strategies to increase the intake of fibre in our country that it was low both in the whole sample and in the plausible reporters, which in turn could prevent and control some health problems such as excess body weight and abdominal obesity. 5. Conclusions The present study demonstrates an insufficient fibre intake among the Spanish adult population, both in the whole sample and in the plausible reporters. The main dietary sources were grains, followed by vegetables, fruits, and pulses for both samples. This study observed an association between the fibre intake and excess body weight and abdominal obesity in the whole sample but not in the plausible reporters. Further studies are needed to confirm the association between the fibre intake and the excess body weight and/or abdominal obesity. Nonetheless, it is advisable to increase the intake of foods rich in fibre in order to prevent diseases related with a low intake. Supplementary Materials: The following are available online at www.mdpi.com/1999-4907/9/04/325/s1, Table S1: Dietary food sources of total fibre (%) in the whole sample of the ANIBES study of the Spanish adult population (18–64 years) by body mass index., Table S2: Dietary food sources of total fibre (%) in the plausible reporters of the ANIBES study of the Spanish adult population (18–64 years) by body mass index, Table S3: Dietary food sources of total fibre (%) in the whole sample of the ANIBES Study Spanish adult population (18–64 years) by the presence or absence of abdominal obesity using the waist to height ratio, Table S4: Dietary food sources of total fibre (%) in the plausible reporters of the ANIBES Study Spanish adult population (18–64 years) by the presence or absence of abdominal obesity using the waist to height ratio, Table S5: Dietary food sources of total fibre (%) in the whole sample of the ANIBES study of the Spanish adult population (18–64 years) by the presence or absence of excess body weight and/or abdominal obesity using the body mass index and the waist to height ratio, Table S6: Dietary food sources of total fibre (%) in the plausible reporters of the ANIBES study of the Spanish adult population (18–64 years) by the presence or absence of excess body weight and/or abdominal obesity using the body mass index and the waist to height ratio. Acknowledgments: The authors would like to thank Coca-Cola Iberia and IPSOS for its support and technical advice, particularly Rafael Urrialde and Javier Ruiz. Author Contributions: L.G.G.-R., J.M.P.S and R.M.O. analysed the data. L.G.G.-R. also drafted and wrote the manuscript. J.M.P.S and R.M.O. contributed to the analysis and wrote the manuscript. J.A.-B., A.G., R.M.O., M.G.-G., and L.S.-M. are members of the Scientific Advisory Board of the ANIBES study and were responsible for careful review of the protocol, design, and methodology. These authors provided continuous scientific advice for the study and for the interpretation of results. These authors also critically reviewed the manuscript. G.V.-M., Principal Investigator of the ANIBES study, was responsible for the design, protocol, methodology, and follow-up checks of the study. All authors approved the final version of the manuscript. Conflicts of Interest: The ANIBES study was financially supported by a grant from Coca Cola Iberia through an agreement with the Spanish Nutrition Foundation (FEN). The funding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of the data; in the writing of the manuscript, and in the decision to publish the results. The authors declare no conflict of interest. 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Undeveloped till soils in scree areas are an overlooked important phosphorus source for waters in alpine catchments

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Jiří Kaňa 1,2*, Eva Kaštovská 2, Michal Choma 2, Petr Čapek 2, Karolina Tahovská 2 & Jiří Kopáček 1 Jiří Kaňa 1,2*, Eva Kaštovská 2, Michal Choma 2, Petr Čapek 2, Karolina Tahovská 2 & Jiří Kopáček 1 Scree deposits in alpine catchments contain undeveloped till soils that are “hidden” between and under stones. These scree areas have no vegetation except for sparse lichen patches on stone surfaces, but the soils exhibit biological activity and active cycling of nitrogen (N), phosphorus (P), and organic carbon (C). We compared the chemical and biochemical properties of till soils in the scree areas (scree soils) with developed soils in alpine meadows (meadow soils) of 14 catchments in the alpine zone of the Tatra Mountains. The data showed that scree soils served as an important source of mobile P forms for waters in high elevation catchments. We then conducted a detailed soil survey focused on four selected alpine catchments with scree cover proportions > 30%. This study confirmed that scree soils have significantly higher concentrations of mobile P forms compared to meadow soils, and a high specific microbial activity directed towards the extraction of P with rapid turnover in the microbial biomass. The combination of these properties and the amounts of scree soils in high-elevation areas highlight their importance in overall biogeochemical P cycling in alpine catchments, and the terrestrial P export to receiving waters. Alpine catchments in the Tatra Mountains (Slovak-Polish border; central Europe) represent relatively remote ecosystems without direct human impacts, except for acidification and N-saturation caused by the long-distance transport of sulphur and nitrogen (N) compounds that peaked in the 1980s and has been dramatically decreasing since the early ­1990s1. As a response to the decreasing acidic deposition, lake water chemistry and biota (regularly studied since 1984) have been successfully ­recovering2–4. At the same time, increases in dissolved organic carbon (DOC), total organic N (TON), and total phosphorus (P) concentrations have been recently observed in the Tatra Mountain ­lakes5. Similarly, P concentrations have been rising in some other ­European6 and North ­American7–9 mountain waters. P usually limits the primary production of mountain lakes, and elevated P inputs thus increase their algal concentrations and change the composition of zooplankton and benthos, altering anticipated trajecto- ries during recovery from their original pre-acidification ­states4. www.nature.com/scientificreports www.nature.com/scientificreports 1Institute of Hydrobiology, Biology Centre CAS, Na Sádkách 7, 37005  České Budějovice, Czech Republic. 2Department of Ecosystem Biology, Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 1645/31a, 370 05 České Budějovice, Czech Republic. *email: [email protected] Undeveloped till soils in scree areas are an overlooked important phosphorus source for waters in alpine catchments OPEN Jiří Kaňa 1,2*, Eva Kaštovská 2, Michal Choma 2, Petr Čapek 2, Karolina Tahovská 2 & Jiří Kopáček 1 www.nature.com/scientificreports/ soils have higher concentrations of extractable P ­forms14 and a higher ability to liberate phosphate during their pH increase than soils in alpine meadows (hereafter meadow soils)10. soils have higher concentrations of extractable P ­forms14 and a higher ability to liberate phosphate during their pH increase than soils in alpine meadows (hereafter meadow soils)10. Direct atmospheric deposition on surfaces is an important source of P for lakes, especially for those with a small ratio of catchment to lake area. The P concentrations in atmospheric deposition are an order of magnitude higher than in most of the Tatra Mountain alpine ­lakes15. This atmospherically deposited P is more effectively immobilized in soils than in bare rock areas. Consequently, the proportion of rocks, meadows and scree deposits in catchments is also important for the overall catchment ability to immobilize atmospherically deposited P. y y Besides physico-chemical processes, phosphorus in soils is also immobilized by plants and transformed into organic forms (e.g. Ref.16) or into the microbial biomass (e.g. Refs.17,18); both of these processes are expected to be higher in vegetated parts of the catchment.h g g p The aim of this study was to characterize the chemical and microbial characteristics of scree soils and compare them with alpine meadow soils, with an emphasis on their potential role in P cycling. Besides their lower ability of scree soils to adsorb atmospherically deposited P and higher ability of P desorption during pH increases, we hypothesized that higher P leaching from scree than meadow soils resulted from their lower potential to immo- bilize P in soil organic matter and microbial biomass. Materials and methodsh The Tatra Mountains (the highest part of Carpathian chain) are situated in central Europe along the Polish-Slovak border (Fig. 1). Bedrock is granodiorite in the central part, the western part also consists of gneiss and mica ­schist19. For more details on the Tatra Mountain characteristics see Ref.12. The soils in alpine zone are dominated by shallow undeveloped leptosol and regosol in alpine meadows, and by sparse scree soils below surface stones in scree ­deposits14. Scree deposits investigated in this study were formed by unconsolidated bedrock material originated from physical weathering of rocks (Fig. SI-1). They were to a large extent similar to talus areas as defined by Williams et al.20, but were without vegetation. i y g Soils were sampled in the alpine zone of the Tatra Mountains (elevation of 1700–2200 m a.s.l.), where land cover is comprised mostly of scree or rocks (bare and/or covered with lichens; commonly Rhizocarpon, Acaros- pora oxytona, and Dermatocarpon luridum) and with sparse vegetation (dry alpine tundra dominated by Calama- grostis villosa, Festuca picta, and Luzula luzuloides). Two sampling surveys were conducted (Fig. 1). The first soil sampling survey aiming to compare the basic soil chemistry of scree areas (covering 5–67% of ­catchments12) and alpine meadows was performed in September 2015. The second survey was done in September 2020 and focused on comparisons of the two soil types in four catchments with the same granodiorite bedrock and with high proportions of scree deposits (> 30%). The comparison included more detailed soil analyses, including microbial biomass and its enzymatic activity. Soil sampling and analyses. The 2015 soil survey. In each catchment, one soil sample from a scree de- posit and one sample from a meadow were taken. Figure 1. Location of sampling sites in 2015 and 2020. Full-red squares denote catchments sampled in 2015 only, red squares with yellow fill denote positions of catchments with high proportions of scree deposits analyzed in more detail in 2020 (VS-02 was sampled in 2020 only). Codes of lake catchments: BV-22 (Vyšné Žabie Bielovodské), BY-01 (Veľké Bystré), FU-01 (Vyšné Wahlenbergovo), GA-03 (Zadni Staw Gąsienicowy), JM-05 (Vyšné Jamnícke), ME-01 (Veľké Hincovo), ME-02 (Malé Hincovo), MO-06 (Wyżni Mnichowy Stawek), NE-01 (Vyšné Terianske), NE-03 (Nižné Terianské), RA-01 (Vyšné Račkové), RO-01 (Horné Roháčske), TE-01 (Veľké Temnosmrečinské), VS-02 (Pusté), and VS-04 (Ľadové). Map data came from OpenStreetMap. Figure 1. Location of sampling sites in 2015 and 2020. Jiří Kaňa 1,2*, Eva Kaštovská 2, Michal Choma 2, Petr Čapek 2, Karolina Tahovská 2 & Jiří Kopáček 1 The changes in P inputs to the Tatra Mountain lakes are probably associated with the recovery of till soils in scree areas (hereafter scree soils) from acidification (increasing P co-export with DOC)10 and climatic changes, affecting both the physico-chemical weathering of rocks and soil microbial processes in the ­catchments11–13.f Differences in the recovery rates and trajectories of chemical and biological parameters among the Tatra Mountain lakes reflect the importance of the terrain cover of their catchments such as areal proportions of meadows and scree ­deposits5. The chemical composition of lake water reflects (and integrates) biogeochemical processes in the catchment, which serves as a “transfer medium” of elements (originating from atmospheric deposition and weathering) during their transport from terrestrial to aquatic ecosystems. Alpine catchments widely differ in their proportions of meadows, scree deposits, and bare rocks. Recently, lakes within catchments with abundant scree areas have exhibited faster increases in pH and in concentrations of bicarbonate, calcium (Ca), P, DOC, and TON than lakes in catchments with larger proportions of alpine ­meadows5,12. This indicates that scree areas may be an important source of Ca, bicarbonate and P for lakes, while patchy areas of meadows may more effectively immobilize nutrients entering the catchments with atmospheric deposition or originating from weathering. In accord with this, previous studies on alpine Tatra Mountain soils have shown that scree 1Institute of Hydrobiology, Biology Centre CAS, Na Sádkách 7, 37005  České Budějovice, Czech Republic. 2Department of Ecosystem Biology, Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 1645/31a, 370 05 České Budějovice, Czech Republic. *email: [email protected] Scientific Reports | (2023) 13:14725 | https://doi.org/10.1038/s41598-023-42013-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Meadow soils were taken from 0.25 ­m2 pits (50 × 50 cm), in which two distinguishable upper horizons were excavated quantitatively. For the purpose of the study, we further use the following classification: the uppermost soil horizon rich in organic matter (A-horizon), and the underlying mineral horizon (M). Soil from each horizon was separately mixed and weighed. Scree soils were quantitatively sampled from pits (0.25–0.5 ­m2) made by the removal of stones in scree deposits to a depth of ~ 0.5 m. Fresh soil samples were then stored one week at 4 °C in the dark until further processing prior to analyses.h p g p y To remove coarse particles, soil samples were sieved through a 5-mm stainless-steel sieve. Then the < 5 mm fraction was air dried (AD) for 14–21 days between two sheets of filter paper at laboratory temperature, and finally passed through a stainless-steel < 2-mm sieve (hereafter referred as the AD soil). Before the elemental analyses, the AD soil was finely ground and homogenized. Part of the field-moist < 5 mm soil fraction was frozen at − 20 °C and stored for analyses of potential enzymatic activity. y p y y Elements in the finely ground soil were analyzed as follows: Dry weight (DW) and loss on ignition (LOI) were obtained by drying at 105 °C for 2 h and combustion at 550 °C for 2 h in an oven, respectively. Total phosphorus (P) was determined by ­HNO3 and ­HClO4 digestion according to Ref.21, and total soil organic C and N contents were measured in dried (60 °C) and milled samples using an elemental analyzer (Vario MICRO cube, Elementar, Germany). The total content of metals was analysed by flame atomic absorption spectrometry (Ca, Mg, Na, K, Fe, Mn, Li, and Ti) and/or volumetric titration (Al) after the mineralization of finely ground AD soil with ­H2SO4, ­HNO3, and HF (200 °C, 2 h). The concentration of Si was calculated from the concentration of ­SiO2, calculated as the difference between dry weight and loss on ignition (LOI) and the concentration of metal oxides (CaO, MgO, ­Na2O, ­K2O, ­Al2O3, ­Fe2O3, MnO, ­Li2O and ­TiO2). www.nature.com/scientificreports/ ghfi g EC EN EP Basal soil respiration was characterized by the rate of ­CO2 production from the soil at 10 °C. 10 g of soil in 100-ml flasks were preincubated for 10 days at 10 °C, which allowed the microbial activity to stabilize after res- piratory flushes following sieving pretreatment. Then, the samples were sealed air-tight and the ­CO2 accumulation was measured after 24 h using an Agilent 6850 GC system (Agilent Technologies, Santa Clara, CA, USA). The specific respiration activity was calculated as the ratio of ­CO2-C production to ­CMB29.i pi p y p Potential activities of five hydrolytic enzymes characterized the potential of microbial organic nutrient acquisi- tion. The activity of C-mining (β-glucosidase and cellobiosidase), N-mining (Ala-aminopeptidase and chitinase), and P-mining enzymes (phosphatase) were determined using a microplate fluorometric ­assay30. Thawed soils (1 g) were mixed into 100 ml of distilled water and sonicated for 4 min; 200 μl of the soil suspension was added to 50 μl of methylumbelliferyl solution to measure the potential activities of β-glucosidase, cellobiosidase, phos- phatase, and chitinase, the potential activity of Ala-aminopeptidase was measured after addition of 200 μl of the soil suspension to 50 μl of 7-aminomethyl-4-coumarin substrate solution. From pre-tested concentrations of each fluorogenic substrate (50, 100, and 300 μM), the one with the highest enzymatic activity was chosen. Plates were incubated at 20 °C for 2 ­h31, and then the fluorescence was measured with an INFINITE F200 microplate reader (TECAN, Crailsheim, Germany) at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. The activities of C-, N- and P- mining enzymes, respectively, were summed and expressed as propor- tions (%) of the total hydrolytic ­activity32. The 2020 soil survey. In 2020, soil sampling was performed to compare scree and meadow soils in four catch- ments with granodiorite bedrock (Fig. 1). Their respective catchment codes, areas and percentages of scree cover are as follows: Ľadové (VS-04, 13 ha; 64%), Vyšné Wahlenbergovo (FU-1, 32 ha; 46%), Pusté (VS-02, 20 ha; 43%), and Veľké Hincovo (ME-01, 127 ha; 32%). The relative proportions of scree cover was estimated using aerial geo- referenced photographs, details are given in Ref.11. www.nature.com/scientificreports/ g 2 2 2 3 2 3 2 2) Oxalate-extractable Fe ­(Feox), Al ­(Alox), P ­(Pox) and soluble reactive oxalate-extractable P ­(SRPox) were deter- mined by the extraction of 0.5 g of AD < 2 mm soil with 50 ml of acid ammonium oxalate solution (0.2 M ­H2C2O4 + 0.2 M ­(NH4)2C2O4 at pH 3) according to Ref.22, with extraction process modified by Kopáček et al.14. The ­Feox, ­Alox, and ­Pox concentrations were determined using the method by Kopáček et al.21, and ­SRPox colori- metrically according to Wolf and ­Baker23. Concentrations of bioavailable orthophosphates according to Olsen and ­Sommers24 ­(POlsen) were determined after the extraction of field-moist soil with 0.5 M ­NaHCO3 (1:15; 45 min). The effective cation exchange capacity (CEC) was calculated as the sum of exchangeable base cations ti The effective cation exchange capacity (CEC) was calculated as the sum of exchangeable base cations ­(BCEX = sum of ­Ca2+ EX, ­Mg2+ EX, ­Na+ EX, ­K+ EX) and exchangeable acidity (the sum of ­Al3+ EX and ­H+ EX), measured by extracting of 2.5 g of dried < 2 mm AD soil with 50 ml of 1 M ­NH4Cl and 1 M KCl, ­respectively14. Concentrations of ­BCEX were determined by ICP-MS and exchangeable acidity was determined by titration (phenolphthalein, 0.1 M NaOH) according to ­Thomas25. Base saturation (BS) was the percentage of ­BCEX in CEC. gh p g Concentrations of C, N, and P in the soil microbial biomass (CMB, NMB, PMB) were measured by a chlo- roform fumigation-extraction ­method26–28. Fresh samples (< 4 mm, 5 g), were extracted either directly or after fumigation with ethanol-free chloroform for 24 h with 40 ml of 0.5 M ­K2SO4 for CMB and NMB, and with 75 ml of 0.5 M ­NaHCO3 for PMB, respectively, for 45 min, and then filtrated (Whatman, No 42). The sulfate extracts were analyzed for concentrations of organic C and total N using a TOC-L analyzer equipped with a total N meas- uring unit (TNM-L, Shimadzu, Japan). Bicarbonate extracts were acidified with ­H2SO4 and analyzed for reactive P concentrations spectrophotometrically according to Brookes et al.26. The CMB, NMB, and PMB were calcu- lated as differences in the concentrations of organic C, total N, and extractable P in soil extracts before and after fumigation. The values were corrected for extraction efficiencies using ­kEC = 0.4128, ­kEN = 0.4527, and ­kEP = 0.426. Materials and methodsh Full-red squares denote catchments sampled in 2015 only, red squares with yellow fill denote positions of catchments with high proportions of scree deposits analyzed in more detail in 2020 (VS-02 was sampled in 2020 only). Codes of lake catchments: BV-22 (Vyšné Žabie Bielovodské), BY-01 (Veľké Bystré), FU-01 (Vyšné Wahlenbergovo), GA-03 (Zadni Staw Gąsienicowy), JM-05 (Vyšné Jamnícke), ME-01 (Veľké Hincovo), ME-02 (Malé Hincovo), MO-06 (Wyżni Mnichowy Stawek), NE-01 (Vyšné Terianske), NE-03 (Nižné Terianské), RA-01 (Vyšné Račkové), RO-01 (Horné Roháčske), TE-01 (Veľké Temnosmrečinské), VS-02 (Pusté), and VS-04 (Ľadové). Map data came from OpenStreetMap. https://doi.org/10.1038/s41598-023-42013-4 Scientific Reports | (2023) 13:14725 | Results i The main significant differences between these soil types were the higher total concentrations of Ca, Na, and Mn in scree soils than in the meadow M horizon (for more details see Table SI-2).hif The 2020 soil survey in the four selected catchments confirmed the general pattern of differences between meadow and scree soils observed in 2015 (Table 2; details for individual catchments are given in Table SI-3). Scree soils significantly differed from meadow soils in the A horizon in concentrations of P species. Scree soils exhibited markedly higher concentrations of extractable P forms compared to meadow soils despite their slightly lower content of total P (21.8 vs. 26 µmol ­g−1; p < 0.05) and no significant differences in ­Pox concentrations. The ­SRPox concentrations were three times higher in the scree than meadow soils (10 vs. 3 µmol ­g−1; p < 0.001), and their ­POlsen concentrations were even one order of magnitude higher (1.5 vs. 0.11 µmol ­g−1; p < 0.001).hi g g µ g p The concentrations of ­Pox were significantly correlated with concentrations of ­Alox and ­Feox, with stronger relationships in scree than in meadow soils. Scree soils had similar ­Feox concentrations to meadow soils (58 and 67 µmol ­g−1) but lower ­Alox contents (87 vs. 163 µmol ­g−1; p < 0. 01).h µ g ox µ g p The meadow soils had higher total C contents than scree soils, and a lower δ13C (− 25.96‰ compa to − 24.12 ‰ in scree soils, Fig. 3) indicating different origins of organic matter for these soil types.fh f Concentrations of water-extractable C, N, and P forms also differed between the soil types. The meadow soils had higher concentrations of DOC, DN, DON, and ­NH4-N, but lower ­NO3-N (72 vs. 36 µmol ­kg−1; p < 0.001) and ­SRPH2O concentrations (8.3 vs. 3.4 µmol ­kg−1; p < 0.001) than scree deposit soils (Fig. 4). Soil microbial and biochemical parameters. The microbial biomass in scree soils was similar to that in the M horizon in meadows, but substantially lower compared to the meadow A horizon (Table 3). The CMB and NMB concentrations were about 5 times lower in scree soils than in the meadow A horizon (34 vs. 168 µmol ­g−1 and 2.8 vs. 15.4 µmol ­g−1, respectively), and the PMB concentration was ~ 3 times lower (1.7 vs. 5.6 µmol ­kg−1; p < 0.001). Results i Basic soil characteristics. Soils in alpine meadows were generally shallow. The upper organic-rich hori- zon A was on average 14 cm deep (5–36 cm), densely rooted and rich in stones (the average ratio of removed coarse stones >  ~ 5 cm to unsieved soil ratio was 1.4 on site). In places with deeper soils, the A horizon gradually turned into the lighter colored M horizon. The amount of soils (dry weight < 2 mm fraction) varied between 8–85 kg ­m−2 in the A horizon, and 0–125 kg ­m−2 in the M horizon (Table 1). In the 2020 soil survey, we observed an average depth of the A horizon of 11 cm, underlain by regolith (Table 2).h g p y g The diameter of stones in scree deposits varied from centimeters to meters. Scree soils below the upper layer of stones were deposited in gaps between stones or on surfaces of deeper stones (Fig. SI-1). Hence, the scree soils did not form a continuous layer, but appeared in isolated deposits weighing from grams to kilograms. The estimated amount of soil (< 2 mm fraction; dry weight) ranged between 4 and 16 kg ­m−2, with an average of 9 kg ­m−2 in the 0.5 m upper layer of scree. Similar scree soil amounts were also observed in the 2020 survey, with an average of 9 kg ­m−2 in the upper 0.5 m scree layer, varying from 4 in the VS-02 to 14 kg ­m−2 in the FU-01 catchment; details are given in Supplementary information (Table SI-1). Soil chemistry. All the soils were acidic, with ­pHH2O ranging between 4.1–5.1 and ­pHCaCl2 between 3.35–4.1 (Tables 1, 2), with no significant difference between meadow and scree soils. The chemical composition of scree soils was similar to soils in the M horizon of alpine meadows. Both these soil types contained less C and ­BCEX than the meadow A horizon (Table 2). However, there were several important differences between the scree and M horizon soils. Scree soils had lower concentrations of total N, ­Alox, ­Al3+ EX, and CEC than the M horizons, but higher concentrations of all extractable P forms ­(Pox, ­SRPox, ­POlsen) (Table 1), and also higher proportions of ­SRPox and ­POlsen in total P concentrations (Fig. 2). g Total concentrations of metals and Si were generally similar in the M horizons of scree and meadow soils (Table SI-2). www.nature.com/scientificreports/ www.nature.com/scientificreports/ The chemical and microbiological analyses were performed the same as in 2015, with the following exceptions: an additional determination of nutrient (C, N, P) availability by the extraction of fresh soil samples with water was performed by extracting field-moist soils with distilled water (1:10, w/v) with 1 h shaking on a horizontal shaker; the extracts were filtered (Whatman GF/C filters) and analyzed for DOC, total dissolved nitrogen (DN), ­NO3-N, ­NH4-N, and soluble reactive P ­(SRPH2O). DOC and DN were analyzed using a TOC-L analyzer equipped with the TNM-L total N measuring unit (Shimadzu, Japan). Concentrations of ­NO3-N, ­NH4-N, and SRP were determined using a flow injection analyzer (FIA Lachat QC8500, Lachat Instruments, Milwaukee, WI, USA) by the following methods: ascorbic acid reduction of phosphomolybdic acid for the analysis of SRP, a phenol- hypochlorite assay with sodium nitroprussite as the catalyst (Berthelot reaction) for ­NH4-N determination, and the diazotisation of sulfanilic acid with subsequent coupling with N-(1-naphthyl)-ethylenediamine in a strongly acid solution for ­NO3-N determination. Dissolved organic N (DON) was calculated by subtraction of ­NO3-N and ­NH4-N from DN (DON = DN–NH4–N–NO3–N).hh 4 4 3 The isotopic composition of soil C was characterized using an NC Elemental analyzer (ThermoQuest, Ger- many) connected to an isotope ratio mass spectrometer (IR-MS Delta X Plus, Finnigan, Germany). The measured ratio of 13C/12C was expressed as δ13C using a Vienna PDB standard. All data on chemistry and biochemistry in this study are given as related to the DW (105 °C) fraction. Due to a non-normal data distribution as tested by the Shapiro–Wilk test (p < 0.01), the non-parametric Mann–Whitney U was chosen for testing differences between soil types. All statistical tests were performed using XLSTAT 2022 software (Addinsoft). www.nature.com/scientificreports/ p g p g Meadow soils were sampled only from the upper organic-rich horizon (here called the A horizon) from pits of 0.023 ­m2 (15 × 15 cm) at nine sites within each catchment (3 samples were randomly combined by weigh to gain three representative samples per catchment). Scree soils were sampled from three representative plots in each catchment, using the same approach as in 2015. Scientific Reports | (2023) 13:14725 | https://doi.org/10.1038/s41598-023-42013-4 Results i Meadow A Meadow M Scree n = 14 n = 9 n = 14 Depth cm 5–36 (14) 0–36 ­(21†) n.m Soil (< 2 mm) kg ­m−2 8–85 (32) 0–125 ­(71†) 4–16 (9)†† pHH2O 4.24–4.88 (4.63) 4.58–5.1 (4.81) 4.39–4.88 (4.61) pHCaCl2 3.35–4.10 (3.77) 3.66–4.23 (4.0) 3.57–4.05 (3.79) LOI % 13–41 (23)b 6–18 (11)a 5.8–17.5 (11.1)a C mmol ­g−1 6.2–18.2 (9.6)b 2.8–8.2 (4)a 2.2–8.2 (5)a N mmol ­g−1 0.34–1.04 (0.6)c 0.16–0.48 (0.19)a 0.12–0.44 (0.32)b P µmol ­g−1 17.4–59 (32.2)b 10.7–45.9 (21.1)a 18.2–39.43 (27.3)ab C:P molar ratio 64–222 (311)b 135–387 (224)a 79–266 (197)a C:N molar ratio 13.4–20 (16.5)a 14.7–24.1 (19.2)b 13.9–18.6 (16.4)a Pox µmol ­g−1 8.2–26 (14.2)ab 5.2–29.1 (10.8)a 8.4–22 (15.4)b SRPox µmol ­g−1 3–8.8 (4.1)a 1.4–8.8 (5.7)a 5.3–16.3 (10)b Alox µmol ­g−1 47–345 (151)b 88–377 (209)b 42–159 (86)a Feox µmol ­g−1 24–148 (83) 24–213 (96) 42–103 (61) (Al + Fe)ox µmol ­g−1 91–492 (223)b 112–509 (309)b 85–219 (147)a POlsen µmol ­g−1 0.1–0.3 (0.17)a 0.04–0.34 (0.18)a 0.16–3.5 (1.95)b Na+ EX µeq ­g−1 0.2–1.3 (0.6)b 0.1–1 (0.3)a 0.1–0.3 (0.2)a K+ EX µeq ­g−1 2.2–9.1 (4.9)b 0.4–2.4 (1.3)a 0.7–2.0 (1.3)a Mg2+ EX µeq ­g−1 1.4–7.1 (4.0)b 0.3–1.8 (0.7)a 0.3–2.1 (0.9)a Ca2+ EX µeq ­g−1 2.1–18 (6.3)b 0.4–4.3 (2.6)a 1.1–5.3 (1.8)a H+ EX µeq ­g−1 8.5–77 (22.5)b 2.4–20.7 (14.5)a 3.3–21.3 (10.7)a Al3+ EX µeq ­g−1 72–152 (99)b 49–123 (94)b 31–89 (66)a BCEX µeq ­g−1 6.6–95 (15)b 1.7–9.2 (4.4)a 2.3–8.4 (4.4)a BS % 5–45 (12.6)a 1.2–13 (5.3)a 3.6–20 (5.9)a CEC µeq ­g−1 107–212 (152)c 55–145 (110)b 43–109 (79)a Table 1. Basic chemical characteristics of alpine meadow (horizons A and M), and scree soils sampled in 2015 from 14 Tatra Mountains catchments (minimum–maximum values with medians in parentheses). n.m. not measured. Different upper-case letters indicate statistically significantly (p < 0.05) different chemical parameters. † The median was calculated only for cases if the M horizon was present. †† Up to 0.5 m under the surface. In concert with their lower microbial biomass, scree soils exhibited almost one order of magnitude lower respiration rates than soils in the A horizon of meadows (0.15 vs. 1.3 µmol C ­g−1 ­d−1) and a lower total activity of hydrolytic enzymes (Fig. 5). However, microbial communities in scree soils were more active compared to those in meadow soils, evidenced by their higher biomass-specific respiration rate ­(CO2-C:CMB of 11 vs. 8 µmol ­mol−1; p < 0.05) and enzymatic activity. Results i The relative enzymatic investments into P-gaining was higher but that into C-gaining lower in scree soils than in meadow soils (Fig. 5). Results i The detailed 2020 soil survey thus confirmed that scree soils contained a lower microbial biomass than meadow soils (Table 3). https://doi.org/10.1038/s41598-023-42013-4 Scientific Reports | (2023) 13:14725 | www.nature.com/scientificreports/ Table 1. Basic chemical characteristics of alpine meadow (horizons A and M), and scree soils sampled in 2015 from 14 Tatra Mountains catchments (minimum–maximum values with medians in parentheses). n.m. not measured. Different upper-case letters indicate statistically significantly (p < 0.05) different chemical parameters. † The median was calculated only for cases if the M horizon was present. †† Up to 0.5 m under the surface. Meadow A Meadow M Scree n = 14 n = 9 n = 14 Depth cm 5–36 (14) 0–36 ­(21†) n.m Soil (< 2 mm) kg ­m−2 8–85 (32) 0–125 ­(71†) 4–16 (9)†† pHH2O 4.24–4.88 (4.63) 4.58–5.1 (4.81) 4.39–4.88 (4.61) pHCaCl2 3.35–4.10 (3.77) 3.66–4.23 (4.0) 3.57–4.05 (3.79) LOI % 13–41 (23)b 6–18 (11)a 5.8–17.5 (11.1)a C mmol ­g−1 6.2–18.2 (9.6)b 2.8–8.2 (4)a 2.2–8.2 (5)a N mmol ­g−1 0.34–1.04 (0.6)c 0.16–0.48 (0.19)a 0.12–0.44 (0.32)b P µmol ­g−1 17.4–59 (32.2)b 10.7–45.9 (21.1)a 18.2–39.43 (27.3)ab C:P molar ratio 64–222 (311)b 135–387 (224)a 79–266 (197)a C:N molar ratio 13.4–20 (16.5)a 14.7–24.1 (19.2)b 13.9–18.6 (16.4)a Pox µmol ­g−1 8.2–26 (14.2)ab 5.2–29.1 (10.8)a 8.4–22 (15.4)b SRPox µmol ­g−1 3–8.8 (4.1)a 1.4–8.8 (5.7)a 5.3–16.3 (10)b Alox µmol ­g−1 47–345 (151)b 88–377 (209)b 42–159 (86)a Feox µmol ­g−1 24–148 (83) 24–213 (96) 42–103 (61) (Al + Fe)ox µmol ­g−1 91–492 (223)b 112–509 (309)b 85–219 (147)a POlsen µmol ­g−1 0.1–0.3 (0.17)a 0.04–0.34 (0.18)a 0.16–3.5 (1.95)b Na+ EX µeq ­g−1 0.2–1.3 (0.6)b 0.1–1 (0.3)a 0.1–0.3 (0.2)a K+ EX µeq ­g−1 2.2–9.1 (4.9)b 0.4–2.4 (1.3)a 0.7–2.0 (1.3)a Mg2+ EX µeq ­g−1 1.4–7.1 (4.0)b 0.3–1.8 (0.7)a 0.3–2.1 (0.9)a Ca2+ EX µeq ­g−1 2.1–18 (6.3)b 0.4–4.3 (2.6)a 1.1–5.3 (1.8)a H+ EX µeq ­g−1 8.5–77 (22.5)b 2.4–20.7 (14.5)a 3.3–21.3 (10.7)a Al3+ EX µeq ­g−1 72–152 (99)b 49–123 (94)b 31–89 (66)a BCEX µeq ­g−1 6.6–95 (15)b 1.7–9.2 (4.4)a 2.3–8.4 (4.4)a BS % 5–45 (12.6)a 1.2–13 (5.3)a 3.6–20 (5.9)a CEC µeq ­g−1 107–212 (152)c 55–145 (110)b 43–109 (79)a Table 1. Basic chemical characteristics of alpine meadow (horizons A and 2015 from 14 Tatra Mountains catchments (minimum–maximum values w not measured. Different upper-case letters indicate statistically significantly parameters. † The median was calculated only for cases if the M horizon was surface. Discussion To assess the role of scree deposit soils in the biogeochemistry of alpine catchments, and to gain insights into their role in terrestrial P cycling and supply to lakes, we estimated the amounts and properties of scree soils in the alpine zone of the Tatra Mountains. We found 4–16 kg ­m−2 of scree soils in 14 alpine catchments in 2015, as well as in 4 scree-rich catchments in 2020. These scree soil pools were similar to previous ­estimates14 which reported on average 13 kg ­m−2 of dry weight < 2 mm soils in the upper 0.5 m layer of scree areas of six Tatra Mountain catchments. We are aware that these values probably underestimate the total amount of scree soils due to the relatively shallow sampling resulting from the physical impossibility of reaching deeper levels by manual removing stones (no mechanization or terrain disturbance is allowed in the protected areas). Hence, we suppose that the total amount of soil accumulated in the whole profile of scree areas is likely higher than reported here for the uppermost 0.5 m. Our estimates should thus be considered as a lower limit for scree soils in the Tatra Mountain catchments.h The comparison of scree and meadow soils revealed general similarities in their basic elemental composition, reflecting their common origin in the physico-chemical weathering of parent granodiorite bedrock. Both soils are relatively rich in P, which is, however, present primarily in forms inaccessible for organisms. On the other hand, these soil types largely differ in microbial and biochemical parameters, primarily connected with inputs of organic matter having different quantity, quality and origin.fh g gf q y q y g Alpine meadow soils are substantially affected by plant activity. The presence of higher plants is connected with the input of organic matter via litter and rhizodeposition, and its transformation mediated by the activity of soil ­microorganisms33. The accumulation of soil organic matter leads to the formation of an A horizon, whose Scientific Reports | (2023) 13:14725 | https://doi.org/10.1038/s41598-023-42013-4 www.nature.com/scientificreports/ Table 2. Basic chemical characteristics of alpine meadow (A horizon) and scree soils sampled in 2020 in 4 selected Tatra Mountain catchments with a high proportion of scree deposits (Fig. 1). Ranges and medians (in brackets) are given. Asterisks denote significant differences: *p < 0.05, **p < 0.01, ***p < 0.001. Discussion Meadow Scree n = 12 n = 12 pHH2O 4.12–4.73 (4.38) 4.24–4.16 (4.46) pHCaCl2 3.48–3.91 (3.69) 3.73–4.13 (3.87)** LOI % 4.9–43 (15.4) 4.3–11.8 (6.2)** C mmol ­g−1 1.6–17.9 (6) 1.2–4.7 (2.6)** N mmol ­g−1 0.11–1.1 (0.39) 0.09–0.28 (0.17)** P µmol ­g−1 18–52 (26) 11–32 (21.8)* Pox µmol ­g−1 8–28.2 (17.6) 5.2–21.1 (16.1) SRPox µmol ­g−1 2.1–4.4 (3) 3.5–16.3 (10)*** Alox µmol ­g−1 57–210 (163) 48–203 (87)** Feox µmol ­g−1 27–101 (58) 34–87 (67) (Al + Fe)ox µmol ­g−1 102–275 (228) 68–213 (119)* POlsen µmol ­g−1 0.02–0.28 (0.11) 0.5–2.4 (1.5)*** C:P Molar ratio 0.05–0.34 (0.24) 0.06–0.29 (0.12)* C:N Molar ratio 13.2–18.6 (15.9) 12.8–16.9 (15.4) Na+ EX µeq ­g−1 0.3–1 (0.8) 0.12–0.48 (0.3)*** K+ EX µeq ­g−1 2.3–8.4 (3.9) 0–1.3 (0.7)*** Mg2+ EX µeq ­g−1 2.1–8.2 (4) 0.3–0.9 (0.6)*** Ca2+ EX µeq ­g−1 2.4–21.1 (5.9) 0.5–3.4 (1)*** H+ EX µeq ­g−1 15.6–54 (28) 9.2–18 (13)*** Al3+ EX µeq ­g−1 62–113 (88) 30–60 (49)*** BCEX µeq ­g−1 8.4–34 (15.2) 1.5–4.1 (2.6)*** BS % 6.7–23 (10.7) 2.2–9.1 (4.1)*** CEC µeq ­g−1 95–178 (138) 43–79 (65)*** Meadow Scree n = 12 n = 12 pHH2O 4.12–4.73 (4.38) 4.24–4.16 (4.46) pHCaCl2 3.48–3.91 (3.69) 3.73–4.13 (3.87)** LOI % 4.9–43 (15.4) 4.3–11.8 (6.2)** C mmol ­g−1 1.6–17.9 (6) 1.2–4.7 (2.6)** N mmol ­g−1 0.11–1.1 (0.39) 0.09–0.28 (0.17)** P µmol ­g−1 18–52 (26) 11–32 (21.8)* Pox µmol ­g−1 8–28.2 (17.6) 5.2–21.1 (16.1) SRPox µmol ­g−1 2.1–4.4 (3) 3.5–16.3 (10)*** Alox µmol ­g−1 57–210 (163) 48–203 (87)** Feox µmol ­g−1 27–101 (58) 34–87 (67) (Al + Fe)ox µmol ­g−1 102–275 (228) 68–213 (119)* POlsen µmol ­g−1 0.02–0.28 (0.11) 0.5–2.4 (1.5)*** C:P Molar ratio 0.05–0.34 (0.24) 0.06–0.29 (0.12)* C:N Molar ratio 13.2–18.6 (15.9) 12.8–16.9 (15.4) Na+ EX µeq ­g−1 0.3–1 (0.8) 0.12–0.48 (0.3)*** K+ EX µeq ­g−1 2.3–8.4 (3.9) 0–1.3 (0.7)*** Mg2+ EX µeq ­g−1 2.1–8.2 (4) 0.3–0.9 (0.6)*** Ca2+ EX µeq ­g−1 2.4–21.1 (5.9) 0.5–3.4 (1)*** H+ EX µeq ­g−1 15.6–54 (28) 9.2–18 (13)*** Al3+ EX µeq ­g−1 62–113 (88) 30–60 (49)*** BCEX µeq ­g−1 8.4–34 (15.2) 1.5–4.1 (2.6)*** BS % 6.7–23 (10.7) 2.2–9.1 (4.1)*** CEC µeq ­g−1 95–178 (138) 43–79 (65)*** Table 2. Basic chemical characteristics of alpine meadow (A horizon) and scree soils sampled in 2020 in 4 selected Tatra Mountain catchments with a high proportion of scree deposits (Fig. 1). Ranges and medians (in brackets) are given. Asterisks denote significant differences: *p < 0.05, **p < 0.01, ***p < 0.001. Table 2. Discussion Basic chemical characteristics of alpine meadow (A horizon) and scree soils sampled in 2020 in 4 selected Tatra Mountain catchments with a high proportion of scree deposits (Fig. 1). Ranges and medians (in brackets) are given. Asterisks denote significant differences: *p < 0.05, **p < 0.01, ***p < 0.001. Figure 2. Comparison of the proportion (%) of extractable P forms in total P in alpine meadow soils (horizons A and M), and scree soils. ­Pox—oxalate-extractable P, ­SRPox—oxalate-extractable soluble reactive P, ­POlsen—P measured in ­HCO3 - extract according to Olsen and ­Sommers24. Ranges, outliers, and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, red crosses denote averages. Different letters denote statistically significant differences. Figure 2. Comparison of the proportion (%) of extractable P forms in total P in alpine meadow soils (horizons A and M), and scree soils. ­Pox—oxalate-extractable P, ­SRPox—oxalate-extractable soluble reactive P, ­POlsen—P measured in ­HCO3 - extract according to Olsen and ­Sommers24. Ranges, outliers, and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, red crosses denote averages. Different letters denote statistically significant differences. chemistry differs from the deeper mineral M horizon as well as from scree soils. Larger contents of organic C, N, and P in meadow A horizons than in scree soils is connected with a larger microbial biomass, which is evidenced by higher rates of exoenzymatic activities, and a higher C mineralization rate. Larger relative invest- ments into C-mining enzymes in meadow compared to scree soils (Fig. 5b) indicate that microbial metabolism targets the decomposition of complex C-rich organic compounds of plant origin. The high biological activity in the rhizosphere, consisting of root and microbial respiration and organic acid production, likely also intensified chemical ­weathering34,35. This enriched the meadow soil with extractable forms of base cations in the A horizon, https://doi.org/10.1038/s41598-023-42013-4 Scientific Reports | (2023) 13:14725 | www.nature.com/scientificreports/ tificreports/ Figure 3. Comparison of the isotopic composition of soil C in alpine meadow soils (horizon A) and scree soils from 4 catchments sampled in 2020 (FU-1, ME-01, VS-02, VS-04). In each catchment, three soil samples for each of both soil types were analyzed. Ranges and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. ntificreports/ Figure 3. Discussion and increased ­Alox and ­Feox ­concentrations36 in both A and M horizons (Table 2) compared to the scree soil, which enhanced the potential of P retention in alpine meadow ­soils37. The high biological activity within the organic layer also influenced the mineral horizons of meadow soils. Intensified weathering and element leaching with organic acids during pedogenesis probably decreased the contents of total Ca and Na in the M horizons of meadow soils in comparison to the scree soils, which were otherwise similar in many parameters (Table 2, Table SI-2).fh and increased ­Alox and ­Feox ­concentrations36 in both A and M horizons (Table 2) compared to the scree soil, which enhanced the potential of P retention in alpine meadow ­soils37. The high biological activity within the organic layer also influenced the mineral horizons of meadow soils. Intensified weathering and element leaching with organic acids during pedogenesis probably decreased the contents of total Ca and Na in the M horizons of meadow soils in comparison to the scree soils, which were otherwise similar in many parameters (Table 2, Table SI-2).fh ) In contrast to meadow soils, the effect of vegetation is absent during the development of scree soils. They contained organic C in similarly low amounts as the M horizons of meadow soils (< 10 mol ­kg−1). The C concen- tration in scree soils was markedly enriched in 13C compared to the isotopic composition of plant-derived C in meadow soils (Fig. 3), indicating a different origin. The organic matter in scree soils probably originates mainly from the activity of prokaryotic autotrophs (cyanobacteria and other autotrophic bacteria, e.g., nitrifiers), which have a higher δ13C signature of bulk biomass compared to C3 ­plants38,39 (Alexander et al. 2006; Wada et al. 2012) with a minor potential contribution of allochtonous organic C sources like lichens, mosses, and wind-delivered organic litter and dust, including snow dust as described ­by40 Ley et al. (2004). Prokaryotes likely dominate the microbial communities in scree soils. A similar situation was observed in thin “soils” of non-vegetated areas in the low ­Arctic41 in habitats similar to the scree soils in alpine regions. The dominance of bacteria in the microbial community of scree soils could also explain the lower biomass C:N ratio (Table SI-3) and higher biomass-specific enzymatic and respiratory activity compared to meadow soils (Table 3). The low proportion of hydrolytic activ- ity targeted to C-mining (Fig. Discussion 5b) is in accord with the likely high degradability (due to the absence of plant structural polymers) of organic matter of microbial origin. In addition, the investment into N-mining in both soil types was very low (Fig. 5c), which can be explained by the N saturation of thin soils in alpine catchments due to the high long-term atmospheric N deposition in the Tatra ­Mountains1. In contrast, the high proportion of phosphatase within the hydrolytic activity in scree soils (~ 60% of the total hydrolytic activity, Fig. 5d), was significantly higher than in meadow soils, pointing to targeted fast “P-mining” from organic matter. We suggest that these unique properties of scree soils (i.e., the high biomass-specific activity of the microbial community connected with rapid turnover of the small pool of soil organic matter, and targeted P-mining from this pool) may contribute to a higher potential for P leaching from scree than meadow soils.hf y g p g The differences in soil chemistry also pointed to a higher P mobility in scree soils compared to meadow soils. Scree soils contained more than two-times higher amounts of the most mobile P form (i.e., ­SRPH2O), indicat- ing a higher potential for P loss. As ­shown10, the concentrations of ­SRPH2O extracted from the Tatra Mountain soils increase when soil water pH increases from 3 to 3.5 to higher values. However, even at the similarly low pH range in both meadow and scree soils, the ­SRPH2O release was about four-times higher from scree ­soils10. The present pH values are > 3.5 (Table 3) and significantly higher in scree soils, which favors the release of P and further enhances the potential for P losses. Scree soils further showed a higher ratio of mobile ­SRPox to ­Pox than meadow soils. Concentrations of ­Alox and ­Feox were lower in scree than in meadow soils (Table 3), which is in concert with their about two-times lower ability to retain ­phosphate10. A higher potential for P loss from scree soils is also indicated by the order of magnitude higher ­POlsen concentrations. In contrast to meadow soils, concentrations of ­POlsen in scree soils were well above the threshold value of 20 mg ­kg−1 (0.65 µmol ­g−1) above which the release of P from soils during rainfall has been suggested to ­increase42. Discussion Comparison of the isotopic composition of soil C in alpine meadow soils (horizon A) and scree soils from 4 catchments sampled in 2020 (FU-1, ME-01, VS-02, VS-04). In each catchment, three soil samples for each of both soil types were analyzed. Ranges and 25% and 75% quartiles are given, horizontal lines within the Figure 3. Comparison of the isotopic composition of soil C in alpine meadow soils (horizon A) and scree soils from 4 catchments sampled in 2020 (FU-1, ME-01, VS-02, VS-04). In each catchment, three soil samples for each of both soil types were analyzed. Ranges and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. Figure 3. Comparison of the isotopic composition of soil C in alpine meadow soils (horizon A) and scree soils from 4 catchments sampled in 2020 (FU-1, ME-01, VS-02, VS-04). In each catchment, three soil samples for each of both soil types were analyzed. Ranges and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. Figure 3. Comparison of the isotopic composition of soil C in alpine meadow soils (horizon A) and scree soils from 4 catchments sampled in 2020 (FU-1, ME-01, VS-02, VS-04). In each catchment, three soil samples for each of both soil types were analyzed. Ranges and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. Figure 4. Concentrations of water-extractable forms of C, N, and P in alpine meadow soils (horizons A) and scree soils sampled in four scree-rich catchments in 2020. Ranges, outliers, and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. Figure 4. Concentrations of water-extractable forms of C, N, and P in alpine meadow soils (horizons A) and scree soils sampled in four scree-rich catchments in 2020. Ranges, outliers, and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. Scientific Reports | (2023) 13:14725 | https://doi.org/10.1038/s41598-023-42013-4 www.nature.com/scientificreports/ Table 3. Microbial characteristics of alpine meadow and scree deposit soils sampled in 2015 from 14 catchments, and in 2020 from 4 catchments in the Tatra Mountains (data show ranges, averages are in parenthesis). CMB and NMB C and N in the microbial biomass, respectively. Resp. respiration ­(CO2-C production), CO2-C CMB specific respiration. Discussion Different letters in the 2015 survey section denote statistically significantly different values. In the 2020 survey section, asterisks indicate statistically significant differences: *p < 0.05, **p < 0.01, ***p < 0.001. The 2015 soil survey The 2020 soil survey A horizon (n = 14) M horizon (n = 9) Scree soil (n = 14) A horizon (n = 12) Scree soil (n = 12) CMB µmol ­g−1 46–480 (168)b 16–66 (33)a 19–51 (34)a 39–476 (185) 7–21 (14)*** NMB µmol ­g−1 4–42 (15.4)b 0.9–8.2 (3.0)a 0.8–5.5 (2.8)a 4–54 (23) 1.3–4 (2.6)*** PMB µmol ­g−1 1.8–13.5 (5.6)b 0.3–2.5 (1.2)a 0.3–4.3 (1.7)a 1.1–18.3 (5.7) 0.2–1.3 (0.7)*** Resp. µmol C ­g−1 ­d−1 0.53–1.7 (1.04)b 0.03–0.3 (0.2)a 0.13–0.46 (0.3)a 0.6–2.4 (1.3) 0.07–0.29 (0.15)*** CO2-C:CMB µmol ­mol−1 3.2–12.4 (7.2) 1–10.2 (6.8) 4.7–11.3 (8.6) 5–16 (8.1) 4.9–26.5 (11)* The 2015 soil survey The 2020 soil survey A horizon (n = 14) M horizon (n = 9) Scree soil (n = 14) A horizon (n = 12) Scree soil (n = 12) CMB µmol ­g−1 46–480 (168)b 16–66 (33)a 19–51 (34)a 39–476 (185) 7–21 (14)*** NMB µmol ­g−1 4–42 (15.4)b 0.9–8.2 (3.0)a 0.8–5.5 (2.8)a 4–54 (23) 1.3–4 (2.6)*** PMB µmol ­g−1 1.8–13.5 (5.6)b 0.3–2.5 (1.2)a 0.3–4.3 (1.7)a 1.1–18.3 (5.7) 0.2–1.3 (0.7)*** Resp. µmol C ­g−1 ­d−1 0.53–1.7 (1.04)b 0.03–0.3 (0.2)a 0.13–0.46 (0.3)a 0.6–2.4 (1.3) 0.07–0.29 (0.15)*** CO2-C:CMB µmol ­mol−1 3.2–12.4 (7.2) 1–10.2 (6.8) 4.7–11.3 (8.6) 5–16 (8.1) 4.9–26.5 (11)* Table 3. Microbial characteristics of alpine meadow and scree deposit soils sampled in 2015 from 14 catchments, and in 2020 from 4 catchments in the Tatra Mountains (data show ranges, averages are in parenthesis). CMB and NMB C and N in the microbial biomass, respectively. Resp. respiration ­(CO2-C production), CO2-C CMB specific respiration. Different letters in the 2015 survey section denote statistically significantly different values. In the 2020 survey section, asterisks indicate statistically significant differences: *p < 0.05, **p < 0.01, ***p < 0.001. Table 3. Microbial characteristics of alpine meadow and scree deposit soils sampled in 2015 from 14 catchments, and in 2020 from 4 catchments in the Tatra Mountains (data show ranges, averages are in parenthesis). CMB and NMB C and N in the microbial biomass, respectively. Resp. respiration ­(CO2-C production), CO2-C CMB specific respiration. Different letters in the 2015 survey section denote statistically significantly different values. In the 2020 survey section, asterisks indicate statistically significant differences: *p < 0.05, **p < 0.01, ***p < 0.001. Discussion Because the majority of scree deposits reaches the banks of the study lakes, there are absent meadow soil areas potentially able to reduce the direct nutrient fluxes from the scree slopes to lakes. l Scree soils thus had a lower potential than alpine meadows to immobilize P in organic matter due to the absence of plants and lower microbial biomass, but also due to their lower P retention capacity related to their lower ­Alox and ­Feox ­concentrations10,37, and had higher concentrations of mobile P forms. Leaching of P from soils to waters is therefore more probable in scree than meadow soils. P concentrations have been recently increasing in some high elevation Tatra Mountain lakes, which typically have catchments with large proportions of scree ­deposits12. All four catchments studied in 2020 belonged to the group of rocky or meadow-rocky catchments, representing 25 from 30 long-term studied alpine catchments in the Tatra ­Mountains5. In these scree-rich catch- ments, higher annual increases in the in-lake total P (and chlorophyll a) concentrations were documented from 1992 to 2018 compared to catchments with higher meadow ­proportions12. For example, concentrations of total Scientific Reports | (2023) 13:14725 | https://doi.org/10.1038/s41598-023-42013-4 www.nature.com/scientificreports/ 1 Figure 5. Comparisons of total hydrolytic activity (a) and relative enzymatic investments into C-mining (b), N-mining (c), and P mining (d) within the total activity of hydrolases in meadow alpine soils (A horizon) and scree soils in four selected Tatra Mountain catchments (Fig. 1) in 2020. Ranges, and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. Figure 5. Comparisons of total hydrolytic activity (a) and relative enzymatic investments into C-mining (b), N-mining (c), and P mining (d) within the total activity of hydrolases in meadow alpine soils (A horizon) and scree soils in four selected Tatra Mountain catchments (Fig. 1) in 2020. Ranges, and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages Figure 5. Comparisons of total hydrolytic activity (a) and relative enzymatic investments into C-mining (b) N-mining (c), and P mining (d) within the total activity of hydrolases in meadow alpine soils (A horizon) an scree soils in four selected Tatra Mountain catchments (Fig. 1) in 2020. Ranges, and 25% and 75% quartiles a given, horizontal lines within the boxes denote medians, and red crosses denote averages. Figure 5. Conclusions h d We characterized the chemical properties of scree soils, a relatively unknown aspect of alpine catchment ecosys- tems. We compared their biochemical and chemical parameters with meadow soils in alpine catchments in the Tatra Mountains, focusing on their potential to release P. Scree deposit soils had a lower microbial biomass with a higher biomass-specific activity, and a higher proportion of P-mining enzymes compared to meadow soils. The chemical and microbial composition of meadow soils is affected by large inputs of plant-derived complex organic matter, transformed by large microbial communities with hydrolytic activity targeting C-mining. These differences in the microbial activity and biochemical processes of scree and meadow soils affect their ability to retain/release P. Scree deposit soils exhibited higher concentrations of mobile P forms ­(SRPox, ­POlsen, ­SRPH2O), and lower concentrations of ­Alox and ­Feox, which together with higher pH, low organic matter and a low micro- bial biomass, led to a lower ability of scree soils to retain P. The leaching of P from scree deposit soils may thus represent an important P source for alpine lakes. The combination of climate change, recovery from atmospheric acidification, and specific characteristics of scree areas and their soils is probably responsible for the increasing input of P to alpine lakes in the Tatra Mountains. Similar changes also could be expected in other alpine areas recovering from acidification, especially in lakes with scree-rich catchments. Discussion Comparisons of total hydrolytic activity (a) and relative enzymatic investments into C-mining (b), N-mining (c), and P mining (d) within the total activity of hydrolases in meadow alpine soils (A horizon) and scree soils in four selected Tatra Mountain catchments (Fig. 1) in 2020. Ranges, and 25% and 75% quartiles are given, horizontal lines within the boxes denote medians, and red crosses denote averages. P in four lakes belonging to the group of scree-rich catchments increased from 0.05 to 0.08 µmol ­L−1 in 2000 to 0.07–0.14 µmol ­L−1 in ­201410. Because atmospheric P inputs are stable in this ­area15, this increase is probably associated with elevated P losses from the catchments. This may be associated with either an elevated terrestrial P source or a decreasing ability of soils to immobilize deposited P. A climate-driven increase in the physical weathering of rocks, which is more pronounced in scree deposits than in alpine meadows with rocks insulated by ­soil12 may also provide additional P sources. Physical (followed by chemical) weathering of unstable scree rocks could be accelerated by increasing frequency of heavy rains, number of days without snow cover, and number of days with temperature fluctuations around zero (i.e., the number of freeze–thaw cycles), which were documented in the Tatra Mountains during the last 30 ­years12. Decrease in the ability of soils to retain P is associated with their recovery from atmospheric acidification, and this process is more pronounced for scree than alpine ­soils10. In addition, we further show that soil microbial processes are able to mobilize more P available for leaching in scree soils, while more effectively immobilizing P in alpine meadows. All three processes probably contribute to the recent more rapid P loading of lakes in catchments with a large proportion of scree than in catchments dominated by alpine meadows. https://doi.org/10.1038/s41598-023-42013-4 Scientific Reports | (2023) 13:14725 | www.nature.com/scientificreports/ References 1. Kopáček, J. et al. Catchment biogeochemistry modifies long-term effects of acidic deposition on chemistry of mountain lakes. Biogeochemistry 125, 315–335 (2015).i 1. Kopáček, J. et al. 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Biochem. 14(4), 319–329 (1982). https://doi.org/10.1038/s41598-023-42013-4 Scientific Reports | (2023) 13:14725 | Fundingh Funding This study was funded by the Grant Agency of the Czech Republic (project No. P503/20/19284 S), J.K. is the principal investigator. Competing interests h p g The authors declare no competing interests. Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​42013-4. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​42013-4. Correspondence and requests for materials should be addressed to J.K. Correspondence and requests for materials should be addressed to J.K. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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Development and characterization of 20 polymorphic microsatellite markers for Epinephelus marginatus (Lowe, 1834) (Perciformes: Epinephelidae) using 454 pyrosequencing

Genetics and Molecular Biology

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Abstract The dusky grouper, Epinephelus marginatus, is a well-known and widespread marine fish assessed as endangered by the International Union for the Conservation of Nature. Analyzing the genetic diversity of this species is, therefore, of utmost importance and necessary for conservation purposes. Microsatellites are molecular tools with advantages that are ideal for population analyses. This study provides the first set of species-specific microsatellite loci for E. marginatus that can be applied when assessing both intra- and interpopulation genetic variation. Twenty micro- satellite loci were isolated and characterized for the dusky grouper by genotyping 20 individuals obtained from the North Eastern Atlantic Ocean (n = 4) and from the South Western Atlantic Ocean (n = 16). The number of alleles per locus varied from 2 to 11, while the observed and expected heterozygosities ranged from 0.25 to 0.94 and 0.34 to 0.89, respectively. The polymorphic information content varied from moderately to highly informative. This suite of markers provides the first specific nuclear tools for E. marginatus and, thus, allows to assess with more specificity dif- ferent populations’ structures. Keywords: Short tandem repeat, population genetics, conservation, fishery, marine resources. Received: March 05, 2018; Accepted: April 26, 2018. Keywords: Short tandem repeat, population genetics, conservation, fishery, marine resources. Received: March 05 2018; Accepted: April 26 2018 South Africa, including the Macaronesian Archipelagos of Azores, Madeira, Canaries, and Cape Verde (Heemstra, 1991; Heemstra and Randall, 1993). On the Atlantic coast of South America, this species occurs from Rio de Janeiro (Brazil) to Patagonia (Argentina) (Rico and Acha, 2003; Irigoyen et al., 2005). Fishing data have shown a 50% de- cline in the overall dusky grouper catches from European countries between 1994 (7699 metric tons) and 2011 (869 metric tons) (Harmelin-Vivien and Craig, 2015). This pop- ulation reduction combined with their life-history has led to an assessment of Endangered A2d status by the Interna- tional Union for the Conservation of Nature (Cornish and Harmelin-Vivien, 2004; Harmelin-Vivien and Craig, 2015). Epinephelidae, known as groupers, are considered commercially important marine resources by commercial and recreational coastal fisheries (Begossi and Silvano, 2008; Schunter et al., 2011). In 2009, approximately 275,000 metric tons of the global catch were groupers (Epinephelidae), which represented approximately 90 mil- lion fish (Sadovy de Mitcheson et al., 2013). Groupers play an important role in trophic foodwebs in coral and rocky reef ecosystems (Condini et al., 2015). Send correspondence to Alexandre Wagner Silva Hilsdorf. Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, Caixa Postal 411, 08701-970, Mogi das Cruzes, SP, Brazil. Genetics and Molecular Biology, 42, 1, 74-79 (2019) Copyright © 2019, Sociedade Brasileira de Genética. Printed in Brazil DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2018-0067 Short Communication Development and characterization of 20 polymorphic microsatellite markers for Epinephelus marginatus (Lowe, 1834) (Perciformes: Epinephelidae) using 454 pyrosequencing Jussara Oliveira Vaini1 , Kenneth Gabriel Mota1, Alejandra Paola Ojeda1, João Pedro Barreiros2 , Renata Guimarães Moreira3 and Alexandre Wagner Silva Hilsdorf1 Jussara Oliveira Vaini1 , Kenneth Gabriel Mota1, Alejandra Paola Ojeda1, João Pedro Barreiros2 , Renata Guimarães Moreira3 and Alexandre Wagner Silva Hilsdorf1 1Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil. 2Centro de Ecologia, Evolução e Alterações Ambientais, Universidade dos Açores, Angra do Heroísmo, Portugal. 3Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil. nstituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil. Abstract Of these, 165 sequences presented simple and per- fect repetitions with a minimum of five repeat motifs, where 125 resulted in dinucleotides, 26 in tri-, 13 in tetra- nucleotides and one pentanucleotide. Forty-eight micro- satellite loci were initially selected for polymorphism assessment, and 20 of these loci amplified reliably and showed evidence of polymorphism (Table 1). Primer sets were designed from the microsatellite flanking regions us- ing the QDD software. Twenty individuals of E. marginatus were analyzed to validate the panel of spe- cies-specific microsatellite markers, with 16 individuals being from the Southwestern Atlantic Ocean near Brazil (São Paulo, n = 4; Rio de Janeiro, n = 4; Parana, n = 4 and Santa Catarina coast, n = 4) and 4 individuals from the Northeastern Atlantic Ocean (Spain / Mallorca (n = 2), Greece / Cyclades Islands (n = 1) and Azores Archipelago (n = 1)). For each primer set, the forward primer was 5’-end- tailed with the M13 universal sequence (5’- TGTAAAACGACGGCCAGT-3; Schuelke, 2000). The polymerase chain reactions (PCR) were performed in 20 L reaction volumes (~100 ng genomic DNA, 1X PCR buffer, 0.25 mM dNTPs, 1.5-3.0 mM MgCl2, 0.5 U Taq DNA polymerase, 0.2 M of the IRDye700 fluorescently labeled universal M13 primer, 10 M of the forward primer, and 10 M of the reverse primer) in a Veriti thermal cycler (Applied Biosystems). The amplification thermal profile for all markers had an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 40 s at 94 °C, 1 min at the primer set annealing temperature (Table 1), and 40 s at 72 °C, with a final extension of 10 min at 72 °C. Amplicons were separated on 6.0% polyacrylamide gels using an auto- mated DNA sequencer (DNA Analyzer 4300; LI-COR Bio- sciences, Lincoln, NE, USA). Alleles were sized using a 50-350 bp standard (LI-COR Biosciences), and genotypes were scored using SAGA v.3.3 software (LI-COR Biosci- ences). Now that next generation sequencing (NGS) has be- come more accessible, the development of species-specific microsatellite loci is much faster and less labor intensive (Kumar and Kocour, 2017). In addition, microsatellites are generally found in non-coding regions, where the substitu- tion rate is higher than in the coding regions, and hence, the flanking regions of the microsatellites, where the primers are designed, are prone to mutations (Primmer et al., 2005). Abstract They are protogynous hermaphrodites characterized by high site fi- delity, slow growth, delayed sexual maturation in males, and large body size (Marino et al., 2001; Koeck et al., 2014). This group of Perciformes is especially susceptible to overfishing (Morris et al., 2000). The dusky grouper, Epinephelus marginatus (Lowe, 1834), is broadly distributed in the Atlantic Ocean from the Mediterranean Sea to southern Africa, in the Indian Ocean northwards to Madagascar, and from the British Isles to Knowledge of intra- and intergenic diversity is im- portant for planning the long-term conservation and recov- ery of marine fish resources through legal environmental protection and ecosystem-based fisheries management (Zhou et al., 2010). Therefore, molecular markers are needed to acquire knowledge on the genetic diversity of a given species, and microsatellite markers are the most fre- quently used tool in marine fish (Cuéllar-Pinzón et al., Vaini et al. 75 2016). Although, species-specific microsatellite markers have been developed for different species of the genus Epinephelus: E. quernus – 9 loci (Rivera et al., 2003); E. septemfasciatus – 12 loci (Zhao et al., 2009a) and 22 loci (An et al., 2014); E. awoara – 12 loci (Zhao et al., 2009b); E. fuscoguttatus – 10 loci (Mokhtar et al., 2011); E. merra – 13 loci (Muths and Bourjea, 2011); E. akaara – 12 loci (Watanabe et al., 2011) and 10 loci (Xie et al., 2015); E. lanceolatus – 32 loci (Yang et al., 2011) and 24 loci (Kim et al., 2016); E. striatus – 15 loci (Bernard et al., 2012); E. bruneus – 28 loci (Kang et al., 2013); E. polyphekadion – 12 loci (Ma et al., 2013); E. itajara and E. quinquefasciatus, 29 and 25 loci, respectively (Seyoum et al., 2013); and E. ongus – 18 loci (Nanami et al., 2017). As species-specific microsatellite marker have not yet been de- veloped for E. marginatus, previous studies of population genetic diversity used microsatellites by cross-amplification (Sola et al., 1999; De Innocentiis et al., 2001; De Innocentiis et al., 2008; Schunter et al., 2011; Elglid et al., 2015; Buchholz-Sørensen and Vella, 2016; Reid et al., 2016). ACTC. The software QDD (Meglécz et al., 2010) was used to identify the microsatellites from the raw sequences. A to- tal of 5526 sequences were recovered with microsatellite motifs. Abstract Mutations in these regions may result in null alleles that af- fect the cross-amplification success rate (Maduna et al., 2014). Another issue regarding microsatellite cross-ampli- fication is that the ascertainment bias phenomenon may be operating, that is, the chance of the median allele length of microsatellites being longer in one species in which it was first developed than in other cognate species (Crawford et al., 1998; Barbará et al., 2007; Li and Kimmel, 2013). With this in mind we have now used NGS to develop a novel set of 20 specie-specific microsatellite markers to provide sup- port to conservation management programs for the dusky grouper. The number of alleles per locus, estimates of ob- served (Ho) and expected heterozygosity (He), and devia- tions from Hardy-Weinberg expectations were determined with an exact test using the Markov chain-randomization approach (Guo and Thompson 1992) with HW-Quickcheck (Kalinowski, 2006). Linkage disequilibrium was assessed using GENEPOP v.4.2 (Rousset, 2008), and Micro- Checker v.2.2.3 (Van Oosterhout et al., 2004) was used to test for null alleles and allele dropout. The polymorphic in- formation content (PIC) was analyzed using Cervus v.3.0.7 (Kalinowski et al., 2007). Genomic DNA was extracted from the caudal fins of five Epinephelus marginatus specimens using QIAGEN DNeasy blood and tissue extraction kits (QIAGEN Inc., Valencia, CA) following the manufacturer protocols, and these samples were forwarded to GenoScreen (Lille, Fran- ce) for commercial microsatellite library production. The extracted DNA was fragmented (~1500 bp) by sonication (S220 Focused-ultrasonicator; Covaris, Newtown, CT, USA) and used to construct a shotgun library (GS-FLX Ti- tanium kit; Roche Diagnostics Corporation, Branford, CT, USA), which was then sequenced in a 454 GS-FLX Tita- nium pyrosequencer (Roche Diagnostics Corporation). The following DNA probes were used to perform the enrich- ment: TG, CT, AAC, AAG, AGG, ACG, ACAT, and Table 1 summarizes the information for each primer sequence, providing GenBank accession number, repeat motif, number of alleles, diversity estimates (Ho and He), 76 Microsatellites for dusky grouper Table 1 - Genetic characteristic of 20 microsatellite loci in Epinephelus marginatus. Abstract Locus GenBank accession Primer sequence (5-3) Repeat motif Ta (°C) N A Size range (bp) Ho He Ema01 KU762341 F: ACAGCAAACCATGTGAGCAG R: TGGAGTGATAGTCCTCTGTTGG (AC)14 60 20 09 158-180 0.70 0.82 Ema02 KU762342 F: CAGACGTATGCACTGGACCT R: ATATGTCAGCCTCCACCTCC (TG)7 62 20 07 123-165 0.85 0.78 Ema03 KU762343 F: CCACCATGCCCTCCAATA R: GAGCCAGGTGAATGACCACT (ATCT)15 58 19 11 125-165 0.89 0.89 Ema04 KU762344 F: CTGATGGAATCCACAAATTTAATC R: TCTGACTGACAGATAACAAGAGAA (GATG)9 52 20 09 183-223 0.80 0.87 Ema05 KU762345 F: TGCTCAGGGAGACTGACAGA R: GATGAGCAAAGAGGGCAGAG (GA)7 56 18 09 177-195 0.83 0.81 Ema06 KU762346 F: TTGTACGTTTGCTAATGCTGTG R: CTGAACTGTACTCATGAACCTGC (CAA)5 55 20 05 204-216 0.75 0.70 Ema07 KU762347 F: CCTCTACTGCTATCATGACTTCTCC R: ACAGTTGAAATAGTGGTGCAGA (TAC)5 52 12 02 205-208 0.25 0.34 Ema12 KU762348 F: AAAGTGCACTGTAGCCAACG R: TGATGTGGACAACGGAAAGA (TCC)7 58 17 03 221-230 0.94 0.56 Ema17 KU762350 F: GGGCAGTGACGGTAGACATT R: CGAAGACGCAATGAAACTGA (CTAT)13 56 20 09 142-182 0.80 0.82 Ema18 KU762351 F: GGACAAGTGGACATTTTGGC R: AACCAGGAGCTTATGTGGCT (CTAT)16 60 20 09 175-207 0.80 0.89 Ema20 KU762352 F: TGATTATGAATGCAAGAAGTGATG R: AGGGCCGATGATCAAATGT (CATC)7 60 18 04 150-162 0.78 0.63 Ema22 KU762353 F: GTTTGCAGTGTTGCAGTGCT R: TAGGGTGGGATTTCAGATGC (TATG)7 58 20 07 111-135 0.85 0.81 Ema23 KU762354 F: AACATGATCCGAATAGGCTGA R: CGAAGGCTCCAGGTCAGTAT (ACAG)7 60 19 07 214-234 0.84 0.74 Ema26 KU762355 F: CAGGTGGAGTGATTTCAGCA R: TTCACCCATGGGAAGTATGA (TTC)8 52 20 06 128-143 0.65 0.76 Ema35 MG640563 F: ACTCCCACTCTGCCTCTCAG R: ACGTGCAAATTTCTTGGACA (AC)14 56 20 11 169-197 0.55 0.84 Ema38 MG640564 F: TGTCTGTGACGAACTCTGCC R: CCCATCTACTGCTGGTGTCTC (TG)11 60 20 07 160-172 0.35 0.82 Ema42 MG640565 F: AATATGACTGATAATTTGACCACCA R: CACCCCTAGACCAGCACAAT (CTG)7 54 20 06 147-162 0.55 0.66 Ema43 MG640566 F: TGGGAGAAATGTGTCCTCAG R: CTGCTGCATGTTCTAGCCAA (GT)9 58 15 04 189-195 0.33 0.71 Ema45 MG640567 F: GGAGTTGCTAGAACCAAGCC R: CAGCAGTCACAGAAACACGC (TGT)6 56 17 04 158-167 0.47 0.62 Ema48 MG640568 F: TCCAAGTTACCACCTAGCCTTC R: ATGGATAGATGATAGATGGATGC (ATCC)5 50 19 04 113-125 0.68 0.57 Ta: Annealing temperature; N: number of individuals; A: number of alleles; Allele size in base pairs; Ho: observed heterozygosity; He: expected heterozygosity; PIC: polymorphic information content; HWE: Hardy-Weinberg Equilibrium p-values. *p < 0 05 significant departure from Hardy-Weinberg Equilibrium 77 Vaini et al. PIC, and probability of Hardy-Weinberg equilibrium (HWE). septemfasciatus developed using a 454 pyrosequencing ap- proach. Conserv Genet Resour 6:665-667. Barbará T, Palma-Silva C, Paggi GM, Bered F, Fay MF and Lexer C (2007) Cross-species transfer of nuclear microsatellite markers: potential and limitations. Mol Ecol 16:3759-3767. Abstract ( ) Among the 20 individuals, the number of alleles per locus ranged from 2 (Ema07) to 11 (Ema03 and Ema35), with an average of 6.65, while the observed and expected heterozygosities ranged from 0.25 (Ema07) to 0.94 (Ema12) and 0.34 (Ema07) to 0.89 (Ema03 and Ema18), respectively (Table 1). Sixteen loci (Ema01, Ema02, Ema03, Ema04, Ema05, Ema06, Ema07, Ema17, Ema18, Ema20, Ema22, Ema23, Ema26, Ema42, Ema45, and Ema48) were in HWE (p > 0.05), while four loci signifi- cantly deviated (p < 0.05; Ema12, Ema35, Ema38, and Ema43). Three loci (Ema35, Ema38, and Ema43) showed evidence of null alleles, but no allele dropout was detected. No statistical evidence for linkage disequilibrium was found between any of the 20 loci pairwise comparisons. The number of alleles was high and exhibited moderate to high levels of polymorphism (PIC), ranging from 0.27 (Ema07) to 0.85 (Ema02 and Ema18), with an average of 0.67. When separated in di- (0.74), tri- (0.54) and tetranucleotides (0.72), the loci that presented the highest levels of polymorphism (PIC) were the dinucleotides. Begossi A and Silvano RAM (2008) Ecology and ethnoecology of dusky grouper [garoupa, Epinephelus marginatus (Lowe, 1834)] along the coast of Brazil. J Ethnobiol Ethnomed 4:20. Bernard AM, Feldheim KA, Richards VP, Nemeth RS and Shivji MS (2012) Development and characterization of fifteen novel microsatellite loci for the Nassau grouper (Epinephelus striatus) and their utility for cross- amplification on a suite of closely related species. Conserv Genet Resour 4:983-986. Buchholz-Sørensen M and Vella A (2016) Population structure, genetic diversity, effective population size, demographic history and regional connectivity patterns of the endangered dusky grouper, Epinephelus marginatus (Teleostei: Serranidae), within Malta’s fisheries management zone. PLoS One 11:e0159864. Condini MV, Hoeinghaus DJ and Garcia AM (2015) Trophic ecology of dusky grouper Epinephelus marginatus (Actinopterygii, Epinephelidae) in littoral and neritic habi- tats of southern Brazil as elucidated by stomach contentes and stable isotope analyses. Hydrobiologia 743:109-125. These novel polymorphic microsatellite loci devel- oped using NGS technology will aid in achieving better res- olution when assessing stock structure and population connectivity for the dusky grouper’s long-term conserva- tion and the sustainable use of this valuable marine re- source. Cornish A and Harmelin-Vivien M (Grouper & Wrasse Specialist Group) (2004) Epinephelus marginatus. The IUCN Red List of Threatened Species 2004:e.T7859A12857009. Crawford AM,Kappes SM,Paterson KA,de Gotari MJ,Dodds KG, Freking BA,Stone RTand Beattie CW (1998) Microsatellite evolution: Testing the ascertainment bias hypothesis. Conflict of interest Elglid A, Crouau-Roy B, Bradai MN and Fadhlaoui-Zid K (2015) Population genetic structure of Epinephelus marginatus in the Central Mediterranean Sea (Gulf of Gabès and the coast of Libya). J Adv Biol 8:1571-1580. The authors declare that there is no conflict of interest associated with this study. Abstract J Mol Evol 46:256-260. Author contributions Guo SW and Thompson EA (1992) Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48:361-372. J.O.V., K.G.M. and A.P.O. conducted the experi- ments; J.O.V. and A.W.S.H. analyzed the data and wrote the manuscript; J.P.B. and R.G.M. contributed to data anal- ysis and revising the manuscript; A.W.S.H. supervised the whole project. 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De Innocentiis S, Sola L, Cataudella S and Bentzen P (2001) Allozyme and microsatellite loci provide discordant esti- mates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea. Mol Ecol 10:2163-2175. De Innocentiis S, Longobardi A and Marino G (2008) Molecular tools in a marine restocking program for the endangered Dusky Grouper, Epinephelus marginatus. Rev Fish Sci 16:269-277. References Koeck B, Pastor J, Saragoni G, Dalias N, Payrot J and Lefant P (2014) Diel and seasonal movement pattern of the dusky grouper Epinephelus marginatus inside a marine reserve. Mar Environ Res 94:38-47. Schuelke M (2000) An economic method for the fluorescent la- beling of PCR fragments. Nat Biotechnol 18:233-234. Kumar G and Kocour M (2017) Applications of next-generation sequencing in fisheries research: A review. Fish Res 186:11-22. 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Linguistic Neutrosophic Topology

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Linguistic Neutrosophic Topology N. Gayathri1 2 1Research scholar; Nirmala college for women; Coimbatore ;Tamil Nadu; India 2Associate professor; Nirmala college for women;Coimbatore; Tamil Nadu; India ∗ Correspondence: [email protected] and M. Helen N. Gayathri1 2 1Research scholar; Nirmala college for women; Coimbatore ;Tamil Nadu; India 2Associate professor; Nirmala college for women;Coimbatore; Tamil Nadu; India ∗ Correspondence: [email protected] and M. Helen Abstract. By utilizing linguistic neutrosophic sets and topological spaces, we construct and develop a new notion called ”linguistic neutrosophic topological spaces”, in this article. Many basic definitions, theorems and properties were defined with suitable examples. Keywords: Linguistic Neutrosophic topology; Linguistic Neutrosophic open set; Linguistic Neutrosophic closed set; Linguistic Neutrosophic interior; Linguistic Neutrosophic closure; Linguistic Neutrosophic continuous func- tion; Linguistic Neutrosophic neighborhood; Linguistic Neutrosophic derived sets; Linguistic Neutrosophic dense sets; L L Neutrosophic Sets and Systems, Vol. 46, 2021 Neutrosophic Sets and Systems, Vol. 46, 2021 University of New Mexico N. Gayathri, M. Helen, Linguistic Neutrosophic topology 1. Introduction Fang, Zebo etc.,al [6] found linguistic neutrosophic numbers in 2017, with a concrete definition.This paper is categorized as follows: Section 2 deals with the basic definitions of LNNs. In Section 3, the idea of linguistic neutrosophic topology is introduced and some properties are discussed. Lin- guistic neutrosophic derived set is discussed in section 4. In last section, the notion of linguistic neutrosophic continuity and linguistic neutrosophic dense sets are defined and discussed with suitable examples. 1. Introduction Many investigators in business, science, economy and a variety of other branches deal with modeling unknown data on a regular basis. For these ambiguous and uncertainties, traditional techniques are not always successful. LotfiA. Zadeh [16] instigated the idea of membership or truth value to the elements of collection of well-defined objects called, sets. These systems can handle a variety of inputs, including ambiguous, distorted, or inaccurate data. The idea of fuzzy topology was initially developed by Chang [2] in 1967. Many topological structures and generalizations have developed in time utilizing fuzzy sets. In addition to the degree of truth membership, Atanassov [1] paired non-membership value called false membership, which was the generalization of fuzzy sets, called intuitionistic fuzzy sets. In 1997, intuitionistic fuzzy topology was found by Coker [4]. Along with the two membership values, Smarandache [11] introduced the idea of indeterminacy membership function in 1999. Neutrosophic sets play an important part in many aspects like, decision making, medical diagnosis, etc., Wang and Smarandache [13] introduced the notion of interval valued neutrosophic sets. Qualitative attributes can be easily expressed in linguistic terms, which was developed by Zadeh [17]. The idea of linguistic variables was applied in decision making by Herrara etc.,al [9] in 2000 and Herrara-Viedma, Vergegay [8] in 1996. Su [12] used linguistic preference informa- tion in group decision making. Chen, Liu, etc.,al [3] introduced linguistic intuitionistic fuzzy number(LIFN) in 2015. As LIFN lacks indeterminacy, Ye [15] in 2015, proposed the notion of 255 Neutrosophic Sets and Systems, Vol. 46, 2021 single valued neutrosophic linguistic numbers and developed an extended TOPSIS model for MAGDM approach utilizing SVNLNs. An extended COPRAS model for MAGDM based on SVN 2-tuple neutrosophic environment was developed by Wei, Wu, etc.,al [14]. Fang, Zebo etc.,al [6] found linguistic neutrosophic numbers in 2017, with a concrete definition.This paper is categorized as follows: Section 2 deals with the basic definitions of LNNs. In Section 3, the idea of linguistic neutrosophic topology is introduced and some properties are discussed. Lin- guistic neutrosophic derived set is discussed in section 4. In last section, the notion of linguistic neutrosophic continuity and linguistic neutrosophic dense sets are defined and discussed with suitable examples. single valued neutrosophic linguistic numbers and developed an extended TOPSIS model for MAGDM approach utilizing SVNLNs. An extended COPRAS model for MAGDM based on SVN 2-tuple neutrosophic environment was developed by Wei, Wu, etc.,al [14]. Neutrosophic Sets and Systems, Vol. 46, 2021 A = {⟨x, sθ(x), sψ(x), sσ(x)⟩|x ∈S}, where sθ(x), sψ(x), sσ(x) ∈Q represent the linguistic truth, linguistic indeterminacy and linguistic falsity degrees of S to A, respectively, with con- dition 0 ≤θ+ψ+σ ≤3t. This triplet (sθ, sψ, sσ) is called a linguistic single valued neutrosophic number. Definition 2.5. [6] Let α = (sθ, sψ, sσ), α1 = (sθ1, sψ1, sσ1), α2 = (sθ2, sψ2, sσ2) be three LSVNNs, then Definition 2.5. [6] Let α = (sθ, sψ, sσ), α1 = (sθ1, sψ1, sσ1), α2 = (sθ2, sψ2, sσ2) be three LSVNNs, then Definition 2.5. [6] Let α = (sθ, sψ, sσ), α1 = (sθ1, sψ1, sσ1), α2 = (sθ2, sψ2, sσ2) be three LSVNNs, then LSVNNs, then (1) αc = (sσ, sψ, sθ); (2) α1 ∪α2 = (max(θ1, θ2), min(ψ1, ψ2), min(σ1, σ2)); (3) α1 ∩α2 = (min(θ1, θ2), max(ψ1, ψ2), max(σ1, σ2)); (4) α1 = α2 iffθ1 = θ2, ψ1 = ψ2, σ1 = σ2; (1) αc = (sσ, sψ, sθ); (2) α1 ∪α2 = (max(θ1, θ2), min(ψ1, ψ2), min(σ1, σ2)); (3) α1 ∩α2 = (min(θ1, θ2), max(ψ1, ψ2), max(σ1, σ2)); (4) α1 = α2 iffθ1 = θ2, ψ1 = ψ2, σ1 = σ2; Definition 2.6. Let α = (lθ, lψ, lσ) be a LSVNN. The set of all labels is, L = {l0, l1, l2, ....., lt}. Then the unit linguistic neutrosophic set (1LN) is defined as 1LN = (lt, l0, l0), which is the truth membership,and the zero linguistic neutrosophic set (0LN) is defined as 0LN = (l0, lt, lt), which is the falsehood membership. Example 2.7. For the linguistic neutrosophic set, L = {very bad, bad, fair, very fair, good, very good}, the set of all labels be, L = {l0, l1, l2, l3, l4, l5}. Then the unit LNs is defined as 1LN = (l5, l0, l0), and the zero LNs is defined as 0LN = (l0 l5 l5) Example 2.7. For the linguistic neutrosophic set, L = {very bad, bad, fair, very fair, good, very good}, the set of all labels be, L = {l0, l1, l2, l3, l4, l5}. Then the unit LNs is defined as 1LN = (l5, l0, l0), and the zero LNs is defined as 0LN = (l0, l5, l5). 2. Preambles Definition 2.1. [11] Let S be a space of points (objects), with a generic element in x denoted by S. A neutrosophic set A in S is characterized by a truth-membership function TA, an indeterminacy membership function IA and a falsity-membership function FA. TA(x), IA(x) and FA(x) are real standard or non-standard subsets of ]0−, 1+[. That is TA : S →]0−, 1+[, IA : S →]0−, 1+[, FA : S →]0−, 1+[ There is no restriction on the sum of TA(x), IA(x) and FA(x), so 0−≤sup TA(x)+ sup IA(x)+ sup FA(x) ≤3+. There is no restriction on the sum of TA(x), IA(x) and FA(x), so 0−≤sup TA(x)+ sup IA(x)+ sup FA(x) ≤3+. Definition 2.2. [11] Let S be a space of points (objects), with a generic element in x denoted by S. A single valued neutrosophic set (SVNS) A in S is characterized by truth-membership function TA, indeterminacy-membership function IA and falsity-membership function FA. For each point S in S, TA(x), IA(x), FA(x) ∈[0, 1]. When S is continuous, a SVNS A can be written as A = R ⟨T(x), I(x), F(x)⟩/x ∈S. When S is discrete, a SVNS A can be written as A = P⟨T(xi), I(xi), F(xi)⟩/xi ∈S. When S is continuous, a SVNS A can be written as A = R ⟨T(x), I(x), F(x)⟩/x ∈S. When S is discrete, a SVNS A can be written as A = P⟨T(xi), I(xi), F(xi)⟩/xi ∈S. Definition 2.3. [6] Let S = {sθ|θ = 0, 1, 2, ....., τ} be a finite and totally ordered discrete term set, where τ is the even value and sθ represents a possible value for a linguistic variable. For example, when τ = 6, S can be expressed as, S = {very bad, bad, fair, very fair, good, very good}. Su [12] extended the discrete linguistic term set S into a continuous term set S = {sθ|θ ∈ [0, q]}, where, if sθ ∈S, then we call sθ the original term, otherwise it is called as a virtual term. Definition 2.4. [6] Let Q = {s0, s1, s2, ..., st} be a linguistic term set (LTS) with odd cardi- nality t+1 and Q = {sh/s0 ≤sh ≤st, h ∈[0, t]}. Then, a linguistic single valued neutrosophic set A is defined by, N. Gayathri, M. Helen, Linguistic Neutrosophic topology 256 Neutrosophic Sets and Systems, Vol. 46, 2021 N. Gayathri, M. Helen, Linguistic Neutrosophic topology 3. Linguistic Neutrosophic Topology In this chapter, we introduce the concepts of linguistic neutrosophic topological spaces. In this chapter, we introduce the concepts of linguistic neutrosophic topological Definition 3.1. For a linguistic neutrosophic topology π, the collection of linguistic neutro- sophic sets should obey, (1) 0LN, 1LN ∈π (2) K1 T K2 ∈π for any K1, K2 ∈π (3) S Ki ∈π, ∀{Ki : i ∈J} ⊆π (1) 0LN, 1LN ∈π (2) K1 T K2 ∈π for any K1, K2 ∈π (3) S Ki ∈π, ∀{Ki : i ∈J} ⊆π (1) 0LN, 1LN ∈π (2) K1 T K2 ∈π for any K1, K2 ∈π (3) S Ki ∈π, ∀{Ki : i ∈J} ⊆π We call, the pair (SLN, πLN), a linguistic neutrosophic topological space. We call, the pair (SLN, πLN), a linguistic neutrosophic topological space. We call, the pair (SLN, πLN), a linguistic neutrosophic topological space. Remark 3.2. Let (SLN, πLN) be a linguistic neutrosophic topological space (LNTS). Then, (SLN, πLN)c is the dual LN topology, whose elements are KcLN for KLN ∈(SLN, πLN). Any open set in πLN is known as linguistic neutrosophic open set(LNOsS). Any closed set in πLN is known as linguistic neutrosophic closed set(LNCS) iffit’s complement is linguistic neutrosophic open set. N. Gayathri, M. Helen, Linguistic Neutrosophic topology 257 Neutrosophic Sets and Systems, Vol. 46, 2021 Example 3.3. Let ULN be the universe of discourse ULN = {u, v, w, z} and SLN = {u, v} and the linguistic term set be, L = { very poor, poor, very bad, bad, fair, good, very good} Then L can be taken as, L = {l0, l1, l2, l3, l4, l5, l6}. Let KLN = {⟨u, (l5, l3, l4)⟩, ⟨v, (l4, l2, l3)⟩}, hat is, the element u’s degree of appurtenance to the set KLN is good(l5) That is, the element u’s degree of appurtenance to the set KLN is good(l5) e element u’s degree of indeterminate-appurtenance to the set KLN is bad(l3) the element u’s degree of indeterminate-appurtenance to the set KLN is ba the element u’s degree of non-appurtenance to the set KLN is fair(l4). And the element v’s degree of appurtenance to the set KLN is fair(l4) the element v’s degree of indeterminate-appurtenance to the set KLN is very bad(l2) the element v’s degree of non-appurtenance to the set KLN is bad(l3). the element v’s degree of non-appurtenance to the set KLN is bad(l3). N. Gayathri, M. Helen, Linguistic Neutrosophic topology 3. Linguistic Neutrosophic Topology Let, HLN = {⟨u, (l6, l2, l2)⟩, ⟨v, (l6, l1, l0)⟩} That is, the element u’s degree of appurtenance to the set HLN is very good(l6) the element u’s degree of indeterminate-appurtenance to the set HLN is very bad(l2) the element u’s degree of non-appurtenance to the set HLN is very bad(l2). And the element v’s degree of appurtenance to the set HLN is very good(l6) the element v’s degree of indeterminate-appurtenance to the set HLN is poor(l1) the element v’s degree of non-appurtenance to the set HLN is very poor(l0). Similarly, let MLN = {⟨u, (l6, l3, l2)⟩, ⟨v, (l6, l2, l0)⟩} That is, the element u’s degree of appurtenance to the set MLN is very good(l6) the element u’s degree of indeterminate-appurtenance to the set MLN is bad(l3) the element u’s degree of non-appurtenance to the set MLN is very bad(l2). And the element v’s degree of appurtenance to the set MLN is very good(l6) the element v’s degree of indeterminate-appurtenance to the set MLN is very bad(l2) the element v’s degree of non-appurtenance to the set MLN is very poor(l0). Then the collection πLN = {0LN, KLN, HLN, MLN, KLN ∨HLN, 1LN} forms a LN topology on (SLN, πLN). Then the collection πLN = {0LN, KLN, HLN, MLN, KLN ∨HLN, 1LN} forms a LN topology on (SLN, πLN). Definition 3.4. The linguistic neutrosophic closure and linguistic neutrosophic interior are given by, Definition 3.4. The linguistic neutrosophic closure and linguistic neutrosophic interior are given by, (i) LNint(KLN) = S{OLN/OLN is a LNOSinSLN where OLN ⊆KLN} and it is the largest LN open subset of KLN. (ii) LNcl(HLN) = T{JLN/JLN is a LNCSinSLN where HLN ⊆JLN} and it is the smallest LN closed set containing HLN. (ii) LNcl(HLN) = T{JLN/JLN is a LNCSinSLN where HLN ⊆JLN} and it is the smallest LN closed set containing HLN. LN closed set containing HLN. Example 3.5. In example 3.3, LNint(KLN) = NLN and LNcl(KLN) = 1LN xample 3.5. In example 3.3, LNint(KLN) = NLN and LNcl(KLN) = 1LN Theorem 3.6. Let (SLN, πLN) be a LNTS and KLN, HLN ∈SLN. Then N. Gayathri, M. Helen, Linguistic Neutrosophic topology Theorem 3.6. Let (SLN, πLN) be a LNTS and KLN, HLN ∈SLN. Then N. Gayathri, M. Helen, Linguistic Neutrosophic topology Neutrosophic Sets and Systems, Vol. 46, 2021 258 (i) From the definition, KLN ∈LNcl(KLN) (i) From the definition, KLN ∈LNcl(KLN) (ii) If KLN is LN closed, then KLN is the smallest LN closed set containing KLN. So, KLN = LNcl(KLN). Conversely, if KLN = LNcl(KLN), then KLN is the smallest LN closed set containing KLN and hence KLN is LN closed. (ii) If KLN is LN closed, then KLN is the smallest LN closed set containing KLN. So, KLN = LNcl(KLN). Conversely, if KLN = LNcl(KLN), then KLN is the smallest LN closed set containing KLN and hence KLN is LN closed. KLN and hence KLN is LN closed. KLN and hence KLN is LN closed. (iii) If KLN is LN closed, then KLN = LNcl(KLN). As φLN and SLN are LN closed, LNcl(φLN) = φLN and LNcl(SLN) = SLN. (iv) When KLN ⊆HLN, since HLN ⊆LNcl(HLN) and KLN ⊆LNcl(HLN). That is, LNcl(HLN) is a LN closed set that contains K. But LNcl(KLN) is the smallest LN closed set contains K. Thus, LNcl(KLN) ⊆LNcl(HLN) (v) As KLN ⊆KLN ∩HLN and HLN ⊆KLN ∩HLN, LNcl(KLN) ⊆LNcl(KLN ∩HLN) and LNcl(HLN) ⊆LNcl(KLN ∩HLN). Thus, LNcl(KLN) ∩LNcl(HLN) ⊆LNcl(KLN ∩ HLN). Since, KLN ∪HLN ⊆LNcl(KLN)∩LNcl(HLN), and since LNcl(KLN ∪HLN) is the smallest LN closed set containing KLN ∪HLN, LNcl(KLN ∪HLN) ⊆LNcl(KLN)∪ LNcl(HLN). Thus, LNcl(KLN ∪HLN) = LNcl(KLN) ∪LNcl(HLN). (vi) Since (KLN ∩HLN) ⊆KLN and (KLN ∩HLN) ⊆HLN, LNcl(KLN ∩HLN) ⊆ LNcl(HLN) ⊆LNcl(HLN). (vi) Since (KLN ∩HLN) ⊆KLN and (KLN ∩HLN) ⊆HLN, LNcl(KLN ∩HLN) ⊆ LNcl(HLN) ⊆LNcl(HLN). (vii) AS LNcl(KLN) is a LN closed set, LNcl(LNcl(KLN)) = LNcl(KLN). (vii) AS LNcl(KLN) is a LN closed set, LNcl(LNcl(KLN)) = LNcl(KLN). Remark 3.7. If LNint(KLN) is LNcl(KLN) is a LNCS, then we have, Remark 3.7. If LNint(KLN) is LNcl(KLN) is a LNCS, then we have, Neutrosophic Sets and Systems, Vol. 46, 2021 (i) KLN ∈LNcl(KLN) (ii) KLN is LN closed if and only if KLN = LNcl(KLN) (iii) LNcl(φLN) = φLN and LNcl(SLN) = SLN. (iv) KLN ⊆HLN ⇒LNcl(KLN) ⊆LNcl(HLN) (v) LNcl(KLN ∪HLN) = LNcl(KLN) ∪LNcl(HLN) (vi) LNcl(KLN ∩HLN) ⊆LNcl(KLN) ∩LNcl(HLN) (vii) LNcl(LNcl(KLN)) = LNcl(KLN) (i) KLN ∈LNcl(KLN) (ii) KLN is LN closed if and only if KLN = LNcl(KLN) (iii) LNcl(φLN) = φLN and LNcl(SLN) = SLN. (iv) KLN ⊆HLN ⇒LNcl(KLN) ⊆LNcl(HLN) (v) LNcl(KLN ∪HLN) = LNcl(KLN) ∪LNcl(HLN) (vi) LNcl(KLN ∩HLN) ⊆LNcl(KLN) ∩LNcl(HLN) (vii) LNcl(LNcl(KLN)) = LNcl(KLN) Proof: Remark 3.7. If LNint(KLN) is LNcl(KLN) is a LNCS, then we have, (i) LNint(KLN) = KLN if and only if KLN is LNOS in (SLN, πLN). (i) LNint(KLN) = KLN if and only if KLN is LNOS in (SLN, πLN). (ii) LNcl(KLN) = KLN if and only if KLN is LNCS in (SLN, πLN). i) LNcl(KLN) = KLN if and only if KLN is LNCS in (SLN, πLN). Theorem 3.8. Let (SLN, πLN) be a LNTS and KLN ∈SLN. Then (i) S −LNint(KLN) = LNint(SLN −KLN) Theorem 3.8. Let (SLN, πLN) be a LNTS and KLN ∈SLN. Then (i) S −LNint(KLN) = LNint(SLN −KLN) (ii) S −LNcl(KLN) = LNcl(SLN −KLN) Neutrosophic Sets and Systems, Vol. 46, 2021 259 (ii) S −LNcl(KLN) = LNcl(SLN −KLN) Neutrosophic Sets and Systems, Vol. 46, 2021 (ii) S −LNcl(KLN) = LNcl(SLN −KLN) Proof: (i): Let S ∈SLN −LNint(KLN) ⇒S /∈LNint(KLN). Thus, G ̸⊆KLN∀LN open set G containing S, (i.e) CLN ∩(S −KLN) ≠= φLN, ∀LN open set G. Hence, S ∈LNcl(SLN −KLN) and SLN −LNint(KLN) ⊆LNcl(SLN −KLN). Proof: (i): Let S ∈SLN −LNint(KLN) ⇒S /∈LNint(KLN). Thus, G ̸⊆KLN∀LN open set G containing S, (i.e) CLN ∩(S −KLN) ≠= φLN, ∀LN open set G. Hence, S ∈LNcl(SLN −KLN) and SLN −LNint(KLN) ⊆LNcl(SLN −KLN). Conversely, if S ∈LNcl(SLN −KLN), then GLN ∩(SLN −KLN) ̸= φLN for every LN open set containing S (i e) G ̸⊆A∀LN open set G containing S That is S /∈LNint(A) ⇒S ∈ Proof: (i): Let S ∈SLN −LNint(KLN) ⇒S /∈LNint(KLN). Thus, G ̸⊆KLN∀LN open set G containing S, (i.e) CLN ∩(S −KLN) ≠= φLN, ∀LN open set G. Hence, S ∈LNcl(SLN −KLN) and SLN −LNint(KLN) ⊆LNcl(SLN −KLN). Conversely, if S ∈LNcl(SLN −KLN), then GLN ∩(SLN −KLN) ̸= φLN for every LN open set containing S, (i.e) G ̸⊆A∀LN open set G containing S. That is, S /∈LNint(A) ⇒S ∈ S −LNint(A). Then, LNcl(SLN −KLN) ⊆SLN −LNint(KLN). Thus, SLN −LNint(KLN) = LNint(SLN −KLN) (ii) Proof is similar to (i). (ii) Proof is similar to (i). Remark 3.9. On taking complements on both sides of SLN −LNint(KLN) = LNint(SLN − KLN) and SLN −LNcl(KLN) = LNcl(SLN −KLN), we have, Remark 3.9. On taking complements on both sides of SLN −LNint(KLN) = LNint(SLN − KLN) and SLN −LNcl(KLN) = LNcl(SLN −KLN), we have, LNi t(K ) S LN l(S K ) d LN l(K ) S LNi t(S K ) LNint(KLN) = SLN −LNcl(SLN −KLN) and LNcl(KLN) = SLN −LNint(SLN − heorem 3.10. Remark 3.7. If LNint(KLN) is LNcl(KLN) is a LNCS, then we have, Let (SLN, πLN) be a LNTS and KLN, HLN ∈SLN. Then (i) LNint(KLN) = KLN if and only if KLN is LN open. (ii) LNint(φLN) = φLN and LNint(SLN) = SLN. (iii) KLN ⊆HLN ⇒LNint(KLN) ⊆LNint(HLN) (iv) LNint(KLN) ∪LNint(HLN) ⊆LNint(KLN ∪HLN) (iv) LNint(KLN ∩HLN) = LNint(KLN) ∩LNint(HLN) (vi) LNint(LNint(KLN)) = LNint(KLN) (vi) LNint(LNint(KLN)) = LNint(KLN) Proof: (i): KLN is LN open if and only if SLN −KLN is LN closed, if and only if, LNcl(SLN −KLN) = SLN −KLN, if and only if, SLN −LNcl(KLN) = KLN iffLNint(KLN) = KLN bT remark. (ii): Since φLN and SLN are LN open, LNint(φLN) = φLN and LNint(SLN) = SLN (iii): KLN ⊆HLN ⇒SLN −HLN ⊆SLN −KLN. Thus, LNcl(SLN −HLN) ⊆ LNcl(SLN −KLN), (i.e)SLN −LNcl(SLN −KLN) ⊆SLN −LNcl(SLN −HLN). Therefore, LNint(KLN) ⊆LNint(HLN) (ii): Since φLN and SLN are LN open, LNint(φLN) = φLN and LNint(SLN) = SLN (iii): KLN ⊆HLN ⇒SLN −HLN ⊆SLN −KLN. Thus, LNcl(SLN −HLN) ⊆ LNcl(SLN −KLN), (i.e)SLN −LNcl(SLN −KLN) ⊆SLN −LNcl(SLN −HLN). Therefore, LNint(KLN) ⊆LNint(HLN). (ii): Since φLN and SLN are LN open, LNint(φLN) = φLN and LNint(SLN) = SLN (iii): KLN ⊆HLN ⇒SLN −HLN ⊆SLN −KLN. Thus, LNcl(SLN −HLN) ⊆ LNcl(SLN −KLN), (i.e)SLN −LNcl(SLN −KLN) ⊆SLN −LNcl(SLN −HLN). Therefore, LNint(KLN) ⊆LNint(HLN). ( LN LN), ( ) LN ( LN LN) ⊆ LN ( LN LN) , LNint(KLN) ⊆LNint(HLN). Definition 3.11. Let SLN be a non-void set and KLN = {⟨S, [TKLN , IKLN , FKLN ]⟩} and HLN = {⟨S, [THLN , IHLN , FHLN ]⟩} are LNSs in LNTS. Definition 3.11. Let SLN be a non-void set and KLN = {⟨S, [TKLN , IKLN , FKLN ]⟩} and HLN = {⟨S, [THLN , IHLN , FHLN ]⟩} are LNSs in LNTS. Definition 3.11. Let SLN be a non-void set and KLN = {⟨S, [TKLN , IKLN , FKLN ]⟩} an HLN = {⟨S, [THLN , IHLN , FHLN ]⟩} are LNSs in LNTS. (I) KLN ∪HLN can be defined as (a) KLN ∪HLN = {⟨S, [TKLN ∧THLN , IKLN ∧IHLN , FKLN ∨FHLN ]⟩} (II) KLN ∩HLN can be defined as (a) KLN ∩HLN = {⟨S, [TKLN ∧THLN , IKLN ∧IHLN , FKLN ∨FHLN ]⟩} N. Gayathri, M. N. Gayathri, M. Helen, Linguistic Neutrosophic topology Remark 3.7. If LNint(KLN) is LNcl(KLN) is a LNCS, then we have, Helen, Linguistic Neutrosophic topology (I) KLN ∪HLN can be defined as (I) KLN ∪HLN can be defined as (a) KLN ∪HLN = {⟨S, [TKLN ∧THLN , IKLN ∧IHLN , FKLN ∨FHLN ]⟩} (II) KLN ∩HLN can be defined as (a) KLN ∪HLN = {⟨S, [TKLN ∧THLN , IKLN ∧IHLN , FKLN ∨FHLN ]⟩} (II) KLN ∩HLN can be defined as (a) KLN ∩HLN = {⟨S, [TKLN ∧THLN , IKLN ∧IHLN , FKLN ∨FHLN ]⟩} (a) KLN ∩HLN = {⟨S, [TKLN ∧THLN , IKLN ∧IHLN , FKLN ∨FHLN ]⟩} N. Gayathri, M. Helen, Linguistic Neutrosophic topology 260 Neutrosophic Sets and Systems, Vol. 46, 2021 Proof: If ULN is a LN open set and if S ∈LNcl(KLN), thenSLN−ULN is LN closed. If KLN∩ULN = φLN, then KLN ⊆SLN −ULN. That is, SLN −ULN is LN closed set containing KLN. Therefore, LNcl(KLN) ⊆SLN −ULN, which is a contradiction, since S ∈LNcl(KLN) but S /∈SLN−ULN. Hence, KLN∩ULN ̸= φLN, for every LN open set ULN containing S. Conversely, if KLN ∩ULN ̸= φLN, for every LN open set ULN containing S and if S /∈ LNcl(KLN), S ∈SLN −LNcl(KLN) which is LN open. Hence, (SLN −LNcl(KLN)) ∩KLN ̸= φLN. But KLN ⊆LNcl(KLN) and hence SLN − LNcl(KLN) ⊆SLN −KLN, that implies (SLN −LNcl(KLN)) ∩KLN ⊆(SLN −KLN) ∩KLN. Thus, (SLN −KLN) ∩KLN ̸= φLN, which is a contradiction. Hence, S ∈LNcl(KLN). Hence, (SLN −LNcl(KLN)) ∩KLN ̸= φLN. But KLN ⊆LNcl(KLN) and hence SLN − LNcl(KLN) ⊆SLN −KLN, that implies (SLN −LNcl(KLN)) ∩KLN ⊆(SLN −KLN) ∩KLN. Thus, (SLN −KLN) ∩KLN ̸= φLN, which is a contradiction. Hence, S ∈LNcl(KLN). Definition 3.13. Let (SLN, πLN) be a LNTS and πLN = {0, SLN}. Then, π is called the LN indiscrete topology over S. Definition 3.13. Let (SLN, πLN) be a LNTS and πLN = {0, SLN}. Then, π is called the LN indiscrete topology over S. Definition 3.14. Let π be the collection of all LN sets that can be defined over SLN. Then, (SLN, πLN) is the called the LN discrete topology over SLN. Definition 3.14. Let π be the collection of all LN sets that can be defined over SLN. Then, (SLN, πLN) is the called the LN discrete topology over SLN. Theorem 3.15. Let (SLN, π1LN) and (SLN, π2LN) be two LNTSs, then (SLN, π1 ∩π2LN) is a LNTS on SLN. Theorem 3.15. Let (SLN, π1LN) and (SLN, π2LN) be two LNTSs, then (SLN, π1 ∩π2LN) is a LNTS on SLN. Neutrosophic Sets and Systems, Vol. 46, 2021 (III) The complement of KLN = {⟨S, [TKLN , IKLN , FKLN ]⟩} is defined as, (III) The complement of KLN = {⟨S, [TKLN , IKLN , FKLN ]⟩} is defined as, (a) KLN c = {⟨S, [1 −FKLN , IKLN , 1 −TKLN ]⟩} (b) (KLN c)c = KLN (c) (KLN ∩HLN)c = KLN c ∪HLN c (d) (KLN ∪HLN)c = KLN c ∩HLN c Theorem 3.12. Let (SLN, πLN) be a LNTS. S ∈LNcl(KLN) iffULN ∩KLN ̸= φLN for every LN open set ULN containing S, where KLN ⊆SLN. Theorem 3.12. Let (SLN, πLN) be a LNTS. S ∈LNcl(KLN) iffULN ∩KLN ̸= φLN for every LN open set ULN containing S, where KLN ⊆SLN. Proof: (1) clearly, 0LN and 1LN ∈π1LN ∩π2LN (1) clearly, 0LN and 1LN ∈π1LN ∩π2LN (2) Let Fi ∈π1LN ∩π2LN. Then, Fi ∈π1LN and Fi ∈π2LN∀i ∈I. Therefore, ∪i∈IFi ∈π1LN and ∪i∈IFi ∈π2LN. Thus, S i∈I Fi ∈π1LN ∩π2LN. (2) Let Fi ∈π1LN ∩π2LN. Then, Fi ∈π1LN and Fi ∈π2LN∀i ∈I. Therefore, ∪i∈IFi ∈π1LN and ∪i∈IFi ∈π2LN. Thus, S i∈I Fi ∈π1LN ∩π2LN. ( ) , Therefore, ∪i∈IFi ∈π1LN and ∪i∈IFi ∈π2LN. Thus, S i∈I Fi ∈π1LN ∩π2LN. (3) Let KLN and HLN ∈ π1LN ∩π2LN, which implies, KLN, HLN ∈ π1LN and KLN, HLN ∈π2LN. Since, KLN ∩HLN ∈π1LN and KLN ∩HLN ∈π2LN, KLN ∩HLN ∈ π1LN ∩π2LN (3) Let KLN and HLN ∈ π1LN ∩π2LN, which implies, KLN, HLN ∈ π1LN and KLN, HLN ∈π2LN. Since, KLN ∩HLN ∈π1LN and KLN ∩HLN ∈π2LN, KLN ∩HLN ∈ π1LN ∩π2LN Thus, (SLN, π1LN T π2LN) is a LNTS on SLN.. Thus, (SLN, π1LN T π2LN) is a LNTS on SLN.. Thus, (SLN, π1LN T π2LN) is a LNTS on SLN.. Remark 3.16. Union of two LNTSs may not be a LN topology over SLN.. N. Gayathri, M. Helen, Linguistic Neutrosophic topology N. Gayathri, M. Helen, Linguistic Neutrosophic topology 261 Neutrosophic Sets and Systems, Vol. 46, 2021 Example 3.17. Let the universe of discourse be U = {a, b, c} and S = {a}. The set of all linguistic term is, L = { very salt(l0), salt(l1), very sour(l2), sour(l3), bitter(l4), sweet(l5), very sweet(l6)}. Example 3.17. Let the universe of discourse be U = {a, b, c} and S = {a}. The set of all linguistic term is, L = { very salt(l0), salt(l1), very sour(l2), sour(l3), bitter(l4), sweet(l5), very sweet(l6)}. And π1LN = {0LN, 1LN, KLN} where KLN = {⟨a, (l6, l3, l3)⟩}, where the element a’s degree of appurtenance to the set KLN is very sweet(l6), the element a’s degree of indeterminate- appurtenance to the set KLN is sour(l3), the element a’s degree of non-appurtenance to the set KLN is bitter(l4). Let π2LN = {0LN, 1LN, HLN} where HLN = {⟨a, (l4, l5, l2)⟩}, where the element a’s de- gree of appurtenance to the set HLN is bitter(l4), the element a’s degree of indeterminate- appurtenance to the set HLN is sweet(l5), the element a’s degree of non-appurtenance to the set HLN is very sour(l2). Let π1LN and π2LN be two LN topologies on SLN. Proof: Then, π1LN ∪π2LN = {0LN, 1LN, KLN, HLN} = {0LN, 1LN, {⟨a, (l6, l3, l3)⟩}, {⟨a, (l6, l5, l2)⟩}}. Now, KLN ∪HLN = {⟨a, (l6, l5, l2)⟩} /∈π1LN ∪π2LN. KLN ∩HLN = {⟨a, (l4, l3, l3)⟩} /∈π1LN ∪π2LN. Therefore, union of any two linguistic neutrosophic topologies need not be a linguistic neutro- sophic topology. Definition 3.18. Let (SLN, πLN) be a LNTS and ULN be a LN set over SLN.. Then any point S is a LN interior point of ULN, if there exists a LN open set VLN such that S ∈ULN ⊆VLN. Definition 3.18. Let (SLN, πLN) be a LNTS and ULN be a LN set over SLN.. Then any point S is a LN interior point of ULN, if there exists a LN open set VLN such that S ∈ULN ⊆VLN. Definition 3.19. Let (SLN, πLN) be a LNTS and ULN be a LN set over SLN.. Then, VLN is called a LN neighborhood if there exists a LN open set VLN such that S ∈ULN ⊆VLN. Definition 3.19. Let (SLN, πLN) be a LNTS and ULN be a LN set over SLN.. Then, VLN is called a LN neighborhood if there exists a LN open set VLN such that S ∈ULN ⊆VLN. Theorem 3.20. Let (SLN, πLN) be a LNTS, then (1) each s ∈S has a neighborhood (1) each s ∈S has a neighborhood. (2) If ULN and VLN are LN neighborhoods of some x ∈SLN, then ULN ∩VLN is also a LN neighborhood of s. (3) If ULN is a LN neighborhood of S and ULN ∩VLN, then VLN is also a LN neighborhood of s ∈SLN. Proof: Proof: Proof: Let KLN ∈πLN. For any S ∈∩i∈IKLN i, we have S ∈Ai∀i ∈I. Thus, S ∈KLN and hence KLN is a LN neighborhood of S. Let KLN ∈πLN. For any S ∈∩i∈IKLN i, we have S ∈Ai∀i ∈I. Thus, S ∈KLN and hence KLN is a LN neighborhood of S. Neutrosophic Sets and Systems, Vol. 46, 2021 Theorem 3.21. Let (SLN, πLN) be a LNTS. For any LN open set KLN over S, KLN is a LN neighborhood of each point of ∩i∈IAi. Theorem 3.21. Let (SLN, πLN) be a LNTS. For any LN open set KLN over S, KLN is a LN neighborhood of each point of ∩i∈IAi. Proof: KLN is LN closed if and only if LNcl(KLN) = KLN, iffKLN ∪LND(KLN) = KLN, iff LND(KLN) ⊆KLN. Theorem 4.4. If KLN is a singleton subset of SLN, then LND(KLN) = LNcl(KLN) −KLN. Theorem 4.4. If KLN is a singleton subset of SLN, then LND(KLN) = LNcl(KLN) −KLN. Proof: 4. Linguistic neutrosophic derived sets Definition 4.1. Let (SLN, πLN) be a LNTS and KLN ⊆SLN. Let s ∈SLN. s is called as a LN limit point of KLN if ELN ∩(KLN −{s}) ̸= φ for every LN open set ELN containing s. The collection of all LN limit points of KLN is the LN derived set (LND(KLN))ofKLN. Theorem 4.2. LNcl(KLN) = KLN ∪LND(KLN) where KLN ⊆SLN Theorem 4.2. LNcl(KLN) = KLN ∪LND(KLN) where KLN ⊆SLN Proof: (1) : (2): Let ULN and VLN are LN neighborhoods of some s ∈S, then there exists U 1LN and V 1LN ∈τ such that S ∈U 1LN ⊆ULN and Now, S ∈ULN and S ∈VLN implies that S ∈U 1LN ∩V 1LN and U 1LN ∩V 1LN ∈τ. So w have S ∈U1LN ∩V1LN ⊆ULN ∩VLN. Thus, ULN T VLN is a LN neighborhood of s. (3): Let ULN is a LN neighborhood of s and ULN T VLN. By definition, there exists a LN open N. Gayathri, M. Helen, Linguistic Neutrosophic topology (3): Let ULN is a LN neighborhood of s and ULN T VLN. By definition, there exists a LN open N. Gayathri, M. Helen, Linguistic Neutrosophic topology 262 Proof: If s ∈KLN ∪LND(KLN), then s ∈KLN or S ∈LND(KLN). If s ∈KLN, then s ∈ LNcl(KLN). Therefore, let s /∈KLN. That is, s ∈LND(KLN). Then, ∀LN open set ELN containing s, ELN ∩(KLN −s) ̸= φ. Since s /∈KLN, ELN ∩KLN /∈φ. Thus, s ∈LNcl(KLN). Hence, KLN ∪LND(KLN) ⊆LNcl(KLN). If s ∈LNcl(KLN) and s ∈KLN, then s ∈ KLN ∪LND(KLN). If s ∈LNcl(KLN) but s /∈KLN, then ELN ∩KLN /∈φ for every LN open set ELN containing s and hence ELN ∩(KLN −s) /∈φ. Therefore, s ∈LND(KLN), (i.e) s ∈KLN ∪LND(KLN). Thus, LNcl(KLN) ⊆KLN ∪LND(KLN). Therefore, LNcl(KLN) = KLN ∪LND(KLN). Theorem 4.3. If the derived set of KLN is a subset of KLN, then KLN is LN closed. Proof: Definition 4.5. (2) Linguistic Neutrosophic semi-open set if KLN ⊆LNcl(LNint(KLN)) (3) Linguistic Neutrosophic semi-pre closed if LNint(LNcl(LNint(KLN))⊆KLN (4) Linguistic Neutrosophic semi-pre open if KLN ⊆LNcl(LNint(LNcl(KLN))) (5) Linguistic Neutrosophic pre-closed if LNcl(LNint(KLN) ⊆KLN -doubt (6) Linguistic Neutrosophic pre-open if KLN ⊆LNint(LNcl(KLN)) (7) Linguistic Neutrosophic regular closed if KLN = LNint(LNcl(KLN)) (8) Linguistic Neutrosophic regular open if KLN = LNcl(LNint(KLN)) (2) Linguistic Neutrosophic semi-open set if KLN ⊆LNcl(LNint(KLN)) (3) Linguistic Neutrosophic semi-pre closed if LNint(LNcl(LNint(KLN))⊆KLN (4) Linguistic Neutrosophic semi-pre open if KLN ⊆LNcl(LNint(LNcl(KLN))) (5) Linguistic Neutrosophic pre-closed if LNcl(LNint(KLN) ⊆KLN -doubt (6) Linguistic Neutrosophic pre-open if KLN ⊆LNint(LNcl(KLN)) (7) Linguistic Neutrosophic regular closed if KLN = LNint(LNcl(KLN)) (8) Linguistic Neutrosophic regular open if KLN = LNcl(LNint(KLN)) (8) Linguistic Neutrosophic regular open if KLN = LNcl(LNint(KLN)) Proof: If s ∈LND(KLN), then for every LN open set ELN containing , ELN ∩(KLN −s) ̸= φ. Then s /∈KLN. Suppose if s ∈KLN, then KLN = {s}, and ELN ∩(KLN −s) = φ. It is true that, LND(KLN) ⊆LNcl(KLN). Then, s ∈LNcl(KLN) but s /∈KLN, when s ∈LND(KLN). Thus, LND(KLN) ⊆LNcl(KLN) −KLN. Thus, s ∈LNcl(KLN) −KLN, s ∈LNcl(KLN) N. Gayathri, M. Helen, Linguistic Neutrosophic topology 263 Neutrosophic Sets and Systems, Vol. 46, 2021 but s /∈KLN. Thus, ELN ∩KLN ̸= φ for every LN open set ELN containing s, (i.e) ELN ∩(KLN −s) ̸= φ for every LN open set ELN containing s. Thus, s ∈LND(KLN). Thus, LNcl(KLN) −KLN ⊆LND(KLN). Hence, LND(KLN) = LNcl(KLN) −KLN, if KLN is a singleton set. but s /∈KLN. Thus, ELN ∩KLN ̸= φ for every LN open set ELN containing s, (i.e) ELN ∩(KLN −s) ̸= φ for every LN open set ELN containing s. Thus, s ∈LND(KLN). Thus, LNcl(KLN) −KLN ⊆LND(KLN). Hence, LND(KLN) = LNcl(KLN) −KLN, if KLN is a singleton set. Definition 4.5. (1) Linguistic Neutrosophic semi-closed set if LNint(LNcl(KLN)) ⊆ KLN Definition 4.5. (1) Linguistic Neutrosophic semi-closed set if LNint(LNcl(KLN)) ⊆ KLN N. Gayathri, M. Helen, Linguistic Neutrosophic topology 5. Linguistic Neutrosophic continuity Definition 5.1. Define the image and pre-image of linguistic neutrosophic sets. Let SLN and TLN be two non-void sets and f : SLN →TLN be a function, then Definition 5.1. Define the image and pre-image of linguistic neutrosophic sets. Let SLN and TLN be two non-void sets and f : SLN →TLN be a function, then TLN be two non-void sets and f : SLN →TLN be a function, then TLN be two non-void sets and f : SLN →TLN be a function, then (i) If ELN = {⟨S, [TELN (S), IELN (S), FELN (S)]⟩} is a LN set in TLN, then the pre image of ELN under f is denoted by, f−1(ELN) is defined by, f−1(ELN) = {⟨S, [f−1(TELN (S)), f−1(IELN (S)), f−1(FELN (S))]⟩} (ii) If FLN = {⟨S, [TFLN (S), IFLN (S), FLN F(S)]⟩; S ∈SLN} is a LN set in SLN, then the image of FLN under f is denoted bT, f(FLN) = {⟨T, [f(TFLN (T)), f(IFLN (T)), f(FFLN (T))]⟩; T ∈TLN} Definition 5.2. A function f : SLN →TLN is called a linguistic neutrosophic continuous func- tion if the inverse image of every linguistic neutrosophic open set FLN is linguistic neutrosophic open in SLN. Example 5.3. Let the universe of discourse be ULN = {a, b, c, d, x, y, z, w} and S1 = {a, b, c} and S2 = {x, y, z}. The set of all linguistic term is, L = { very salt(l0), salt(l1), very sour(l2), sour(l3), bitter(l4), sweet(l5), very sweet(l6)}. Define linguistic neutrosophic sets KLN and HLN as KLN = {s1, (a, ⟨l0, l6, l0⟩), (b, ⟨l4, l0, l2⟩), (c, ⟨l2, l3, l1⟩)}, where the element a’s degree of appurtenance to the set KLN is very sweet(l0), the element a’s degree of indeterminate- appurtenance to the set KLN is very sweet(l6), the element a’s degree of non-appurtenance to the set KLN is very salt(l0). Neutrosophic Sets and Systems, Vol. 46, 2021 264 Similarly, b’s degree of appurtenance to the set KLN is bitter(l4), b’s degree of indeterminate- appurtenance to the set KLN is very salt(l0), b’s degree of non-appurtenance to the set KLN is very sour(l2). And, c’s degree of appurtenance to the set KLN is very sour(l2), c’s degree of indeterminate- appurtenance to the set KLN is sour(l3), c’s degree of non-appurtenance to the set KLN is very salt(l1). 5. Linguistic Neutrosophic continuity Also, let HLN = {s2, (x, ⟨l6, l0, l0⟩), (y, ⟨l0, l4, l2⟩), (z, ⟨l3, l2, l1⟩)}. Then, πLN = {0LN, 1LN, KLN} and ηLN = {0LN, 1LN, HLN} are linguistic neutrosophic topologies on S1, S2 respectively. Let g : (S1, πLN) →(S2, ηLN) be defined by g(a) = b, g(b) = a, g(c) = c. Then, g is linguistic neutrosophic continuous function. Theorem 5.4. A function f : SLN →TLN is linguistic neutrosophic continuous if and only if the pre image of every linguistic neutrosophic closed set in TLN is linguistic neutrosophic closed in SLN. Example 5.6. In example(5.3), g is a linguistic neutrosophic continuous function. Let KLN = {s1, ⟨a, (l0, l6, l0)⟩, ⟨b, (l4, l0, l2)⟩, ⟨c, (l2, l3, l1)⟩} ⊆(S1, πLN). Then, g(LNcl(KLN)) = N. Gayathri, M. Helen, Linguistic Neutrosophic topology Neutrosophic Sets and Systems, Vol. 46, 2021 {s1, ⟨a, (l6, l6, l6)⟩, ⟨b, (l0, l4, l2)⟩, ⟨c, (l3, l5, l4)⟩}. But, LNcl(g(KLN)) = LNcl(HLN) = Bc ̸= {s1, ⟨a, (l6, l6, l6)⟩, ⟨b, (l0, l4, l2)⟩, ⟨c, (l3, l5, l4)⟩}, even though g is linguistic neutrosophic continuous function. Thus, equality is not necessarily holds when g is linguistic neutrosophic continuous function. Theorem 5.7. A function f : SLN →TLN is LN continuous if and only if LNcl(f−1(ELN)) ⊆ f−1(LNcl(ELN)) for each subset ELN of TLN. Proof: Proof: If f is LN continuous and ELN ⊆TLN, then LNcl(ELN) is LN closed in TLN and hence f−1(LNcl(ELN)) is LN closed in SLN. Thus, LNcl(f−1(LNcl(ELN))) = f−1(LNcl(ELN)). Since, ELN ⊆LNcl(ELN), f−1(ELN) ⊆f−1(LNcl(ELN)). Therefore, LNcl(f−1(ELN)) ⊆ LNcl(f−1(LNcl(ELN))) = f−1(LNcl(ELN)), (i.e) LNcl(f−1(ELN)) ⊆f−1(LNcl(ELN)). Conversely, let LNcl(f−1(ELN))f−1(LNcl(ELN)) for all ELN of TLN. If ELN is LN closed, then LNcl(ELN) = ELN. By assumption, LNcl(f−1(ELN)) ⊆f−1(LNcl(ELN)). Thus, LNcl(f−1(ELN)) ⊆ f−1(ELN). But, f−1(ELN) ⊆ LNcl(f−1(ELN)). Thus, LNcl(f−1(ELN)) = f−1(ELN), (i.e) f−1(ELN) is LN closed in SLN for every LN closed set ELN in TLN. Hence, f is LN continuous. Theorem 5.8. A function f : SLN → TLN is LN continuous if and only if f−1(LNint(ELN)) ⊆LNint(f−1(ELN)) for each subset ELN of TLN. Theorem 5.8. A function f : SLN → TLN is LN continuous if and only if f−1(LNint(ELN)) ⊆LNint(f−1(ELN)) for each subset ELN of TLN. Proof: Let f be a LN continuous function and ELN be a LN closed set in TLN, (i.e) TLN −ELN is LN open in TLN. f−1(TLN −ELN) is a LN open set in SLN, as f is LN continuous function. Thus, SLN −f−1(ELN) is LN open set in SLN. That is, f−1(ELN) is LN closed set in SLN. Conversely, let the inverse image of each LN closed set be LN closed. Let FLN be a LN open set in TLN, (i.e) TLN −FLN is LN closed. Then, SLN −f−1(FLN) is LN closed set in SLN, which implies, f−1(FLN) is LN open set in SLN. Thus, f is LN continuous function on SLN. Theorem 5.5. A function f : SLN →TLN is LN continuous if and only if f(LNcl(KLN)) ⊆ LNcl(f(KLN)) for each subset KLN of SLN. Let f be LN continuous function. If KLN ⊆SLN, then f(KLN) ⊆TLN. As f is LN continuous and LNcl(f(KLN)) is LN closed in TLN, f−1(LNcl(f(KLN))) is LN closed set in SLN. Since, f(KLN) ⊆LNcl(f(KLN)), KLN ⊆f−1(LNcl(f(KLN))), which implies, f−1(LNcl(f(KLN))) is the smallest LN closed set that contains KLN. But, LNcl(KLN) is the smallest LN closed set that contains KLN. Hence, LNcl(KLN) ⊆f−1(LNcl(f(KLN))), (i.e) f(LNcl(KLN)) ⊆ LNcl(f(KLN)). Conversely, let f(LNcl(HLN)) ⊆LNcl(f(HLN)). If HLN is LN closed in TLN, f(LNcl(f−1(HLN))) ⊆LNcl(HLN). Thus, LNcl(f−1(HLN)) ⊆f−1((LNcl(HLN))) = f−1((HLN)). But, f−1((HLN)) ⊆LNcl(f−1((HLN))), that implies, LNcl(f−1((HLN))) = f−1((HLN)) ⇒f−1((HLN)) is LN closed set in SLN for each LN closed set HLN in TLN. Therefore, f is LN continuous. Example 5.6. In example(5.3), g is a linguistic neutrosophic continuous function. Let KLN = {s1, ⟨a, (l0, l6, l0)⟩, ⟨b, (l4, l0, l2)⟩, ⟨c, (l2, l3, l1)⟩} ⊆(S1, πLN). Then, g(LNcl(KLN)) = N. Gayathri, M. Helen, Linguistic Neutrosophic topology 265 Neutrosophic Sets and Systems, Vol. 46, 2021 Proof: As ULN is LN dense in SLN, f(LNcl(ULN)) = f(SLN) = TLN, since f is onto. Also, f(LNcl(ULN)) ⊆LNcl(ULN), as f is LN continuous. Thus, TLN = LNcl(f(ULN)). But LNcl(f(ULN)) ⊆TLN. Hence,LNcl(f(ULN)) = TLN, which implies, f(ULN) is LN dense set. C l i Conclusion We have introduced a new type of topology called linguistic neutrosophic topology and it was established with apt examples. Moreover, the basic properties of linguistic neutrosophic were discussed. In addition to this, the ideas of linguistic neutrosophic continuity and linguistic neutrosophic neighborhood were introduced and established. Linguistic neutrosophic derived sets and linguistic neutrosophic dense sets were talked through. Neutrosophic Sets and Systems, Vol. 46, 2021 Theorem 5.11. Let f : SLN →TLN be an onto function and linguistic neutrosophic contin- uous function. If ULN is LN dense in SLN, then f(ULN) is LN dense in TLN. Proof: Let f be LN continuous function and E ⊆TLN. Then, f−1(LNint(ELN)) is LN open in SLN. That means, f−1(LNint(ELN)) = LNint(f−1(LNint(ELN))). As LNint(ELN) ⊆ ELN, implies f−1(LNint(ELN)) ⊆ f−1(ELN). Thus, LNint(f−1(LNint(ELN))) ⊆ LNint(f−1(ELN)). Therefore,f−1(LNint(ELN)) ⊆LNint(f−1(ELN)). Conversely, let f−1(LNint(ELN)) ⊆LNint(f−1(ELN)), for each subset ELN of TLN. If ELN is LN open, then f−1(ELN) ⊆LNint(f−1(ELN)). But, LNint(f−1(ELN)) ⊆f−1(ELN). Thus, f−1(ELN) = LNint(f−1(ELN)). Hence, f is LN continuous. Example 5.9. In example(5.3), HLN = {s2, ⟨x, (l6, l0, l0)⟩, ⟨y, (l0, l4, l2)⟩, ⟨z, (l3, l2, l1)⟩}. Then, g−1(LNcl(HLN)) = g−1(HLN c) = {s2, ⟨z, (l0, l6, l0)⟩, ⟨y, (l4, l4, l6)⟩, ⟨z, (l2, l5, l3)⟩}. And LNcl(g−1(HLN)) = KLN c. Thus, g−1(LNcl(ELN)) ⊆LNcl(g−1(ELN)). Similarly, g−1(LNint(ELN)) ⊆LNint(g−1(ELN)). Even if g is LN continuous, equality does not hold in theorems (5.7) and (5.8). Definition 5.10. Any subset of a LN topological space (SLN, πLN) is a LN dense set if LNcl(KLN) = SLN). N. Gayathri, M. Helen, Linguistic Neutrosophic topology 266 Neutrosophic Sets and Systems, Vol. 46, 2021 N. Gayathri, M. Helen, Linguistic Neutrosophic topology N. Gayathri, M. Helen, Linguistic Neutrosophic topology References [1] Atanassov, K.T. Intuitionistic fuzzy sets. Fuzzy Sets and Systems, 1986; 20, pp. 87–96. [1] Atanassov, K.T. Intuitionistic fuzzy sets. Fuzzy Sets and Systems, 1986; 20, pp. 87–96. [2] Chang, C.L. Fuzzy topological spaces, J Math.Anal.Appl. 1968; 24, pp. 182–190. [3] Chen, Z.C.; Liu, P.H.; Pei, Z. An approach to multiple attribute group decision making based on linguistic intuitionistic fuzzy numbers. Int. J. Comput. Intell. Syst. 2015, 8, 747–760. [3] Chen, Z.C.; Liu, P.H.; Pei, Z. An approach to multiple attribute group decision making based on linguistic intuitionistic fuzzy numbers. Int. J. Comput. Intell. Syst. 2015, 8, 747–760. [4] Coker, D. An introduction to intuitionistic fuzzy topological spaces, Fuzzy Sets and Systems, 1997; 88, pp. 81-89. [4] Coker, D. An introduction to intuitionistic fuzzy topological spaces, Fuzzy Sets and Systems, 1997; 88, pp. 81-89. [5] Fan, Feng, and Hu. (2019). Linguistic Neutrosophic Numbers Einstein Operator and Its Application in Decision Making. Mathematics, 7(5), 389. doi:10.3390/math7050389 [6] Fang, Zebo and Te, Jun. Multiple Attribute Group Decision-Making Method Based on Linguistic Neutro- sophic Numbers.Symmetry,9(7), 2017. 111; https://doi.org/10.3390/sTm9070111. [7] Garg H and Nancy. Linguistic single-valued neutrosophic power aggregation operators and their applica- tions to group decision-making problems. IEEE/CAA J. Autom. Sinica, vol. 7, no. 2, pp. 546–558, Mar. 2020. [8] Herrera, F.; Herrera-Viedma, E.; Verdegay, L. A model of consensus in group decision making under linguistic assessments. Fuzzy Sets Syst. 1996, 79, 73–87. [9] Herrera F.; Herrera-Viedma E. Linguistic decision analysis . Steps for solving decision problems under linguistic information. Fuzzy Sets and Systems, 2000, 115(1): 67–82. [10] Munkres, James R. Topology: a First Course. Englewood Cliffs, N.J.: Prentice-Hall, 1974. [11] Smarandache F. A unifying field in logics. Neutrosophy: Neutrosophic probability, set and logic. American Research Press, Rehoboth, 1999. [12] Su Z S. Deviation measures of linguistic preference relations in group decision making. Omega, 2005, 33(3):249–254. [13] Wang H, Smarandache F, Zhang T Q, Sunderraman R. Interval neutrosophic sets and logic: Theory and applications in computing. Hexis, Phoenis, AZ, 2005. [14] Wei, G., Wu, J., Guo, Y., Wang, J., and Wei, C. (2021). An extended COPRAS model for multiple attribute group decision making based on single-valued neutrosophic 2-tuple linguistic environment. Technological and Economic Development of Economy, 27(2), 353-368. https://doi.org/10.3846/tede.2021.14057. N. Gayathri, M. Helen, Linguistic Neutrosophic topology [15] Ye, J. An extended TOPSIS method for multiple attribute group decision making based on single valued neutrosophic linguistic numbers. J. Intell. Fuzzy Syst. 2015, 28, 247–255. [17] Zadeh, L.A. The concept of a linguistic variable and its application to approximate reasoning Part I. Inf. Sci. 1975, 8, 199–249. ] Zadeh, L.A. Fuzzy Sets. Information and Control, 1965; 8,pp. 338–353. [16] Zadeh, L.A. Fuzzy Sets. Information and Control, 1965; 8,pp. 338–353. Neutrosophic Sets and Systems, Vol. 46, 2021 267 [15] Ye, J. An extended TOPSIS method for multiple attribute group decision making based on single valued neutrosophic linguistic numbers. J. Intell. Fuzzy Syst. 2015, 28, 247–255. [16] Zadeh, L.A. Fuzzy Sets. Information and Control, 1965; 8,pp. 338–353. [17] Zadeh, L.A. The concept of a linguistic variable and its application to approximate reasoning Part I. Inf. Sci. 1975, 8, 199–249. Received: May 29, 2021. Accepted: October 1, 2021

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Knockdown of α2,3-Sialyltransferases Impairs Pancreatic Cancer Cell Migration, Invasion and E-selectin-Dependent Adhesion

International journal of molecular sciences

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Received: 6 August 2020; Accepted: 26 August 2020; Published: 28 August 2020 Abstract: Aberrant sialylation is frequently found in pancreatic ductal adenocarcinoma (PDA). α2,3-Sialyltransferases (α2,3-STs) ST3GAL3 and ST3GAL4 are overexpressed in PDA tissues and are responsible for increased biosynthesis of sialyl-Lewis (sLe) antigens, which play an important role in metastasis. This study addresses the effect of α2,3-STs knockdown on the migratory and invasive phenotype of PDA cells, and on E-selectin-dependent adhesion. Characterization of the cell sialome, the α2,3-STs and fucosyltransferases involved in the biosynthesis of sLe antigens, using a panel of human PDA cells showed differences in the levels of sialylated determinants and α2,3-STs expression, reflecting their phenotypic heterogeneity. Knockdown of ST3GAL3 and ST3GAL4 in BxPC-3 and Capan-1 cells, which expressed moderate to high levels of sLe antigens and α2,3-STs, led to a significant reduction in sLex and in most cases in sLea, with slight increases in the α2,6-sialic acid content. Moreover, ST3GAL3 and ST3GAL4 downregulation resulted in a significant decrease in cell migration and invasion. Binding and rolling to E-selectin, which represent key steps in metastasis, were also markedly impaired in the α2,3-STs knockdown cells. Our results indicate that inhibition of ST3GAL3 and ST3GAL4 may be a novel strategy to block PDA metastasis, which is one of the reasons for its dismal prognosis. Keywords: pancreatic ductal adenocarcinoma; α2,3-sialyltransferases; sialyl-Lewis antigens; E-selectin; cell migration Knockdown of α2,3-Sialyltransferases Impairs Pancreatic Cancer Cell Migration, Invasion and E-selectin-Dependent Adhesion Pedro Enrique Guerrero 1 , Laura Miró 1 , Bin S. Wong 2, Anna Massaguer 1, Neus Martínez-Bosch 3, Rafael de Llorens 1, Pilar Navarro 3,4,5 , Konstantinos Konstantopoulos 2, Esther Llop 1,* and Rosa Peracaula 1,* Pedro Enrique Guerrero 1 , Laura Miró 1 , Bin S. Wong 2, Anna Massaguer 1, Neus Martínez-Bosch 3, Rafael de Llorens 1, Pilar Navarro 3,4,5 , Konstantinos Konstantopoulos 2, Esther Llop 1,* and Rosa Peracaula 1,* Pedro Enrique Guerrero 1 , Laura Miró 1 , Bin S. Wong 2, Anna Massaguer 1, Neus Martínez-Bosch 3, Rafael de Llorens 1, Pilar Navarro 3,4,5 , Konstantinos Konstantopoulos 2, Esther Llop 1,* and Rosa Peracaula 1,* 1 Department of Biology, Biochemistry and Molecular Biology Unit, University of Girona, 17003 Girona, Spain; [email protected] (P.E.G.); [email protected] (L.M.); [email protected] (A.M.); [email protected] (R.d.L.) 1 Department of Biology, Biochemistry and Molecular Biology Unit, University of Girona, 17003 Girona, Spain; [email protected] (P.E.G.); [email protected] (L.M.); [email protected] (A.M.); [email protected] (R.d.L.) 2 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; [email protected] (B.S.W.); [email protected] (K.K.) 08003 Barcelona, Spain; [email protected] (N.M.-B.); [email protected] (P.N.) 4 Institute of Biomedical Research of Barcelona (IIBB)-CSIC, 08036 Barcelona, Spain 5 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain * Correspondence: [email protected] (E.L.); [email protected] (R.P.); Tel.: +972-418370 (R.P.); F +972 41 82 41 (R P) 4 Institute of Biomedical Research of Barcelona (IIBB)-CSIC, 08036 Barcelona, Spain 5 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain * Correspondence: [email protected] (E.L.); [email protected] (R.P.); Tel.: +972-418370 (R.P.); Fax: +972-41-82-41 (R.P.)



 Received: 6 August 2020; Accepted: 26 August 2020; Published: 28 August 2020 International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Pancreatic ductal adenocarcinoma (PDA), the most frequent pancreatic tumor, is considered the one with the direst prognosis among all carcinomas, with the lowest five-year survival rate, of about 5–7% [1]. PDA detection is usually performed at late stages since symptoms are often unnoticed and there is a lack of accurate tumor markers for its detection. In advanced stages, PDA is characterized by its aggressiveness, enhanced by the dense tumor microenvironment that acts as a barrier preventing Int. J. Mol. Sci. 2020, 21, 6239; doi:10.3390/ijms21176239 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 6239 2 of 23 treatment efficiency [2], and by its ability to spread to other organs at early stages of the disease. In fact, more than 80% of PDA patients are diagnosed with metastatic or unresectable disease [3]. PDA is therefore a challenging disease, and a major knowledge of PDA biology is urgently required to shed light onto new therapeutic and diagnostic strategies. In recent years, abnormal glycosylation remains in the scientific spotlight since it is a frequent hallmark of cancer and is involved in key steps during cancer formation, progression and metastasis. Glycosylation alterations include changes in sialylation, fucosylation, increased GlcNAc-branching of N-glycans and overexpression of truncated mucin-type O-glycans [4–6]. Among these changes, an increase in cell sialyation and in particular of sialyl-Lewis (sLe) antigens, such as sLex (Neu5Acα2, 3Galβ1,4[Fucα1,3]GlcNAcβ1-R) and sLea (Neu5Acα2,3Galβ1,3[Fucα1,4]GlcNAcβ1-R), have been shown to be critical to the regulation of cell adhesion, tumor invasion and metastasis [7–12]. SLex/a antigen expression correlates with the aggressiveness and the poor outcome of different tumors [13], such as gastric cancers [14], breast cancer [12], colon cancer [15,16], lung cancer [11] and PDA [17,18]. SLe antigens are ligands of the Ca2+ dependent lectin E-selectin, expressed in activated endothelia, and when expressed in cancer cells, they act as mediators of cancer metastasis by favoring selectin-dependent adhesion during the cancer cell extravasation process [19,20]. SLe antigens’ biosynthesis is regulated by a set of glycosyltransferases, namely α1,3/1,4- fucosyltransferases and α2,3-sialyltransferases, two glycoenzymes that are involved in the late steps of glycosylation. Sialyltransferases are a large family of enzymes located in the Golgi, which catalyze the transfer of the sialic acid (SA) through a nucleotide sugar donor CMP-SA. In particular, α2,3-sialyltransferases (α2,3-STs) catalyze the transfer of a SA in α2,3-linkage on β-linked galactose to different carbohydrate acceptors of N-glycans, O-glycans or glycolipids. 2. Results The expression of sLex and sLea determinants in protein cell lysates and secreted glycoconjugates from conditioned media was analyzed by WB (Figure 2). The results were in line with those obtained for cell membrane glycoconjugates determined by flow cytometry. The highest sLex-expressing cell lines were Capan-1 and BxPC-3, whereas for sLea were Capan-2 and BxPC-3, in both cell lysates and cell conditioned media, with the signal being higher in secreted glycoproteins of the conditioned media. The main differences of sialylated determinants between cell lines were primarily detected in the high molecular weight region, which could correspond to highly glycosylated mucins, among others [18,32]. To identify the most appropriate cell lines to knockdown ST3GAL3 and ST3GAL4, we first determined the mRNA expression levels of the α2,3-ST and the fucosyltransferase genes that code for the enzymes that act in the last steps of SLe antigens’ biosynthesis (Figure 3). Regarding α2,3-ST expression, ST3GAL3, ST3GAL4 and ST3GAL6 mRNA levels were analyzed. ST3GAL6 levels were much lower than ST3GAL3 and ST3GAL4 ones for all cell lines examined in this work. Among all cell lines tested, only Capan-1 and BxPC-3 expressed ST3GAL6 levels above the background. ST3GAL3 expression was also 4–20-fold lower than ST3GAL4 for all cell lines. The cells with the highest ST3GAL3 expression were AsPC-1, BxPC-3, Capan-2 and SW 1990. All cell lines expressed appreciable ST3GAL4 mRNA levels, being AsPC-1, and Capan-2 the cell lines with the highest levels. The expression of α1,3/4-fucosyltransferases, involved in the synthesis of sLex and sLea in mammalian cell lines (FUTs-3, -5, -6 and -7) [36], showed that these cell lines exhibited very low levels of FUT5, FUT6 and FUT7, except for Capan-1 cells that displayed much higher FUT6 levels than the rest of the cells (data not shown). FUT3 was the main fucosyltransferase expressed, which was found in six (Capan-1, SW 1990, Panc 10.05, Capan-2, BxPC-3 and HPAF-II) out of the seven cell lines, with Capan-1 expressing the highest levels (Figure 3). AsPC-1 had very low levels, which could not be quantified. g g p g y g y y g To identify the most appropriate cell lines to knockdown ST3GAL3 and ST3GAL4, we first determined the mRNA expression levels of the α2,3-ST and the fucosyltransferase genes that code for the enzymes that act in the last steps of SLe antigens’ biosynthesis (Figure 3). Regarding α2,3-ST expression, ST3GAL3, ST3GAL4 and ST3GAL6 mRNA levels were analyzed. 1. Introduction α2,3-STs comprise of six enzymes (from ST3Gal I to ST3Gal VI), of which ST3Gal III, ST3Gal IV and ST3Gal VI can form the α2,3-SA linkage in mammals on type 1 (Galβ1,3-GlcNAcβ1-R) or type 2 (Galβ1,4-GlcNAcβ1-R) precursor chains, which after their α1,4 or α1,3-fucosylation, will lead to the sLe antigens sLea or sLex, respectively. Expression of α2,3-sialyltransferases has been found to be deregulated in malignancy in several types of tumors [21], like colorectal, gastric carcinoma [22,23], breast [24–26], ovarian [27], cervix [28] and PDA [10]. Increased mRNA levels of STs are closely related with poor prognosis, reduced overall survival [24,25,29,30] and to cellular resistance to therapies. Previous studies from our group showed that α2,3-STs expression is increased in PDA compared to healthy tissue [10]. Increased expression of type 2 Lewis antigens has been found in PDA tissues when compared with normal pancreas, being sLex neoexpressed in PDA [31,32]. PDA cell lines also exhibit increased expression of the sLe antigens and the sialyltransferases (STs) involved in their biosynthesis [9,10,33]. Furthermore, the overexpression of either ST3GAL3 or ST3GAL4 in PDA cells increased sLex expression in their cell membrane glycoconjugates. The overexpressing clones displayed increased adhesion on E-selectin, increased migration on collagen I, a loss of homotypic cell aggregation and a decrease in the overall mouse survival following intrasplenic injection compared to their corresponding controls, which illustrate an aggressive phenotype [9,10,34,35]. Ectopic expression of genes, such as ST3GAL3/4, leads to an artificial cell phenotype of rather limited (patho)physiological significance. To address the role of the α2,3-STs, ST3GAL3 and ST3GAL4 as potential new therapeutic targets of PDA, we have performed a comprehensive analysis of the effects of their knockdown on PDA cell function. We have focused on key steps of cancer progression, such as cell migration and invasion, as well as E-selectin-dependent binding and cell rolling. To this end, we have first assessed the expression of these STs and the sLe antigens in a panel of seven PDA cell lines. ST3GAL3 and ST3GAL4 were knocked down via shRNA in two pancreatic cancer cell lines, BxPC-3 and Capan-1, whose scramble (SC) control cells display high to moderate levels of sLex. The expression of several sialylated determinants on ST3GAL3/4 knockdown cells was characterized by flow cytometry and Western blot (WB). All silenced cells showed a significant reduction in sLex expression with a concomitant increase in α2,6-sialic acid determinants. 1. Introduction Silenced cells displayed reduced migratory and 3 of 23 Int. J. Mol. Sci. 2020, 21, 6239 invasive potentials as well as impaired E-selectin binding. Our results demonstrate that the inhibition of these α2,3-STs in cancer, specifically in PDA, hampers cancer progression steps, suggesting that these STs represent new therapeutic targets for PDA. invasive potentials as well as impaired E-selectin binding. Our results demonstrate that the inhibition of these α2,3-STs in cancer, specifically in PDA, hampers cancer progression steps, suggesting that these STs represent new therapeutic targets for PDA. 2. Results 2.1. Expression of Sialylated Glycan Determinants, α2,3-Sialyltransferases and α1,3/4-Fucosyltransferases in a Panel of PDA Cells 2.1. Expression of Sialylated Glycan Determinants, α2,3-Sialyltransferases and α1,3/4-Fucosyltransferases in a Panel of PDA Cells To investigate the contribution of the α2,3-sialyltransferases ST3GAL3 and ST3GAL4 to the adhesive and invasive capabilities of PDA cells, we first characterized their expression levels as well as those of sLex, sLea and different sialic acid determinants (α2,3 and/or α2,6) in a panel of seven pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF-II, Panc 10.05 and SW 1990). These cell lines represent varying degrees of pancreatic cancer genetic complexity and different grades of neoplastic differentiation. Cell surface glycan expression was first analyzed by flow cytometry with specific monoclonal antibodies (mAb) against sLex and sLea antigens and lectins: Sambucus nigra Lectin (SNA), which binds preferentially to sialic acid attached to terminal galactose in α2,6-linkage, and Maackia amurensis Lectin II (MAL II) that binds sialic acid in α2,3-linkage. Flow cytometry experiments with anti-sLex mAb showed varying expression levels of sLex and sLea on the cell surface of the different cell lines (Figure 1A,B top panel). Capan-1 and BxPC-3 cells displayed significantly higher sLex levels compared with the rest of the PDA cell lines. On the other hand, BxPC-3 and Capan-2 cells had the highest levels of sLea compared to the rest of the cell lines. Quantitative analyses of the expression of α2,6-sialic acid (SA) determinants using SNA (Figure 1A,B, bottom panel) revealed that Capan-2 and SW 1990 exhibit the highest levels followed by BxPC-3. Analysis of α2,3-SA using MAL II lectin showed that Capan-2 was the cell line with the highest α2,3-SA levels followed by Panc 10.05, BxPC-3 and SW 1990. The expression of sLex and sLea determinants in protein cell lysates and secreted glycoconjugates from conditioned media was analyzed by WB (Figure 2). The results were in line with those obtained for cell membrane glycoconjugates determined by flow cytometry. The highest sLex-expressing cell lines were Capan-1 and BxPC-3, whereas for sLea were Capan-2 and BxPC-3, in both cell lysates and cell conditioned media, with the signal being higher in secreted glycoproteins of the conditioned media. The main differences of sialylated determinants between cell lines were primarily detected in the high molecular weight region, which could correspond to highly glycosylated mucins, among others [18,32]. 2. Results ST3GAL6 levels were much lower than ST3GAL3 and ST3GAL4 ones for all cell lines examined in this work. Among all cell lines tested, only Capan-1 and BxPC-3 expressed ST3GAL6 levels above the background. ST3GAL3 expression was also 4–20-fold lower than ST3GAL4 for all cell lines. The cells with the highest ST3GAL3 expression were AsPC-1, BxPC-3, Capan-2 and SW 1990. All cell lines expressed appreciable ST3GAL4 mRNA levels, being AsPC-1, and Capan-2 the cell lines with the highest levels. The expression of α1,3/4-fucosyltransferases, involved in the synthesis of sLex and sLea in mammalian cell lines (FUTs-3, -5, -6 and -7) [36], showed that these cell lines exhibited very low levels of FUT5, FUT6 and FUT7, except for Capan-1 cells that displayed much higher FUT6 levels than the rest of the cells (data not shown). FUT3 was the main fucosyltransferase expressed, which was found in six (Capan-1, SW 1990, Panc 10.05, Capan-2, BxPC-3 and HPAF-II) out of the seven cell lines, with Capan-1 expressing the highest levels (Figure 3). AsPC-1 had very low levels, which could not be quantified. This comprehensive analysis prompted us to select BxPC-3 and Capan-1 cells, which display high sLex expression and high-to-moderate sLea expression, for knocking down ST3GAL4 and ST3GAL3 in order to assess the effect of these STs on their biosynthesis as well as their influence on other biologically relevant interactions. 4 of 23 Int. J. Mol. Sci. 2020, 21, 6239 Figure 1. Analysis of the cell surface glycan structures in pancreatic ductal adenocarcinoma (PDA) cell lines by flow cytometry. (A): Overlay of the representative cytometry histograms of the different glycan structures of the seven PDA cell lines: sialyl-Lewis x (top left), sialyl-Lewis a (top right), α2,6-sialic acid (bottom left) and α2,3-sialic acid (bottom right). Color legend: Negative control is represented with a continuous dot line: ( . . . ), AsPC-1 (dark blue line), BxPC-3 (green line) Capan-1 (light blue line), Capan-2 (purple line), HPAF-II (red line), Panc 10.05 (pink line) and SW 1990 (orange line). (B): Geomean fluorescence intensity of the different glycan structures of the seven PDA cell lines: sialyl-Lewis x (top left), sialyl-Lewis a (top right), α2,6-sialic acid (bottom left) and α2,3-sialic acid (bottom right). Data represent mean ± SD from three independent experiments, except for Capan-2, HPAF-II, Panc 10.05 and SW 1990, in which two values were used. ANOVA and Tukey’s multiple Figure 1. 2. Results Analysis of the cell surface glycan structures in pancreatic ductal adenocarcinoma (PDA) cell lines by flow cytometry. (A): Overlay of the representative cytometry histograms of the different glycan structures of the seven PDA cell lines: sialyl-Lewis x (top left), sialyl-Lewis a (top right), α2,6-sialic acid (bottom left) and α2,3-sialic acid (bottom right). Color legend: Negative control is represented with a continuous dot line: ( . . . ), AsPC-1 (dark blue line), BxPC-3 (green line) Capan-1 (light blue line), Capan-2 (purple line), HPAF-II (red line), Panc 10.05 (pink line) and SW 1990 (orange line). (B): Geomean fluorescence intensity of the different glycan structures of the seven PDA cell lines: sialyl-Lewis x (top left), sialyl-Lewis a (top right), α2,6-sialic acid (bottom left) and α2,3-sialic acid (bottom right). Data represent mean ± SD from three independent experiments, except for Capan-2, HPAF-II, Panc 10.05 and SW 1990, in which two values were used. ANOVA and Tukey’s multiple comparison post hoc test was performed p < 0 05 * p < 0 01 ** and p < 0 001 *** Figure 1 Analysis of the cell surface glycan structures in pancreatic ductal adenocarcinoma (PDA) cell Figure 1. Analysis of the cell surface glycan structures in pancreatic ductal adenocarcinoma (PDA) cell lines by flow cytometry. (A): Overlay of the representative cytometry histograms of the different glycan structures of the seven PDA cell lines: sialyl-Lewis x (top left), sialyl-Lewis a (top right), α2,6-sialic acid (bottom left) and α2,3-sialic acid (bottom right). Color legend: Negative control is represented with a continuous dot line: ( . . . ), AsPC-1 (dark blue line), BxPC-3 (green line) Capan-1 (light blue line), Capan-2 (purple line), HPAF-II (red line), Panc 10.05 (pink line) and SW 1990 (orange line). (B): Geomean fluorescence intensity of the different glycan structures of the seven PDA cell lines: sialyl-Lewis x (top left), sialyl-Lewis a (top right), α2,6-sialic acid (bottom left) and α2,3-sialic acid (bottom right). Data represent mean ± SD from three independent experiments, except for Capan-2, HPAF-II, Panc 10.05 and SW 1990, in which two values were used. ANOVA and Tukey’s multiple comparison post-hoc test was performed. p < 0.05: *; p < 0.01: ** and p < 0.001:***. 5 of 23 Int. J. Mol. Sci. 2020, 21, 6239 Figure 2. 2.2. Stable Silencing of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells For ST3GAL4 KD cells (Figure 4A), shST3GAL4_1 and shST3GAL4_4 were the ones with the highest knockdown efficiency for Capan-1, showing a decrease of 91 and 87%, respectively. The highest reduction in ST3GAL4 expression in BxPC-3 cells was generated by short hairpins RNAs 1, 4 and 5 with 78%, 88% and 81% decrease, respectively. The five different shRNAs designed to silence ST3GAL3 (Figure 4B) resulted in varying levels of knockdown, all of them greater than 48%. For ST3GAL3 KD cells, shST3GAL3_7 and shST3GAL3_10 showed the highest knockdown efficiencies for BxPC-3 (78% and 77% reduction, respectively) and shST3GAL3_8 and shST3GAL3_10 for Capan-1 (82% and 71%, respectively). To confirm that the knockdown of ST3GAL4 or ST3GAL3 genes did not affect the expression levels of the other ST3GAL genes, we determined the mRNA expression of ST3GAL3, ST3GAL4 and ST3GAL6 genes in all silenced cell lines. As expected, the ST3GAL3 knockdown cells did not show significant changes in ST3GAL4 or ST3GAL6 gene expression. Likewise, the ST3GAL4 knockdown cells did not exhibit significant changes in ST3GAL3 or ST3GAL6 gene expression (data not shown). After one-week puromycin selection, ST3GAL4 and ST3GAL3 mRNA expression levels were determined by quantitative real time PCR (RT-qPCR; Figure 4). No changes in ST3GAL4/3 expression levels were detected between parental cells and scramble (SC) cells, therefore SC cells were used as a negative control for expression changes in subsequent experiments. For ST3GAL4 KD cells (Figure 4A), shST3GAL4_1 and shST3GAL4_4 were the ones with the highest knockdown efficiency for Capan-1, showing a decrease of 91 and 87%, respectively. The highest reduction in ST3GAL4 expression in BxPC-3 cells was generated by short hairpins RNAs 1, 4 and 5 with 78%, 88% and 81% decrease, respectively. The five different shRNAs designed to silence ST3GAL3 (Figure 4B) resulted in varying levels of knockdown, all of them greater than 48%. For ST3GAL3 KD cells, shST3GAL3_7 and shST3GAL3_10 showed the highest knockdown efficiencies for BxPC-3 (78% and 77% reduction, respectively) and shST3GAL3_8 and shST3GAL3_10 for Capan-1 (82% and 71%, respectively). To confirm that the knockdown of ST3GAL4 or ST3GAL3 genes did not affect the expression levels of the other ST3GAL genes, we determined the mRNA expression of ST3GAL3, ST3GAL4 and ST3GAL6 genes in all silenced cell lines. As expected, the ST3GAL3 knockdown cells did not show significant changes in ST3GAL4 or ST3GAL6 gene expression. 2. Results Immunodetection by Western blot of sLex (left) and sLea (right) content in proteins from total cell lysates (top) and conditioned media (bottom) of the PDA cells. Blots were probed with clones CSLEX1 mAb against sialyl-Lewis x and the clone 57/27 mAb against sialyl-Lewis a. Figure 2. Immunodetection by Western blot of sLex (left) and sLea (right) content in proteins from total cell lysates (top) and conditioned media (bottom) of the PDA cells. Blots were probed with clones CSLEX1 mAb against sialyl-Lewis x and the clone 57/27 mAb against sialyl-Lewis a. Figure 3. Relative quantification of α2,3-sialyltransferases and α1,3/1,4-fucosyltransfereases. Quantification of ST3GAL3, ST3GAL4, ST3GAL6 and FUT3 mRNA levels by RT-qPCR in the pancreatic cancer cell panel. RNA levels were normalized using TATA-box binding protein (TBP) as a housekeeping gene. Data represent mean ± SD of triplicates of a biological experiment. ANOVA and Tukey’s multiple comparison post-hoc test was performed. p < 0.05: *; p < 0.01: ** and p < 0.001:***. Figure 3. Relative quantification of α2,3-sialyltransferases and α1,3/1,4-fucosyltransfereases. Quantification of ST3GAL3, ST3GAL4, ST3GAL6 and FUT3 mRNA levels by RT-qPCR in the pancreatic cancer cell panel. RNA levels were normalized using TATA-box binding protein (TBP) as a housekeeping gene. Data represent mean ± SD of triplicates of a biological experiment. ANOVA and Tukey’s multiple comparison post-hoc test was performed. p < 0.05: *; p < 0.01: ** and p < 0.001:***. Int. J. Mol. Sci. 2020, 21, 6239 6 of 23 2.2. Stable Silencing of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells Likewise, the ST3GAL4 knockdown cells did not exhibit significant changes in ST3GAL3 or ST3GAL6 gene expression (data not shown). 2.2. Stable Silencing of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells 2.2. Stable Silencing of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells To investigate the role of ST3GAL4 and ST3GAL3 in PDA malignant progression, we targeted both genes for silencing through shRNA technology in BxPC-3 and Capan-1 cell lines. Knockdown experiments by inserting the pLKO.1-puro vectors containing the shRNAs against the target genes were first attempted with liposome-based transfection reagents and with specific electroporation kits, but the transfection efficiency for these pancreatic cell lines was very low. Therefore, stable knockdown (KD) of ST3GAL4 and ST3GAL3 genes was achieved by lentiviral delivery of the pLKO.1-puro vector containing the shRNAs against the target genes. PDA cells lines (BxPC-3 and Capan-1) were transduced with five different shRNAs for each target sialyltransferase and the respective sequences were designated as sh-1 to sh-5 for shRNAs against ST3GAL4 and as sh-6 to sh-10 for the ST3GAL3 KD cells. Parental cells were simultaneously transduced with a scramble control containing a non-targeting sequence. No changes in cell morphology or proliferation were observed in the stably transduced cells. To investigate the role of ST3GAL4 and ST3GAL3 in PDA malignant progression, we targeted both genes for silencing through shRNA technology in BxPC-3 and Capan-1 cell lines. Knockdown experiments by inserting the pLKO.1-puro vectors containing the shRNAs against the target genes were first attempted with liposome-based transfection reagents and with specific electroporation kits, but the transfection efficiency for these pancreatic cell lines was very low. Therefore, stable knockdown (KD) of ST3GAL4 and ST3GAL3 genes was achieved by lentiviral delivery of the pLKO.1-puro vector containing the shRNAs against the target genes. PDA cells lines (BxPC-3 and Capan-1) were transduced with five different shRNAs for each target sialyltransferase and the respective sequences were designated as sh-1 to sh-5 for shRNAs against ST3GAL4 and as sh-6 to sh-10 for the ST3GAL3 KD cells. Parental cells were simultaneously transduced with a scramble control containing a non-targeting sequence. No changes in cell morphology or proliferation were observed in the stably transduced cells. After one-week puromycin selection, ST3GAL4 and ST3GAL3 mRNA expression levels were determined by quantitative real time PCR (RT-qPCR; Figure 4). No changes in ST3GAL4/3 expression levels were detected between parental cells and scramble (SC) cells, therefore SC cells were used as a negative control for expression changes in subsequent experiments. 2.3. Downregulation of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells Reduces sLex Expression In accord with BxPC-3 cells, a significantly increased signal in α2,6-SA content was additionally detected in shST3GAL4_1 and shST3GAL4_4 Capan-1 (35 and 24% in the median values compared with the SC cells; Figure 5A, Table 2). No significant changes in MAL II detected glycans were found either by flow cytometry or WB. ) g g g y y y y For BxPC-3, shST3GAL3_7 and shST3GAL3_9 cells displayed a 33–37% reduction in sLex relative to SC cells (Figure 5A, Table 1), as also shown by WB of their cell lysates (Figure 5B). ST3GAL3 KD also led to a modest reduction of sLea, in the shST3GAL3_9 cells (Figure 5A, Table 1). Because of the high sLea content of BxPC-3 cells and to overcome the possible saturation in the immunodetection by immunofluorescence, CA19-9 content was also determined by the quantitative immunoassay and showed a 28% reduction in the shST3GAL3_7 and a 78% reduction in the shST3GAL3_9 cells. Regarding α2,6-SA content, flow cytometry results indicated a significant increase up to 42% for shST3GAL3_9 cells (Figure 5A, Table 1). Both ST3GAL3 KD cells showed also a slight increase trend in α2,3-SA content in flow cytometry analyses using MAL II lectin (Figure 5A). This result could be explained in part because MAL II lectin is unable to bind to sLex/sLea structures. For Capan-1, shST3GAL3_7 and shST3GAL3_9 cells also displayed significant reduced sLex levels (61% and 73%, respectively; Figure 5A, Table 2), which was corroborated by WB of protein cell lysates, which revealed that the changes were mainly found in high molecular weight glycoproteins (Figure 5B). Reduction of ST3GAL3 also led to a significant decrease in sLea antigen around 40–50% (Figure 5A, Table 2). Regarding the expression of α2,6-SA, a modest increase trend was detected using SNA (Figure 5A, Table 2). MAL II analyses by flow cytometry did not show differences among clones except for a significant increase (39%) in shST3GAL3_9 cells (Figure 5A), which was also confirmed by WB of the protein cell lysates. Figure 4. Relative quantification of ST3GAL3 and ST3GAL4 mRNA expression in pancreatic cells after shRNA transfection. (A) RT-qPCR validation of the knockdown of ST3GAL4 (shST3GAL4_1-5 cells) in Capan-1 and BxPC-3. Scramble control cells (shScramble) were generated by stable transfection with non-target scramble vector. (B) RT-qPCR validation of the knockdown of ST3GAL3 (shST3GAL3_6-10 cells) in Capan-1 and BxPC-3. RNA levels were normalized using TBP as a housekeeping gene. 2.3. Downregulation of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells Reduces sLex Expression To explore how the reduction in ST3GAL4 and ST3GAL3 expression levels leads to changes in cell sialylated glycans in BxPC-3 and Capan-1 cells, glycan expression pattern was analyzed by flow cytometry and WB in all ten knockdown cells (sh-1 to sh-10), and their respective controls (SC and parental cells). The knockdown cells that displayed major decreases in sLe antigens are described below and were selected for further in vitro analyses. In particular, for BxPC-3, shST3GAL4_1 cells were the ones with the most prominent reduction in sLex membrane expression—up to 68%—when compared to SC median values, as shown by flow cytometry (Figure 5A, Table 1) and WB (Figure 5B). Surprisingly, knockdown of this gene led to a moderate increase of sLea on cell membrane glycoconjugates detected by flow cytometry (Figure 5A, Table 1), even though we expected either the same or reduced sLea levels since ST3GAL4 has been reported to also act on type 1 structures [9]. To confirm these results, CA19-9 levels, which correspond to the sLea antigen, of the total cell lysates were quantified using the mAb 1116-NS-19-9. The levels of CA19-9 in shST3GAL4_1 BxPC-3 cells were indeed increased by 30% relative to the ones of SC BxPC-3 cells. Lectins’ analyses of shST3GAL4_1 BxPC-3 cells also showed an increase of 53% in α2,6-sialic acid using SNA (Figure 5A, Table 1), suggesting a multiple enzymatic competition of α2,3- and α2,6-STs for type 2 chains. This is in agreement with our previous work showing that increased levels of sLex are accompanied by decreased α2,6-sialic acid content of membrane glycoconjugates [9]. MAL II analysis did not reveal any significant differences in the total amount of α2,3-sialic acid content (Figure 5A). Sialic acid determinants of protein lysates from the selected silenced cells were also tested by WB using Int. J. Mol. Sci. 2020, 21, 6239 7 of 23 SNA and MAL II lectins. The changes detected in the specific glycan determinants showed the same tendency as the ones obtained by flow cytometry and were found mainly in high molecular weight glycoproteins (data not shown). For Capan-1, ST3GAL4 KD cells also showed a significant (63–64%) reduction in sLex content accompanied by a decrease of sLea levels (in a 32% and 45%; Figure 5A,B Table 2), suggesting that ST3GAL4 is also involved in sLea biosynthesis in Capan-1 cells as previously described [10]. 2.3. Downregulation of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells Reduces sLex Expression Data are expressed as mean ± SD of three independent experiments * represents p < 0.05; **p < 0.01 (Student’s t test). Figure 4. Relative quantification of ST3GAL3 and ST3GAL4 mRNA expression in pancreatic cells after shRNA transfection. (A) RT-qPCR validation of the knockdown of ST3GAL4 (shST3GAL4_1-5 cells) in Capan-1 and BxPC-3. Scramble control cells (shScramble) were generated by stable transfection with non-target scramble vector. (B) RT-qPCR validation of the knockdown of ST3GAL3 (shST3GAL3_6-10 cells) in Capan-1 and BxPC-3. RNA levels were normalized using TBP as a housekeeping gene. Data are expressed as mean ± SD of three independent experiments * represents p < 0.05; **p < 0.01 (Student’s t test). 8 of 23 Int. J. Mol. Sci. 2020, 21, 6239 Figure 5. Glycan analysis of ST3GAL3 and ST3GAL4 knockdown BxPC-3 and Capan-1 cells. (A) Representative flow cytometry profiles of the cell surface glycans structures detected with sLex and sLea antibodies and the lectins SNA and MAL II in knockdown ST3GAL4 cells (left) and ST3GAL3 cells (right) using different shRNA sequences. Negative controls (without the primary antibodies or lectins) are represented using a continuous black dot line: ( . . . ), shScramble (black line), shST3GAL4_1 (green line), shST3GAL4_4 (orange line), shST3GAL3_7 (pink line) and shST3GAL3_9 (blue line). (B) Immunodetection by WB showing the reduction of sLex antigens in selected cell lysates from total protein on BxPC-3 (top left) and Capan-1 cells (top right) with anti-sLex antibody (clone CSLEX1). Anti-tubulin WB of the corresponding membrane is showed on the bottom panel. Figure 5. Glycan analysis of ST3GAL3 and ST3GAL4 knockdown BxPC-3 and Capan-1 cells. (A) Representative flow cytometry profiles of the cell surface glycans structures detected with sLex and sLea antibodies and the lectins SNA and MAL II in knockdown ST3GAL4 cells (left) and ST3GAL3 cells (right) using different shRNA sequences. Negative controls (without the primary antibodies or lectins) are represented using a continuous black dot line: ( . . . ), shScramble (black line), shST3GAL4_1 (green line), shST3GAL4_4 (orange line), shST3GAL3_7 (pink line) and shST3GAL3_9 (blue line). (B) Immunodetection by WB showing the reduction of sLex antigens in selected cell lysates from total protein on BxPC-3 (top left) and Capan-1 cells (top right) with anti-sLex antibody (clone CSLEX1). Anti-tubulin WB of the corresponding membrane is showed on the bottom panel. Int. J. Mol. Sci. 2020, 21, 6239 9 of 23 Table 1. 2.3. Downregulation of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells Reduces sLex Expression Percentage of decrease (↓) or increase (↑) of sialylated glycan determinants in BxPC-3 KD cells vs. the corresponding control (scramble, SC) cells determined by flow cytometry. Cells % change in sLex Sig.1 Sig.1 % change in sLea Sig.1 % change in α2,6-SA Sig.1 Sig.1 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL3_7 shST3GAL4_1 BxPC-3 ↓68% (***) - ↑30%2 ↑53% (***) - shST3GAL3_7 BxPC-3 ↓33% (***) *** - ↓2% (ns) ↑21% (ns) * - shST3GAL3_9 BxPC-3 ↓37% (***) *** ns ↓34% (*) * ↑41% (**) ns ns 1 ANOVA and Tukey’s multiple comparison post-hoc test was performed. ns: not significant; p < 0.05: *; p < 0.01: ** and p < 0.001:***. 2 Determined by CA19.9 immunoassay. Table 2. Percentage of decrease (↓) or increase (↑) of sialylated glycan determinants in Capan-1 KD cells vs. the corresponding control (scramble) cells determined by flow cytometry. Table 1. Percentage of decrease (↓) or increase (↑) of sialylated glycan determinants in BxPC-3 KD cells vs. the corresponding control (scramble, SC) cells determined by flow cytometry. Cells % change in sLex Sig.1 Sig.1 % change in sLea Sig.1 % change in α2,6-SA Sig.1 Sig.1 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL3_7 shST3GAL4_1 BxPC-3 ↓68% (***) - ↑30%2 ↑53% (***) - shST3GAL3_7 BxPC-3 ↓33% (***) *** - ↓2% (ns) ↑21% (ns) * - shST3GAL3_9 BxPC-3 ↓37% (***) *** ns ↓34% (*) * ↑41% (**) ns ns 1 ANOVA and Tukey’s multiple comparison post-hoc test was performed. ns: not significant; p < 0.05: *; p < 0.01: ** and p < 0.001:***. 2 Determined by CA19.9 immunoassay. Table 2. Percentage of decrease (↓) or increase (↑) of sialylated glycan determinants in Capan-1 KD cells vs. the corresponding control (scramble) cells determined by flow cytometry. Cells % change in sLex Sig.1 Sig.1 Sig.1 % change in sLea Sig.1 Sig.1 Sig.1 % change in α2,6-SA Sig.1 Sig.1 Sig.1 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL4_4 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL4_4 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL4_4 vs. 2.3. Downregulation of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells Reduces sLex Expression shST3GAL3_7 shST3GAL4_1 Capan-1 ↓64% (***) - ↓32% (*) - ↑35% (**) - shST3GAL4_4 Capan-1 ↓63% (***) ns - ↓45 (**) ns - ↑24% (*) ns - shST3GAL3_7 Capan-1 ↓61% (***) ns ns - ↓43% (**) ns ns - ↑20% (ns) ns ns - shST3GAL3_9 Capan 1 ↓73% (***) ns ns ns ↓52% (***) ns ns ns ↑12% (ns) * ns ns Table 1. Percentage of decrease (↓) or increase (↑) of sialylated glycan determinants in BxPC-3 KD cells vs. the corresponding control (scramble, SC) cells determined by flow cytometry. increase (↑) of sialylated glycan determinants in Capan-1 KD cells vs. the corresponding control (scramble) ted glycan determinants in Capan-1 KD cells vs. the corresponding control (scramble) cells determined by g ( ) ( ) y g y p p g ( ) y flow cytometry. Cells % change in sLex Sig.1 Sig.1 Sig.1 % change in sLea Sig.1 Sig.1 Sig.1 % change in α2,6-SA Sig.1 Sig.1 Sig.1 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL4_4 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL4_4 vs. shST3GAL3_7 vs. SC cells 1 vs. shST3GAL4_1 vs. shST3GAL4_4 vs. shST3GAL3_7 shST3GAL4_1 Capan-1 ↓64% (***) - ↓32% (*) - ↑35% (**) - shST3GAL4_4 Capan-1 ↓63% (***) ns - ↓45 (**) ns - ↑24% (*) ns - shST3GAL3_7 Capan-1 ↓61% (***) ns ns - ↓43% (**) ns ns - ↑20% (ns) ns ns - shST3GAL3_9 Capan-1 ↓73% (***) ns ns ns ↓52% (***) ns ns ns ↑12% (ns) * ns ns 1 ANOVA and Tukey’s multiple comparison post-hoc test was performed. ns: not significant; p < 0.05: *; p < 0.01: ** and p < 0.001:***. Int. J. Mol. Sci. 2020, 21, 6239 10 of 23 Overall, the knockdown of ST3GAL4 and ST3GAL3 significantly decreased sLex levels to a similar degree for all Capan-1 knockdown cells, whereas in the case of BxPC-3, the reduction of sLex expression was more pronounced for the ST3GAL4 KD cells (Table 1; Table 2). ST3Gal III enzyme is mostly related with the addition of α2,3-sialic acid upon terminal galactose on type 1 chains (sLea precursor), while ST3Gal IV plays the major role in the synthesis of sialylated type 2 chains (sLex precursor). However, our results show the important role of ST3GAL3 in the sLex biosynthesis, too. The decrease in sLex was more pronounced in Capan-1 ST3GAL3 knockdown cells than in the corresponding BxPC-3 ones. ST3GAL4 and ST3GAL3 Knockdown in BxPC-3 and Capan-1 Impaired Pancreatic Cancer Cell Migration Sialyltransferases have been described to play an important role in mediating migration and dissemination events in diverse cancer cells. In addition, sLe antigens overexpressed on cell membrane glycoconjugates enhance and modulate a wide variety of pathologically relevant processes in cancer, including migration [9,10]. To determine whether the reduction of those sialylated antigens had a phenotypic effect on PDA cells, we have characterized the role in cell adhesion and migration events of the ST3GAL4 and ST3GAL3 KD cells with reduced levels of sLex on cell surface glycoconjugates. g y j g ST3GAL4 KD cells (shST3GAL4_1 for BxPC-3 and shST3GAL4_1 and shST3GAL4_4 for Capan-1), and ST3GAL3 KD cells (shST3GAL3_7 and shST3GAL3_9, for both BxPC-3 and Capan-1) were allowed to migrate in modified Boyden chambers using FBS as a chemoattractant. Downregulation of α2,3-STs significantly suppressed cancer cell migration in PDA cells in comparison with SC cells for both cell lines, in decrease percentages that ranged from 41% to 57% for BxPC-3 and from 42% to 51% for Capan-1 cells (Figure 6). Figure 6. SLex reduction resulted in impaired cell migration. Relative quantification of cell migration over shScramble cells of BxPC-3 knockdown cells (shST3GAL4_1, shST3GAL3_7 and shST3GAL3_9) (left) and Capan-1 cells (shST3GAL4_1, shST3GAL4_4, shST3GAL3_7 and shST3GAL3_9) (right). Cells were allowed to migrate through 8 µm-pores-type 1-collagen coated Boyden chambers. Migrated cells were fixed and counted. Results are presented as mean ± SEM of three independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed. * p < 0.05, ** p <0.01. Figure 6. SLex reduction resulted in impaired cell migration. Relative quantification of cell migration Figure 6. SLex reduction resulted in impaired cell migration. Relative quantification of cell migration over shScramble cells of BxPC-3 knockdown cells (shST3GAL4_1, shST3GAL3_7 and shST3GAL3_9) (left) and Capan-1 cells (shST3GAL4_1, shST3GAL4_4, shST3GAL3_7 and shST3GAL3_9) (right). Cells were allowed to migrate through 8 µm-pores-type 1-collagen coated Boyden chambers. Migrated cells were fixed and counted. Results are presented as mean ± SEM of three independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed. * p < 0.05, ** p <0.01. Figure 6. SLex reduction resulted in impaired cell migration. Relative quantification of cell migration over shScramble cells of BxPC-3 knockdown cells (shST3GAL4_1, shST3GAL3_7 and shST3GAL3_9) (left) and Capan-1 cells (shST3GAL4_1, shST3GAL4_4, shST3GAL3_7 and shST3GAL3_9) (right). Cells were allowed to migrate through 8 µm-pores-type 1-collagen coated Boyden chambers. Migrated cells were fixed and counted. 2.3. Downregulation of ST3GAL4 and ST3GAL3 in BxPC-3 and Capan-1 Cells Reduces sLex Expression This difference between cell lines could be explained by the lower expression levels of this gene in Capan-1 compared to BxPC-3. p p A significant reduction of sLea was observed in most of the ST3GAL3 knockdown cells, being also more pronounced for Capan-1 cells (Tables 1 and 2). ST3GAL4 knockdown also resulted in a decrease of sLea but only in the Capan-1 cells. BxPC-3, exhibiting very high levels of sLea, did not show a decrease in their corresponding ST3GAL4 knockdown cells, but an unexpected, albeit moderate increase. The reduction in the α2,3-sialylated Lewis antigens was compensated by an increase of α2,6-sialylated structures, which can be explained by the competition of the α2,6-STs for the same substrates that the α2,3-STs. Next, we evaluated the behavior of these knockdown cells, which showed the greatest reduction in sLex antigens, in cell adhesion and migration in vitro. ST3GAL4 and ST3GAL3 Knockdown in BxPC-3 and Capan-1 Impaired Pancreatic Cancer Cell Migration Results are presented as mean ± SEM of three independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed. * p < 0.05, ** p <0.01. Int. J. Mol. Sci. 2020, 21, 6239 11 of 23 To assess whether sLex reduction of the ST knockdown cells correlates with decreased cell migration, SC cells were incubated with the anti-sLex mAb CSLEX1 prior to their seeding. Both treated BxPC-3 and Capan-1 SC cells showed a reduction in migration, up to 59% in BxPC-3, and up to 71% in Capan-1 (data not shown). In SC Capan-1 cells treated with anti-sLeX antibody, the decrease in migration was notably higher compared to their corresponding silenced clones. The marked decrease in cell migration in the α2,3-ST knockdown cells as well as in in the SC cells treated with anti-sLex antibody indicates that sLex plays a major part in this process. The role of α2,3-sialyltransferases knockdown in cell migration was also assessed inside microchannels of different dimensions. In vivo, disseminated tumor cells migrate within 3D extracellular matrix (ECM) and through longitudinal tracks of prescribed dimensions ranging from 3 to 30 µm in width created by various anatomical structures [37–39]. By using a microfluidic device consisting of an array of parallel microchannels of different widths (W = 6–50 µm) and prescribed height (H = 10 µm) and length (L = 200 µm) coated with collagen type I [40–42], we characterized the migratory potential of SC and KD BxPC-3 and Capan-1 cells. shST3GAL4_1 BxPC-3 cells, which display the highest reduction in sLex, also showed markedly suppressed migration in all channels (Figure 7). These results demonstrate that ST3GAL4 knockdown reduces BxPC-3 cell motility in a microfluidic system, which mimics aspects of the in vivo microenvironment. Figure 7. ST3GAL4 knockdown in BxPC-3 cells impaired cell migration inside microchannels. (A) Representative time lapse images depicting migration through 10 µm-wide confined microchannels at 10-min intervals of BxPC-3 shScramble cells (top row) and shST3GAL4_1 BxPC-3 cells (bottom row). Arrowheads indicate the leading edge of a migrating cell. (B) Illustrative picture showing differences in cell persistence between BxPC-3 shScramble cells (top row) and shST3GAL4_1 BxPC-3 cells (bottom row) migrating inside 50 µm-wide unconfined microchannels in 20-min time intervals. ST3GAL4 and ST3GAL3 Knockdown in BxPC-3 and Capan-1 Impaired Pancreatic Cancer Cell Migration 2020, 21, 6239 12 of 23 12 of 23 2.5. ST3GAL4 and ST3GAL3 Knockdown Reduced Cell Invasion In Vitro 2.5. ST3GAL4 and ST3GAL3 Knockdown Reduced Cell Invasion In Vitro To determine whether the decrease in ST3GAL4 and ST3GAL3 expression and their concomitant reduction in sLex also impaired the invasion of pancreatic cancer cells, transwell invasion assays were performed by coating chambers with Matrigel. ST3GAL4 was more effective than ST3GAL3 knockdown (49% for shST3GAL4_1 versus 30% for shST3GAL3_7) in reducing the invasive potential of BxPC-3 knockdown cells relative to SC cells (Figure 8). This difference between ST3GAL4 and ST3GAL3 depleted BxPC-3 cells could be attributed to the significantly higher reduction of sLex in the former ones (68% vs. 33–37%; Table 1). Along these lines, ST3GAL4 or ST3GAL3 knockdown diminished Capan-1 cell invasion to a roughly similar level (Figure 8), which is in line with their equivalent reduction in sLex expression. Figure 8. SLex reduction impaired cell Matrigel invasion in vitro. Tumor cell invasion was evaluated using Boyden chambers in 24-well plates with Matrigel coated inserts. Invading capacity was assessed by quantifying the number of cells that invade in BxPC-3 knockdown (left) and Capan-1 knockdown cells (right) relative to scramble control cells. Data represent the mean ± SEM from at least 3 independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed. * p < 0.05, ** p <0.01. Figure 8. SLex reduction impaired cell Matrigel invasion in vitro. Tumor cell invasion was evaluated Figure 8. SLex reduction impaired cell Matrigel invasion in vitro. Tumor cell invasion was evaluated using Boyden chambers in 24-well plates with Matrigel coated inserts. Invading capacity was assessed by quantifying the number of cells that invade in BxPC-3 knockdown (left) and Capan-1 knockdown cells (right) relative to scramble control cells. Data represent the mean ± SEM from at least 3 independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed. * p < 0.05, ** p <0.01. To validate the role of sialylation in cell invasion in vitro, we used a blocking antibody against sLex antigens. Treatment of SC Capan-1 and BxPC-3 cells with anti sLex antibody impaired invasion by 39% and 55%, respectively (data not shown). Altogether, these results indicate a positive link between the expression of sLex expression and the invasive capability of PDA cells. To validate the role of sialylation in cell invasion in vitro, we used a blocking antibody against sLex antigens. ST3GAL4 and ST3GAL3 Knockdown in BxPC-3 and Capan-1 Impaired Pancreatic Cancer Cell Migration (C) Migration velocity (left panel), speed (middle panel) and persistence (right panel) of scramble control and shST3GAL4_1 BxPC-3 cells in PDMS-based microchannels of 10 µm in height, 200 µm in length, and either 6, 10, 20 or 50 µm in width. Data represent the mean ± SEM from at least 3 independent experiments. Dunnett’s multiple comparisons test was used to compare each of the shRNAs to scramble in migration microdevices * p < 0.05. Figure 7. ST3GAL4 knockdown in BxPC-3 cells impaired cell migration inside microchannels. Figure 7. ST3GAL4 knockdown in BxPC-3 cells impaired cell migration inside microchannels. (A) Representative time lapse images depicting migration through 10 µm-wide confined microchannels at 10-min intervals of BxPC-3 shScramble cells (top row) and shST3GAL4_1 BxPC-3 cells (bottom row). Arrowheads indicate the leading edge of a migrating cell. (B) Illustrative picture showing differences in cell persistence between BxPC-3 shScramble cells (top row) and shST3GAL4_1 BxPC-3 cells (bottom row) migrating inside 50 µm-wide unconfined microchannels in 20-min time intervals. (C) Migration velocity (left panel), speed (middle panel) and persistence (right panel) of scramble control and shST3GAL4_1 BxPC-3 cells in PDMS-based microchannels of 10 µm in height, 200 µm in length, and either 6, 10, 20 or 50 µm in width. Data represent the mean ± SEM from at least 3 independent experiments. Dunnett’s multiple comparisons test was used to compare each of the shRNAs to scramble in migration microdevices * p < 0.05. Figure 7. ST3GAL4 knockdown in BxPC-3 cells impaired cell migration inside microchannels. (A) Representative time lapse images depicting migration through 10 µm-wide confined microchannels at 10-min intervals of BxPC-3 shScramble cells (top row) and shST3GAL4_1 BxPC-3 cells (bottom row). Arrowheads indicate the leading edge of a migrating cell. (B) Illustrative picture showing differences in cell persistence between BxPC-3 shScramble cells (top row) and shST3GAL4_1 BxPC-3 cells (bottom row) migrating inside 50 µm-wide unconfined microchannels in 20-min time intervals. (C) Migration velocity (left panel), speed (middle panel) and persistence (right panel) of scramble control and shST3GAL4_1 BxPC-3 cells in PDMS-based microchannels of 10 µm in height, 200 µm in length, and either 6, 10, 20 or 50 µm in width. Data represent the mean ± SEM from at least 3 independent experiments. Dunnett’s multiple comparisons test was used to compare each of the shRNAs to scramble in migration microdevices * p < 0.05. Int. J. Mol. Sci. 2.5. ST3GAL4 and ST3GAL3 Knockdown Reduced Cell Invasion In Vitro Treatment of SC Capan-1 and BxPC-3 cells with anti sLex antibody impaired invasion by 39% and 55%, respectively (data not shown). Altogether, these results indicate a positive link between the expression of sLex expression and the invasive capability of PDA cells. 2.6. Reduced Levels of sLex in ST3GAL4 and ST3GAL3 Knockdown Cells Led to Decreased Binding to E-Selectin .6. Reduced Levels of sLex in ST3GAL4 and ST3GAL3 Knockdown Cells Led to Decreased Binding to E-Selectin E-selectin expressed on activated endothelial cells binds to sialylated ligands on the surface of circulating tumor cells slowing them down, which is a key step prior to their extravasation from the circulatory system. We have investigated the impact of ST3GAL4 and ST3GAL3 KD on E-selectin-dependent binding and rolling using BxPC-3 and Capan-1 cells as models. As a first step, we measured cell binding to recombinant human E-selectin (rh-E-selectin) under static (no flow) conditions (Figure 9A). All KD cells displayed significantly decreased adhesion to rh-E-selectin compared to their respective BxPC-3 and Capan-1 SC cells, which is attributed to the reduction of sLe antigens’ levels (Tables 1 and 2). In BxPC-3 cells, the higher decrease of ST3GAL4 than ST3GAL3 KD cell binding to E-selectin (84% reduction for shST3GAL4_1 vs. 31% and 64% reduction for shST3GAL3_7 and shST3GAL3_9, respectively) was due to the higher reduction in sLex levels compared to their respective SC cells. In Capan-1 cells, shST3GAL4_1, shST3GAL4_4 and shST3GAL3_9 exhibited similarly reduced levels in E-selectin adhesion and sLex expression. shST3GAL3_7 was an outlier 13 of 23 Int. J. Mol. Sci. 2020, 21, 6239 since the less efficient reduction of E-selectin-dependent binding did not correlate with the extent of decrease in sLex expression. Nevertheless, the importance of sialylated antigens in E-selectin binding was further demonstrated upon incubation of SC BxPC-3 and Capan-1 cells with an anti-sLex mAb, which resulted in >80% inhibition. Taken together, these findings reveal that sLex is critical to the binding of PDA cells to human E-selectin in vitro. Figure 9. ST3GAL3 and ST3GAL4 knockdown inhibited cell adhesion to rh-E-selectin under static conditions and also impaired cell binding to rh-E-selectin under flow. (A) BxPC-3 (left) and Capan-1 cells (right) were incubated over E-selectin coated 96-well plates and allowed to adhere at 37 ◦C. Adherent cells were quantified after 1 h incubation with MTS-based colorimetric assay. Experiments were performed in triplicate. Results are presented as mean ± SEM of adherent cells respect shScramble cells of three independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed * p< 0.05, ** p <0.01. (B) Reduction in BxPC-3 (left) and Capan-1 (right) knockdown cells binding to rh-E-selectin in comparison to SC control cells flowing over immobilized E-selectin. Graphs depict means and SD from at least three independent experiments. Figure 9. ST3GAL3 and ST3GAL4 knockdown inhibited cell adhesion to rh-E-selectin under static Figure 9. 3. Discussion Aberrant glycosylation of glycoconjugates on the tumor cell surface facilitates distinct steps of the metastatic cascade of events [4–6,20]. In this work, we have addressed the impact of the downregulation of the α2,3-sialyltransferases ST3GAL3 and ST3GAL4 (which regulate sLex and sLea biosynthesis) on migration, invasion and E-selectin-dependent adhesion of pancreatic cancer cells. 3.1. Diversity in Sialyl-Lewis Antigens’ and their Corresponding Glycogenes’ Expression in PDA Cells The expression levels of the α2,3-sialyltransferases and α1,3/1,4-fucosytransferases involved in the biosynthesis of sLex and sLea in mammals as well as the levels of these sialylayted antigens on cell surface, in cell lysates and conditioned media were evaluated in seven pancreatic cancer cell lines of different genetic complexity that cover the wide tumor heterogeneity that can be found in PDA. In all PDA cells, ST3GAL4 was more abundant than ST3GAL3 (from 4 to 20-fold mRNA increase depending on the cell line). These results were qualitatively in accord with the higher expression levels of ST3GAL4 compared to ST3GAL3 described in pancreatic adenocarcinoma tissues, where ST3GAL4 and ST3GAL3 levels were higher than the mean of pancreatic control tissue by about 4-fold and 2-fold, respectively [10]. Similar results on higher ST3GAL4 relative to ST3GAL3 expression have also been reported in gastric carcinoma [44]. ST3GAL6 was present at near background levels, and could only be detected in Capan-1 and BxPC-3 cells albeit at much lower levels than for ST3GAL3, which is in agreement with our previous data [33]. The expression levels of the α1,3/α1,4-fucosyltransferases (FUT-3, 5, 6, 7 and 9) that catalyze the transference of fucose residues to the N-acetylglucosamine (GlcNAc) on the α2,3-sialylated type 1 and type 2 precursors were also analyzed. We found that FUT3 was the only α1,3/α1,4-fucosyltransferase, which presented noticeable expression levels in most of the cell lines, being Capan-1 the one with highest level, in agreement with recent published data, which found FUT3 between the top-upregulated genes in the aggressive pancreatic cancer cell line Capan-1 [45]. The levels of sLex and sLea epitopes on the glycoconjugates of the seven PDA cells were variable among them as expected by their different α2,3-sialyltransferases’ and α1,3/1,4-fucosytransferases’ expression. The differences in sLex levels between cell lines could be explained by the combination of the several expressed α1,3/α1,4-FUTs and α2,3-STs on the available type 2 precursor chains. .6. Reduced Levels of sLex in ST3GAL4 and ST3GAL3 Knockdown Cells Led to Decreased Binding to E-Selectin ST3GAL3 and ST3GAL4 knockdown inhibited cell adhesion to rh-E-selectin under static conditions and also impaired cell binding to rh-E-selectin under flow. (A) BxPC-3 (left) and Capan-1 cells (right) were incubated over E-selectin coated 96-well plates and allowed to adhere at 37 ◦C. Adherent cells were quantified after 1 h incubation with MTS-based colorimetric assay. Experiments were performed in triplicate. Results are presented as mean ± SEM of adherent cells respect shScramble cells of three independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed * p< 0.05, ** p <0.01. (B) Reduction in BxPC-3 (left) and Capan-1 (right) knockdown cells binding to rh-E-selectin in comparison to SC control cells flowing over immobilized E-selectin. Graphs depict means and SD from at least three independent experiments. Figure 9. ST3GAL3 and ST3GAL4 knockdown inhibited cell adhesion to rh-E-selectin under static conditions and also impaired cell binding to rh-E-selectin under flow. (A) BxPC-3 (left) and Capan-1 cells (right) were incubated over E-selectin coated 96-well plates and allowed to adhere at 37 ◦C. Adherent cells were quantified after 1 h incubation with MTS-based colorimetric assay. Experiments were performed in triplicate. Results are presented as mean ± SEM of adherent cells respect shScramble cells of three independent experiments. ANOVA and Tukey’s multiple comparison post-hoc test was performed * p< 0.05, ** p <0.01. (B) Reduction in BxPC-3 (left) and Capan-1 (right) knockdown cells binding to rh-E-selectin in comparison to SC control cells flowing over immobilized E-selectin. Graphs depict means and SD from at least three independent experiments. To further investigate the functional importance of ST3GAL4 and ST3GAL3 in the binding of pancreatic tumor cells to E-selectin, we also analyzed this adhesive interaction under dynamic flow conditions using a microfluidic system [43]. Using this system, we perfused SC and KD BxPC-3 and Capan-1 cells under a shear stress level of 1.1 dyn/cm2 over immobilized E-selectin (Figure 9B). SC cells interacted more efficiently than KD cells for both cell lines. In line with static assays, ST3GAL4 knockdown BxPC-3 cells showed the highest inhibition to tethering and rolling on E-selectin. Along these lines, ST3GAL4 and ST3GAL3 knockdown cells displayed reduced binding to E-selectin even though moderate differences were noted among different KD Capan-1 cell lines. Int. J. Mol. Sci. 2020, 21, 6239 14 of 23 14 of 23 3. Discussion AsPC-1 and HPAF-II showed almost undetectable levels of sLex and sLea antigens probably due to the very low levels of α1,3/α1,4-FUT, FUT3, whereas on the other hand Capan-1 that showed the highest sLex levels was the one with the highest levels of FUT3 and FUT6. SNA and MAL II analysis also showed different expression levels of α2,3 and α2,6-sialic acid determinants’ expression among these seven PDA cell lines. The wide diversity in glycan expression of these different PDA cell lines is a reflection of their different phenotype and metastatic potential in agreement with the results obtained from the analyses of N-glycan determinants of several PDA cells [46]. y g y From the seven PDA cell lines, Capan-1 and BxPC-3 were the ones chosen to knockdown α2,3-STs (ST3GAL4 and ST3GAL3) because they presented the highest levels of sLex on their cell surface, in total protein lysates and conditioned media, and also showed high to moderate levels of sLea. 3.2. ST3GAL4 and ST3GAL3 Knockdown Effects on sLex/sLea Cell Levels A significant reduction of sLex was shown in both ST3GAL4 and ST3GAL3 KD cells, in similar percentages for Capan-1 cells while for BxPC-3 cells, the decrease in sLex, was significantly higher for the ST3GAL4 KD cells than for ST3GAL3 ones, reinforcing the role of ST3GAL4 in sLex biosynthesis as previously described [47]. However, ST3GAL3, which encodes the main enzyme involved in the synthesis of α2,3-sialylated type 1 structures that lead to sLea biosynthesis, has also a key role in the sLex biosynthesis in both PDA cell models. These results are also in agreement with other studies on pancreatic, gastric and breast cancer cells where the overexpression of ST3GAL3 led also to an increase of sLex antigen [9,26,48]. Int. J. Mol. Sci. 2020, 21, 6239 15 of 23 Regarding the changes in sLea levels, both ST3GAL4 and ST3GAL3 KD Capan-1 cells showed a decrease in sLea expression. For BxPC-3 cells, the decrease in sLea was only detected in one of the ST3GAL3 KD cells (shST3GAL3_9), which displayed higher decrease in their E-selectin dependent adhesion than the shST3GAL3_7 cells that did not show a reduction in sLea. ST3GAL3 codifies for the enzyme that preferentially acts on the type 1 structures over type 2 structures, while ST3GAL4 is more active on type 2 and type 3 structures than on type 1 structures [49], which explains the decrease in sLea in the ST3GAL3 silenced cells. Reduction of the α2,3 sialylated-Lewis antigens in the knockdown cells also led to an increase in α2,6-sialic acid, which can be explained through enzymatic competition of α2,3 and α2,6-STs for type 1 and 2 chains, determined by SNA, in agreement with our previous work [9]. 3.3. ST3GAL4 and ST3GAL3 Silencing Effects on Migration and Invasion Capabilities of the Tumor Cells KD of either ST3GAL4 or ST3GAL3 suppressed the migratory (42–57%) and invasive propensities (33–67%) in both BxPC-3 and Capan-1. The inhibition of motility and invasion was due to decreased expression of sLex antigen as substantiated by the use of an antibody against sLex. These results are in agreement with previous studies showing that the increase of sLex via ST3GAL4 or ST3GAL3 overexpression in different carcinomas such as pancreas, gastric or breast leads to an increased invasive phenotype [9,10,26,48]. ST3GAL4 KD have also been reported to decrease the ability of cancer cells to adhere to selectins [47] and to invade and migrate in vitro in gastric cancer [50]. 3.2. ST3GAL4 and ST3GAL3 Knockdown Effects on sLex/sLea Cell Levels Recently, KD of FUT3 in Capan-1 cells, which is involved in the linking of terminal fucose monosaccharides in α1,3 and α1,4-linkage in the latest steps of sLex and sLea respectively, resulted in a high reduction in colony formation and migration as assessed by wound closure assays relative to SC cells [45]. The potential mechanism, underlying the increased invasive phenotype in overexpressed ST3GAL4 in gastric cancer cells, which is accompanied by a concomitant increase in sLex, could involve the activation of tyrosine kinase receptors such as c-Met and its associated downstream signaling effectors [7]. This activation has been postulated to be initiated by secreted and membrane glycoproteins carrying sLex. In this regard, we have also shown that an increase in sLex in pancreatic cancer cells overexpressing ST3GAL3 alters the glycosylation pattern and modulates the function of important cell membrane glycoproteins, such as α2β1 integrin and E-cadherin, involved in tumor cell adhesion and invasion mechanisms [34]. Altogether, these studies provide evidence for the importance of the sialic acid determinants, in particular of sLex in the tumor cell progression steps. 3.4. ST3GAL4 and ST3GAL3 Silencing Effects on E-selectin Binding of Tumor Cells .4. ST3GAL4 and ST3GAL3 Silencing Effects on E-selectin Binding of Tumor Cells Metastasis is a multistep process, which is initiated by the dissemination of cancerous cells from a primary tumor, and involves migration until the development of secondary tumors in a distant organ. In between, cancerous cells have to extravasate the endothelium in the blood vessels, being sialyl-Lewis antigens crucial ligands involved in the initial steps of rolling and arresting of the tumor cells on the activated endothelial cells from blood vessels [21]. In this work, we demonstrated significant impaired binding to human recombinant E-selectin of the ST3GAL4 and ST3GAL3 KD BxPC-3 and Capan-1 cells. In general, higher reduction in E-selectin binding was found in the ST3GAL4 KD cells compared to ST3GAL3 silenced cells in both cell models using microplates and microfluidic assays. These results suggest the key involvement of sialylated structures, and in particular of sLex, in the adhesion to E-selectin, involved in the initial steps of pancreatic cancer cell arrestment on endothelial cells. In agreement with this, the overexpression of FUT1, which competes for the same substrate that α2,3-STs, in BxPC-3 pancreatic cancer cells resulted in a decrease in sLex levels, which was associated with a reduction in E-selectin-expressing CHO cells binding and an impaired metastatic potential into xenograft transplantation [51,52]. This mechanism is also shared with other neoplasms like in human lung carcinoma cells, in which the up-regulation of FUT3 by TNF-α resulted in an increase of sLex expression, which mediated enhanced invasion and E-selectin binding [53]. Studies with metastatic prostate cancer cells also showed an increase in Int. J. Mol. Sci. 2020, 21, 6239 16 of 23 the adhesion to E-selectin and in migration and invasion after cell treatment with TNF-α, which led to an increase of sLex [54]. A further cell treatment with an antibody against sLex compromised the prostate cell migration and invasion with similar results to those obtained in our cell models. p g The importance of targeting the sialyltransferases involved in the late steps of sLex/sLea biosynthesis has also been shown in other carcinomas such as in lung cancer. In this regard, Yoshihama et al. [8] also reported that the KD of ST3GAL4 in lung metastatic H1299 cells inhibit sLex expression reducing cell adhesion to HUVEC cells, which suggests its potential as a target for cell metastasis. 3.4. ST3GAL4 and ST3GAL3 Silencing Effects on E-selectin Binding of Tumor Cells In ovarian cancer, ST3GAL3 KD sensitized ovarian cancer cells to cisplatin and paclitaxel induced apoptosis [55,56]. Intriguingly, the induction of epithelial-mesenchymal transition (EMT) in colon cancer led to the upregulation of FUT3, ST3GAL3 and ST3GAL4, which are implicated in the final steps of the biosynthesis of sLex/a, suggesting a significant link between those Lewis antigens and EMT in colon carcinoma [57]. Further investigations are necessary to increase our understanding about the influence of sialytransferase KD in EMT process. We have herein shown that the KD of ST3GAL4 and ST3GAL3 confers a partial reduction but not a complete abrogation of the sialyl-Lewis antigens (sLex/sLea), which is in line with another report using HL-60 leukocytic cells [47]. However, the effects of such reduction were enough to downregulate in vitro some of the malignant properties of PDA cells. Blockade of sialic acid using a cell permeable sialyltransferase inhibitor has shown to reduce significantly the synthesis of sialoglycans both α2,3 and α2,6, and it has diminished the adhesive and migratory capability of melanoma cells [58] and suppressed tumor growth by enhancing T-cell mediated tumor immunity [59]. Another sialyltransferase inhibitor, Soyasaponin I (SsaI), that targets α2,3-STs, in particular ST3Gal I, inhibited tumor cell migration and dissemination in the in vivo mouse model with transplanted ovarian cancer cells [27]. Furthermore, this inhibitor targeted ST3Gal IV in breast cancer cells and modified their invasive behavior. Overall, we have shown that STs, and in particular ST3GAL4 and ST3GAL3 that lead to the sLex/a biosynthesis can be considered as potential therapeutic targets against pancreatic cancer. This opens a new window to the investigation of compounds that inhibit the corresponding enzymes to block sLea and neo-expressed sLex in pancreatic tumor cells to avoid or reduce their metastasis to other organs. The main limitation of this work is the absence of models other than cells. Experiments with animal model systems are needed to corroborate the results described using the human PDA cell lines in order to improve the impact of these studies. 4.1. Cell Lines Human pancreatic cancer cell lines Capan-1, Capan-2, HPAF-II, Panc 10.05 and SW 1990 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). AsPC-1 and BxPC-3 were obtained from the cancer cell repository at Hospital del Mar Medical Research Institute (IMIM) Barcelona, Spain. Cells were cultured at 37 ◦C in a humidified atmosphere of 5% CO2. HPAF-II were cultured in EMEM (Lonza, Walkersville, MD, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Life Technologies Corporation, Grand Island NY, USA), 100 U/mL penicillin, 100 U/mL streptomycin and 2 mM L-glutamine (Gibco). Panc 10.05 cells were grown in RPMI-1640 (Lonza) with FBS to a final concentration of 15% and 10 U/mL of human recombinant insulin. AsPC-1, BxPC-3, Capan-1, Capan-2 and SW 1990 Cells were routinely grown in DMEM (Gibco) supplemented with 10% FBS (20% FBS for Capan-1 cells). Routine tests were performed to confirm the absence of mycoplasma. 4.2. Conditioned Media, Protein Lysates and Western Blot Analysis To obtain conditioned media, human pancreatic cancer cells, AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF-II, Panc 10.05 and SW 1990 were cultured in 125 cm2 flasks. Once cells reached 80% confluency, they were thoroughly washed and left in serum-free media. After 48 h, media was collected and Int. J. Mol. Sci. 2020, 21, 6239 17 of 23 filtered with a 25 mm diameter sterile syringe filter of polyethersulfone membrane of 0.22 µm pore size (Pall, Ann Arbor, MI, USA). Filtered conditioned media was concentrated to a final volume of 300–400 µL by centrifugation with Amicon® Ultra-15 centrifugal filters of 10 K, previously passivated with 5% Brij-35 overnight. Total protein concentration was determined by QuickStartTM Bradford protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) standard (Bio-Rad). p y g Total protein extracts were obtained mechanically, lysing the cells after 10 min incubation with cold RIPA buffer (50 mM Tris-HCl pH 7.4, 0.1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, 1 mM PMSF, 0.2 mM sodium orthovanadate and complete ULTRA tablets (protease inhibitors cocktail tablets, Roche, Mannheim, Germany)). Cell lysates were centrifuged and supernatants were collected for their subsequent quantification by Bradford assay as described above. Western blot (WB) analysis was performed as previously described [60]. Briefly, 50 µg of total protein were loaded into polyacrylamide gels under reducing conditions. The antibodies and solutions used for the immunoblotting are listed below. Blocking buffers: 2% polyvinylpyrrolidone (PVP) in TBST for blocking lectin WB, 3% BSA in TBST for Lewis antigens and 5% powder low-fat milk for tubulin detection. Primary antibodies or lectins: (a) Anti-sialyl-Lewis x (clone CSLEX1, BD Biosciences, San Jose, CA, USA); (b) anti-α-Tubulin (B-7, Santa Cruz Biotechnology, Dallas, TX, USA); (c) Anti-sialyl-Lewis a, (clone 57/27); (d) Biotinylated Sambucus Nigra Lectin (Vector Laboratories, Burlingame, CA, USA) and (e) Biotinylated Maackia Amurensis Lectin II (Vector Laboratories). For the chemiluminescence detection the following peroxidase-conjugated secondary reagents were used: (a) Peroxidase-Conjugated AffiniPure Goat Anti-Mouse IgG + IgM (Jackson immune Research, West Grove, PA, USA); (b) Peroxidase-Conjugate Goat Anti-Mouse IgG (Merck-Millipore, Darmstadt, Germany) and (c) Streptavidin-HRP Conjugate (GE Healthcare, Little Chalfont, UK). 4.3. 4.2. Conditioned Media, Protein Lysates and Western Blot Analysis Lentiviral Generation, Viral Transduction and Silencing by Short Hairpin RNA (shRNA) For shRNA lentiviral infections, five pLKO-1 vectors targeting ST3GAL3: sh-6 (TRCN0000232797), sh-7 (TRCN0000232799), sh-8 (TRCN0000232798), sh-9 (TRCN0000035715) and sh-10 (TRCN0000035716), five vectors targeting ST3GAL4:(sh-1 (TRCN0000005574), sh-2 (TRCN0000297123), sh-3 (TRCN0000277891), sh-4 (TRCN0000277939) and sh-5 (TRCN0000297059), and a nontargeting shRNA (SHC002) (scramble) were used. All vectors were purchased from the Broad Institute MISSION shRNA library (Sigma, St. Louis, MO, USA). BxPC-3 and Capan-1 cells were transduced with these vectors by lentiviral infection following the protocols previously described [61]. For the selection of the cells that contained the vector pLKO.1-puro, a dose response curve for antibiotic selection (kill curve) was performed. Resistant cells were selected by adding puromycin dihydrochloride (Sigma) at a final concentration of 1.5 µg/mL for BxPC-3 and 1 µg/mL for Capan-1 cells. 4.6. E-selectin Adhesion Assays Adhesion of BxPC-3 and Capan-1 cells to E-selectin was assessed as previously described [62] with minor modifications: 96-well maxisorp microplates (ThermoFisher) were coated for 24 h with 5 µg/mL of rhE-selectin (R&D Systems, Minneapolis, MN, USA) in PBS or PBS 1% BSA (as negative control). Wells were aspirated and then blocked with PBS 1% BSA. Next, 1 × 105 BxPC-3 or Capan-1 cells were added and allowed to settle for 1 h at 37 ◦C in the incubator. In selected experiments, cells were previously incubated with anti-sialyl-Lewis x mAb. Plates were then gentle washed with PBS and the remaining adherent cells were estimated with a proliferation assay (MTS, Promega, Madison, WI, USA) following the manufacturer’s instruction. For flow-based assay, a flow chamber of PDMS was coated with rhE-selectin/CD62E Fc Chimera (R&D) at 10 µg/mL after incubation with goat anti-Human IgG Antibody, Fc, FITC conjugated (Millipore) at a final concentration of 10 µg/mL. Once incubated, devices were blocked with a solution of PBS 1% BSA for 1 h at RT. PDA cells (BxPC-3 and Capan-1) were resuspended at 3 × 105 and 2.5 × 105 cells/mL respectively in PBS 0.1% BSA. Cells were perfused over the immobilized E-selectin-coated devices at a wall shear stress level of 1.1 dyn/cm2, using a parallel plate flow chamber as previously described [43,63]. The total number of binding events in a single field of view during a 2-min period was recorded and quantified with a phase contrast 10 × Ph1 objective of a Nikon Inverted microscope. 4.4. Reverse Transcription and Quantitative Real-Time PCR (RT-qPCR) RNA extraction was performed using the RNeasy® Mini kit (Qiagen, Hilden, Germany). RNA columns were treated with RNAse-Free DNase I. Eluted RNA yield and purity were determined using a NanoDrop™instrument (ND-1000, Thermo Fisher Inc., Waltham, MA, USA). Of the total RNA 2 µg was reverse transcribed to single-stranded cDNA using MultiScribe™Reverse Transcriptase and random hexamer primers (Applied Biosystems Inc., Foster City, CA, USA). Quantification was performed as previously described [10] with TaqMan Pre-Designed Gene Expression Assays™. Primers used for the evaluation of each glycosyltransferase and the housekeeping gene: ST3GAL3 (Hs00544033_m1), ST3GAL4 (Hs00920871_m1), ST3GAL6 (Hs00196085_m1), FUT3 (Hs01868572_s1), FUT5 (Hs00704908_s1), FUT7 (Hs00237083_m1) and TBP (Hs99999910_m1). The results were analyzed by the delta–delta Ct method and using the TATA-box binding protein (TBP) gene as a reference for calculation. Three technical replicates were performed for each sample and gene. At least three Int. J. Mol. Sci. 2020, 21, 6239 18 of 23 independent assays for the expression gene analysis of KD cell lines were performed. Results were expressed as mean ± SD. independent assays for the expression gene analysis of KD cell lines were performed. Results were expressed as mean ± SD. 4.5. Flow Cytometry Analysis Detection of sialic acid determinants was performed by indirect fluorescence as previously described [9]. Briefly, 1 × 105 viable cells were incubated in the presence (or absence, for negative controls) of the corresponding primary antibodies or biotinylated lectins. After washing, cells were incubated with the correspondent secondary antibody or with streptavidin conjugated to Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA). Next, the median and geomean fluorescence of the cells was determined using a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) equipped with CellQuest™software (Becton Dickinson). At least three independent assays for each sample were performed. To express the percentage of decrease or increase of sialylated glycan determinants in KD cells vs. the corresponding scramble cells, first the ratio of the median values of each sialylated determinant of the KD cells vs. scramble cells was determined for each experiment, and then the mean of these ratios of the three experiments (ratio mean) was calculated and the relative percentage change was expressed as: (1-ratio mean) ×100. 4.8. Polydimethylsiloxane (PDMS)-Based Microchannel Migration Assay 4.8. Polydimethylsiloxane (PDMS)-Based Microchannel Migration Assay 4.8. Polydimethylsiloxane (PDMS)-Based Microchannel Migration Assay PDMS-based microchannels devices were fabricated as previously described [64]. Devices were coated with 20 µg/mL of collagen type 1 to facilitate cell adhesion. Cell migration was visualized and recorded via time-lapse live microscopy (Nikon) at 37 ◦C in a humidified atmosphere of 5% CO2. Phase contrast time-lapse images were taken for 24 h in a 10 min interval with a 10× Ph1 objective. The spatial x and y positions of all non-dividing and viable cells that entered and migrated in the microchannels were tracked overtime with the Manual Tracking plugin in ImageJ. Motility parameters, namely velocity, speed and persistence, were computed using a custom-written MATLAB code as previously described [41,42]. 4.10. Statistical Analysis All data are presented as the mean ± standard error of the mean (SEM) or standard deviation (SD) from 3 independent experiments, unless otherwise stated. In the analyses of the sialylated determinants expression of the cells’ panel (Figure 1B), two values were used for Capan-2, HPAF-II, Panc 10.05 and SW 1990. Statistical significance was determined between pairs of data with a t-test, or between groups of data with one-way ANOVA and a Tukey’s multiple comparison post-hoc test. Dunnett’s multiple comparisons test was used to compare each of the shRNAs to scramble in migration microdevices. Figures were designed with Prism7-GraphPad. Author Contributions: Conceptualization, A.M., R.d.L., P.N., K.K., E.L. and R.P.; Data curation, P.E.G., L.M., B.S.W., K.K., E.L. and R.P.; Formal analysis, P.E.G., L.M., B.S.W., A.M., K.K., E.L. and R.P.; Funding acquisition, R.d.L., P.N., K.K. and R.P.; Methodology, P.E.G., L.M., B.S.W., A.M., N.M.-B. and E.L.; Supervision, P.N., K.K. and R.P.; Writing—original draft, P.E.G., E.L. and R.P.; Writing—review and editing, P.E.G., A.M., N.M.-B., P.N., K.K., E.L. and R.P. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Spanish Ministry of Science and Innovation (grant BIO 2015-66356-R), by the University of Girona grant (MPCUdG2016/028) to R.P. and by the AGAUR-Generalitat de Catalunya grant (2014SGR0229) to R.d.L., and by grants from the Spanish Ministry of Economy and Competitiveness/ISCIII-FEDER (PI17/00199), and the Generalitat de Catalunya (2017-SGR-225) to P.N. Acknowledgments: P.E.G. acknowledges funding support from the University of Girona for mobility grant and from the Generalitat of Catalunya for a pre-doctoral fellowship F.I. L.M. also acknowledges funding from the Generalitat of Catalunya for a pre-doctoral fellowship F.I. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 4.9. Transwell Invasion Assay Cell invasion was evaluated using modified Boyden chambers in 24 well plates. Prior to starved-FBS cell seeding, chambers were precoated with 25 µg of Matrigel (Corning, Corning, NY, USA). Then, the mixture was allowed to polymerize at 37 ◦C. After trypsinization, detached cells were washed and resuspended in PBS 1% BSA. In selected experiments, cells were previously incubated 20 min with anti-sialyl Lewis X mAb diluted 1:10 (BD Biosciences). Then, 5 × 104 cells for Capan-1 and 4 × 104 for BxPC-3 were seeded in the top chamber. After 30 min, medium containing 10% FBS for BxPC-3 or 20%FBS for Capan-1 cells was added to the bottom as chemoattractant. Cells were let to invade for 24 h at 37 ◦C, and after that inserts were washed, fixed and stained. Non-invaded cells were removed from the top surface. Then, at least 20 random camps were photographed at 10× magnification under the CKX41 inverted microscope (Olympus). 4.7. Transwell Migration Assay Cell migration was evaluated using modified Boyden chambers in 24 well plates as previously described [10]. Briefly, cells were grown in absence of FBS for 24 h before they were harvested. Prior to cell seeding, serum free medium with 0.001% of collagen type 1 from calf skin (Sigma) was placed in the upper part of the 8 µm pore size ThinCerts TM inserts (Greiner bio-one, Kremsmünster Austria). Detached cells were resuspended in PBS 1% BSA. In selected experiments, cells were previously incubated 20 min with anti-sialyl Lewis X mAb diluted 1:10 (BD Biosciences). The cell inserts were seeded with 3.5 × 104 cells for Capan-1 and 2.5 × 104 for BxPC-3 in the top chamber. Cells were let to migrate for 18 h at 37 ◦C. After that, inserts were washed, fixed and stained. Non-migrated cells were removed from the top surface of the insert using cotton swabs. Then at least 20 random camps were photographed at 10× magnification under the microscope with a CKX41 inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan). Int. J. Mol. Sci. 2020, 21, 6239 19 of 23 19 of 23 Author Contributions: Conceptualization, A.M., R.d.L., P.N., K.K., E.L. and R.P.; Data curation, P.E.G., L.M., B.S.W., K.K., E.L. and R.P.; Formal analysis, P.E.G., L.M., B.S.W., A.M., K.K., E.L. and R.P.; Funding acquisition, R.d.L., P.N., K.K. and R.P.; Methodology, P.E.G., L.M., B.S.W., A.M., N.M.-B. and E.L.; Supervision, P.N., K.K. and R.P.; Writing—original draft, P.E.G., E.L. and R.P.; Writing—review and editing, P.E.G., A.M., N.M.-B., P.N., K.K., E.L. and R.P. All authors have read and agreed to the published version of the manuscript. References 1. Adamska, A.; Domenichini, A.; Falasca, M. Pancreatic Ductal Adenocarcinoma: Current and Evolving Therapies. Int. J. Mol. Sci. 2017, 18, 1338. [CrossRef] [PubMed] 1. Adamska, A.; Domenichini, A.; Falasca, M. 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Overexpression of sialyltransferase CMP-sialic acid: Galβ1,3GalNAc-R α6-sialyltransferase is related to poor patient survival in human colorectal carcinomas. Cancer Res. 2001, 61, 4605–4611. 30. Petretti, T.; Kemmner, W.; Schulze, B.; Schlag, P.M. Altered mRNA expression of glycosyltransferases in human colorectal carcinomas and liver metastases. Gut 2000, 46, 359–366. [CrossRef] 31. Satomura, Y.; Sawabu, N.; Takemori, Y.; Ohta, H.; Watanabe, H.; Okai, T.; Watanabe, K.; Matsuno, H.; Konishi, F. Expression of Various Sialylated Carbohydrate Antigens in Malignant and Nonmalignant Pancreatic Tissues. Pancreas 1991, 6, 448–458. [CrossRef] [PubMed] 32. Balmaña, M.; Duran, A.; Gomes, C.; Llop, E.; López-Martos, R.; Ortiz, M.R.; Barrabés, S.; Reis, C.A.; Peracaula, R. Analysis of sialyl-Lewis x on MUC5AC and MUC1 mucins in pancreatic cancer tissues. Int. J. Biol. Macromol. 2018, 112, 33–45. [CrossRef] [PubMed] 33. 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https://openalex.org/W2049053301

https://digital.csic.es/bitstream/10261/221423/1/Allepuz_Street%20Art.pdf

Spanish; Castilian

El Street Art y la (in)cultura urbana: el ejemplo de Córdoba

Arte, individuo y sociedad

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Street Art: urban culture, urban ignorance. The example of Córdoba Pablo Allepuz-García Universidad de Córdoba [email protected] Recibido: 23 de diciembre de 2012 Aprobado: 19 de julio de 2013 Resumen El Street Art es un fenómeno artístico presente en nuestra vida diaria al que se ha condenado a un cierto ostracismo. Tanto la comunidad académica, en este aspecto ajena a lo que ocurre en el mundo real, como la gran mayoría de la sociedad, inmersa en preocupaciones bien distintas, han obviado su existencia ineludible no ya en el arte, sino en el propio paisaje urbano; aun cobra un sentido más crudo en la Ciudad Histórica, donde la ingente cantidad de Patrimonio eclipsa cualquier otra manifestación. Este trabajo abre una nueva línea de investigación en Córdoba, partiendo de un análisis de la sociedad en que se origina y de aquello que significa; documentando sus representaciones más significativas sobre una base cartográfica; aplicando la experiencia estética como fundamento de nuestra reflexión; y, con base en lo anterior, realizando algunas propuestas de interpretación. Todo ello, con posibilidades de ser extrapolado a otras ciudades españolas. Palabras clave: Street Art, Córdoba, Graffiti, Cartografía, Experiencia Estética. Allepuz-García, P. (2013): El Street Art y la (in)cultura urbana: el ejemplo de Córdoba. Arte, Individuo y Sociedad, 26 (1) 137-151 1. Introducción Una sociedad sin arte es una sociedad sin vida (Lima, 2011) “La sociedad urbana, occidental u occidentalizada de hoy en día se ha fragmenta­ do en una serie de subculturas diferentes, aunque solapadas, cada una con una iden­ tidad musical propia” (Cook, 2001: 18). La complejidad de nuestro Zeitgeist radica en la aparente confluencia de estilos, el sincretismo que supone la globalización; sin embargo, no debe considerarse únicamente producto de nuestro tiempo –que algunos coinciden en denominar hipermodernidad, superando el postmoderno de Lipovetsky o Lyotard (Cfr. Thiebaut, 1999)–, sino resultado de la perspectiva sincrónica: ante la imposibilidad de aislar el presente hemos de recurrir al pasado más reciente, que con tanta frecuencia hunde sus raíces en lo más profundo de la Historia. p El siglo XX ha sido especialmente convulso en todos los ámbitos de la vida: es el siglo de las Guerras Mundiales, la bomba atómica y los campos de concentración; del triunfo del capitalismo, el Crack del 29 y la caída del muro de Berlín; del advenimien­ to de la democracia, la información y los mass media; del fin del arte, las vanguardias y las subastas. Un arte que no podía ya recrearse en elitistas mundos idílicos, sino que debía comprometerse con la realidad, mezclarse con nosotros, ponerse a ras de suelo: adoptando formas insospechadas, ha abandonado incluso sus habituales residencias privadas para abrazar la ciudad en tanto que lugar común de la sociedad. En este con­ texto, toda reflexión sobre el arte puede canalizarse a través del Street Art una vez “ha llegado a ser evidente que nada referente al arte es evidente: ni en él mismo, ni en su relación con la totalidad, ni siquiera en su derecho a la existencia” (Adorno, 1983: 9). La manera en que lo tratamos dibuja una radiografía de nuestro estado mental, se establece como un espejo en el que mirarnos; aunque la decisión de hacerlo haya quedado –por unas razones u otras– a título personal. Abstract Street Art is an artistic phenomenon that, although present in our everyday lives, has been con­ demned to a certain ostracism. On the one hand, the Academic Community is –in this field– oblivious to what happens in real world; on the other hand, the vast majority of our society is immersed in very different concerns. Both of them have avoided its inevitable existence not only in art panorama, but also in the urban landscape. The problem takes a harder perspective within the Historical City, where the variety of the Heritage outshines any other manifestation. This study opens a new research line in Córdoba, starting with an analysis of the society from which Street Art is created and what it means, documenting its most significant features on a cartographic map, applying the aesthetic experience as the foundation of our own thinking, and, based on this, making some proposals for interpretation. All of these conclusions have the potential to be applied to other Spanish cities.i p pp p Key words: Street Art, Córdoba, Graffiti, Cartography, Aesthetic-Experience. Allepuz-García, P. (2013): Street Art: urban culture, urban ignorance. The example of Córdoba. Arte, Individuo y Sociedad, 26 (1) 137-151 Sumario: 1. Introducción, 1.1. Concepto de Street Art, 1.2. Statu quo, 2. Córdoba: el dibujo del flâneur, 2.1. Cartografía, 2.2. Interpretación de la cartografía, 3. Estética y Filosofía del Arte (Urbano), 3.1. Erótica, 3.2. Hermenéutica, 3.3. Metaposición analítica, 4. Conclusiones: problemas y posibilidades del Street Art. Referencias Street Art. Referencias Street Art. Referencias 137 ISSN: 1131-5598 http://dx.doi.org/10.5209/rev_ARIS.2014.v26.n1.41107 137 ISSN: 1131-5598 http://dx.doi.org/10.5209/rev_ARIS.2014.v26.n1.41107 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Arte, Individuo y Sociedad 2014, 26(1), 137-151 We declare the world as our canvas! [Declaramos el mundo como nuestro lienzo] (Street Art Utopia) [Declaramos el mundo como nuestro lienzo] (Street Art Utopia) Pero, ¿qué es el Street Art? Para muchos se trata de un mero oxímoron, una irreve­ rente muestra de la degeneración moral que nos atañe; otros, a su vez, aprecian en tal aspecto –la transgresión– la verdadera esencia de todo acto artístico contemporáneo, siendo el Arte Urbano, por tanto, su epígono y máximo exponente (Cfr. Julius, 2002). Este baile sobre la delgada línea que separa arte y política, algo exclusivo de nuestro definiendum, es un arma de doble filo que, no obstante, podría y debería desembocar en una reflexión sobre la sociedad en sí misma. Pero para desarrollar una exégesis de semejante magnitud hemos de conocer siquiera la naturaleza y el contenido de nues­ tro objeto. Lato sensu, el Street Art engloba cualquier actividad artística que tenga como contexto el entorno urbano, hallando concreción en ciertas manifestaciones: 138 Arte, Individuo y Sociedad 2014, 26(1), 137-151 , y 2014, 26(1), 137-151 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García movimientos como Recuperar Las Calles (Cfr. Klein, 2001: 363-376), que llevan a cabo performances en público; flashmobs, smartmobs, happenings; la contracultura del hip-hop con el graffiti, el stencil o el shoeffiti; la música en la calle; intervenciones merecedoras de estudios individuales y exhaustivos que habrán de ser abordados en otro momento. Ahora bien, ¿qué es el arte? A priori parece una pregunta sencilla, pero no lo es en absoluto (Cfr. Jiménez, 2002); aun se complica más al tratar el Street Art, pues no todo lo relativo a dichas prácticas es considerado arte: dado su carácter rebelde e ile­ gal puede incluso tacharse de vandalismo –la valoración más extendida–. Otro aspec­ to a tener en cuenta es su fenomenología efímera, en la que prima el anónimo proceso creativo sobre la obra resultante: ésta puede perdurar indefinidamente o desaparecer al cabo de algunas horas. Ese proceso de producción de obras, tan emparentado con el Capitalismo, se desvanece aquí a favor de la propia expresión artística. The irony is that despite having to scuttle around at night like Jack the Ripper with a marker pen, writing graffiti is about the most honest way you can be an artist. Arte, Individuo y Sociedad 2014, 26 (1), 137-151 1.2 Statu quo Never paint graffiti in a town where they still point at aeroplanes [Nunca pintes grafitis en una localidad donde aún señalen a los aviones] (Banksy, 2001: 43). Never paint graffiti in a town where they still point at aeroplanes [Nunca pintes grafitis en una localidad donde aún señalen a los aviones] (Banksy, 2001: 43). El lenguaje, por sí mismo, nos da claves analíticas del estado de la cuestión: ha­ blamos de Street Art, graffiti, stencil, performance… Tanta cursiva no viene sino a indicar que España sigue una inercia exterior, sin asimilar por completo el conteni­ do de dicho movimiento. En efecto, no es sencillo estudiar este fenómeno, debido a su –digámoslo así– modernidad, heterogeneidad y dispersión. La bibliografía no es abundante y su difusión dista mucho de ser ejemplar, con títulos difíciles de en­ contrar, la mayoría de fuentes digitales y en otros idiomas; en muchas ocasiones, la solución es recurrir a los pocos entendidos en la materia, que suelen prestar su ayuda en pro de un mayor conocimiento de su cultura. Existe, además, una tendencia gene­ ralizada a ponderar en exceso lo visual, a analizar la voluntad del artista en lugar de extraer los valores directamente de la obra de arte para trascenderla. Se han produci­ do avances, pero aún se ha escrito muy poco sobre el tema en nuestro país: podemos destacar algunas tesis doctorales (Figueroa Saavedra, 1999; Abarca Sanchís, 2010), libros (De Diego, 2000; AA.VV, 2005) o artículos específicos (López Jiménez, 1998; Cambil Hernández, 2012); al margen, por supuesto, de otro tipo de acercamiento como programas de televisión (Ritmo Urbano, 2012-2013) o documentales (Las Calles Hablan, 2013). , ) En el caso concreto de Córdoba, podríamos reducirlo hasta una absoluta ignoran­ cia: apenas se limita a esporádicas apariciones en prensa. Pese a no contar con una base de investigación sólida, el Street Art está presente en la urbe: por tanto, merece ser rescatado del olvido para propiciar la reflexión que tanto tiempo se le ha negado; obviar su existencia supone mantener un tabú envuelto en prejuicios. p p j Córdoba es una ciudad un tanto pasiva respecto a su cultura. La institucionaliza­ ción de los principales monumentos, así como el papel de los museos, generan en la población un cierto sentimiento de despreocupación por su Patrimonio: si ya existe un entramado que explota los bienes culturales, la contribución particular no es ne­ cesaria. We declare the world as our canvas! [Declaramos el mundo como nuestro lienzo] (Street Art Utopia) It takes no money to do it, you don’t need an education to understand it, there’s no admission fee and bus stops are far more interesting places to have pictures than in museums [La ironía es que a pesar de tener que merodear por la noche como Jack el Destripador con un rotulador permanente, pintar grafitis viene a ser la forma más honesta de ser artista. No exige dinero para hacerlo, no requiere una educación para entenderlo, no hay que pagar entrada y las marquesinas son lugares mucho más interesantes para ubicar las pinturas que los museos] (Banksy, 2001: 5). Al menos esa es la parte teórica. La popularidad de Banksy, convertido en un icono pop, ha hecho del Street Art materia de la opinión pública internacional gracias a la comercialización de su marca: toda una estrategia de merchandising, disimulada por la ambigüedad de su implicación en los beneficios y su celoso anonimato, en la que encontramos tres libros e, incluso, una película –Exit through the gift shop (2010)– que podría resumir metafóricamente su trayectoria artística, esto es, des­ de unos orígenes humildes y arraigados hasta la fama mundial; para algunos, como Space Hijackers, desde la pureza ética y política de las formas primeras hasta la ac­ tual prostitución de su obra dentro del sistema. Shepard Fairey se encuentra en una situación similar: de su alter ego OBEY a la campaña electoral de Obama, pasando también por la venta de su logo en numerosos productos. En consecuencia, por estos y otros ejemplos, se ha generado una fuerte controversia en el seno del Street Art: el favoritismo y el privilegio del que gozan estos entre sus iguales provoca un rechazo que se transforma en boicot a sus piezas. La polémica está servida, aunque se trata de un debate que aún –y subrayo el complemento temporal– no se ha generado en España. 139 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García Arte, Individuo y Sociedad 2014, 26(1), 137-151 1.2 Statu quo Craso error: la ciudad está perdiendo su espontaneidad, su capacidad de con­ quistar las calles con actividades de cualquier tipo –más allá del flamenco, los patios, las cruces o las terrazas–. A pesar de las puntuales acciones de la reciente platafor­ ma Emplazarte, aún con escasa visibilidad, del intrigante caso del Callejero pirata, desvelado el misterio en las personas de Antonio Blázquez y Cristian Tena (Diario Córdoba, 14/10/12), y de algunos talleres (Diario Córdoba, 26/07/12) y cursos (El Arsenal de Chinales) de reducido público, la noción de performance es una perfecta desconocida; la música en la calle, por desgracia, una bella anécdota; prácticamen­ te lo único que mantiene viva la llama del Street Art es el graffiti, y su variante en stencil. Como trasunto, el trabajo debe inclinarse en lo sucesivo hacia esta vertiente, por su omnipresencia en el paisaje urbano y la insuficiente atención que ha recibido: “Graffiti ultimately wins out over proper art because it becomes part of your city” [El graffiti se impone sobre el arte institucional porque pasa a formar parte de la ciudad] (Banksy, 2001: 5). 140 Arte, Individuo y Sociedad 2014, 26(1), 137-151 Pablo Allepuz-García Pablo Allepuz-García El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Hacemos en este momento un inciso para aclarar la terminología: preferimos el término internacionalmente aceptado graffiti, así como el plural castellanizado “gra­ fitis” para suplir su ambigüedad; sin olvidar, por ello, su más antigua raíz italiana graffito, de la que derivan las demás. 2. Córdoba: el dibujo del flâneur Escúchela; la ciudad respirando… (Black Star, 1998) . Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Escúchela; la ciudad respirando… (Black Star, 1998) . Muchas son las obras repartidas por la geografía cordobesa, aunque pocas han tenido la repercusión del citado Callejero pirata. En verano del 2010, las fachadas de numerosos edificios, la mayoría en estado de abandono, amanecieron cubiertas por letras de papel pintado que proponían nuevos nombres para el intrincado urbanismo de la ciudad. A imitación del representativo estilo de los rótulos institucionales y oficiales, leyendas como “por fin te he encontrado”, “el quinto pino”, “he encontrado un atajo” o “to’ pa’ lante” duplicaron el significado de los caminos, robando, de paso, una sonrisa cómplice a los viandantes. El Ayuntamiento, por clamor popular, decidió mantenerlos en las paredes. Otro caso es el de Lo, quien imprime su particular lectura del Street Art mediante stencils que reproducen libros, depositados amorosamente sobre los dinteles o en el acto de emprender un vuelo hacia no se sabe dónde. Llenan así de cultura, literalmente, todos los rincones. Black-T, por su parte, ha desarrollado un estilo personal e inconfundible, caracterizado por unas figuras humanas que con­ traponen la esquematización de las formas del cuerpo al realismo y la inocencia de los rostros en escala de grises. g Figura 1. Rótulo de Callejero Pirata, libros de Lo junto a un rótulo institucional y niño de Black-T, respectivamente. (Fotografías de elaboración propia) Figura 1. Rótulo de Callejero Pirata, libros de Lo junto a un rótulo institucional y niño de Black-T, respectivamente. (Fotografías de elaboración propia) En esta apología de la ciudad, escenario del gran teatro del mundo, “l’art de vi­ vre dans la ville comme oeuvre d’art” [el arte de vivir la ciudad como obra de arte] (Lefebvre, 1974: 139) se encarna en la figura del flâneur: a cada forma de mover­ 141 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Pablo Allepuz-García El Street Art y la (in)cultura urbana: el ejemplo de Córdoba se corresponde una forma específica de conocer, y la del flâneur es un divagar que memoriza. No le interesa el adónde, sino el dónde (Cfr. Schlögel, 2007: 257-262). Nuestros caminos se cruzan en este punto, ya que hemos de recorrer las calles para documentar los elementos de nuestro estudio; y, con ellos, configurar el paisaje urba­ no del Street Art sobre un plano de Córdoba. Escúchela; la ciudad respirando… (Black Star, 1998) . p Es evidente que no podríamos seleccionar todo lo que haya salido de un bote de spray por el simple hecho de haber empleado dicho procedimiento: identificar técnica y obra perjudica a la consideración artística del graffiti. Si bien cualquier elemento puede ser interpretado como arte –refugio de la mediocridad en demasiadas ocasio­ nes–, hemos de establecer un criterio a la hora de incorporar a nuestro trabajo tal o cual ejemplar; criterio, por cierto, que puede no ser compartido –dentro de aquel relativismo coexisten tantos como personas y formas de entender el arte–. Así pues, se incorporarán al mapa aquellas piezas cuya intencionalidad o valores estéticos des­ taquen sobre el común de “pintadas”: es decir, se discriminarán mediante omisión las restantes, que a los ojos de la sociedad no demuestran ninguna aportación y, por el contrario, sólo parecen contaminar visualmente el entorno. Adoptar esta posición implica imprimir al conjunto una acusada pátina de subjetividad que, no obstante, trataremos de paliar constituyendo un amplio cuerpo calificable como arte –para la subjetividad, véase el apartado 3.1–. El objetivo se aleja así de la documentación sistemática, que quedará apuntada para un estudio posterior, y se utilizarán obras concretas como herramienta didáctica: el ostracismo del que se habla en el resumen es un problema de educación, y resulta ciertamente más sencillo comenzar una expli­ cación por los ejemplos paradigmáticos para poder comprender, al fin, el Street Art en toda su expresión. p Callejear es una especie de lectura de las calles, donde caras, escaparates y vitri­ nas, terrazas, vías, coches, árboles, [grafitis,] todo se convierte con igual derecho en letras que juntas producen palabras, frases y párrafos de un libro siempre nuevo. Para callejear como es debido no vale tener en la cabeza nada demasiado definido (Hessel, 1984: 145). ) La ciudad está repleta de pequeños destellos que cada día se reproducen y desapa­ recen. Haga el ejercicio de buscarlos y lo descubrirá por usted mismo: en palabras del citado Henri Lefebvre, el futuro del arte no es artístico sino urbano (1974: 140). Arte, Individuo y Sociedad 2014, 26(1), 137-151 Mind the map! [¡Cuidado con el mapa!] (London Transport Museum, 2012) Una de las ironías de nuestra época es que ahora, cuando las calles se han conver­ tido en el artículo más valioso de la cultura publicitaria, las manifestaciones de cultu­ ra se hallen bajo amenaza. Los ataques contra los grafitis, los pósters o la mendicidad están criminalizando todo lo que hay de realmente público en la vida de las ciudades (Klein, 2001: 363-364). Si, como decimos, el carácter del Street Art es meramente efímero, puesto que puede desaparecer en cualquier momento, ¿qué sentido tiene presentar una visión sincrónica del mismo? A lo largo de los siglos, el arte ha tratado 142 Arte, Individuo y Sociedad 2014, 26(1), 137-151 Pablo Allepuz-García El Street Art y la (in)cultura urbana: el ejemplo de Córdoba de desentenderse del espacio para trascender su tiempo; el Street Art, en cambio, establece un espacio sin ambicionar la eternidad. Bien es cierto que los grafitis nacen con fecha de caducidad y, a pesar de ello, los emplazamientos donde tienen lugar –y el espíritu de este apartado es el espacio– suelen perpetuarse. De este modo, las obras de arte se superponen, se alteran, se deterioran y mueren, pero el espacio creativo, el paisaje artístico permanece vivo; y en tanto que vivo, cambiante. p j p y q Este apartado, ampliamente subdividido según los barrios en que se organiza Córdoba, trata de recoger la cartografía temática del graffiti en la ciudad. Es, por tanto, una tarea ambiciosa e imprudente a partes iguales, dadas la ingente cantidad de manifestaciones por documentar, la amplia extensión en que se encuentran repartidas y su constante peligro de extinción.l La textura de una ciudad refleja una suma de lugares complementarios que se yuxtaponen, superponen o encadenan. Cada lugar tiene su característica propia, sin pretensión alguna de inmutabilidad. De ahí que pueda leerse la ciudad como collage en que las formas de construcción ponen de manifiesto posturas urbanísticas, crítica social y modos de trato con la Historia (Schlögel, 2007: 303). y ( g ) Los barrios constituyen las distintas piezas de un puzle que terminará ensamblán­ dose una vez que todas sus partes estén detalladas componiendo, pues, un completo esquema del panorama urbano de Córdoba. Sin embargo, la enorme complejidad de una ciudad, con infinitud de factores históricos, económicos y sociales distribuidos de manera desigual, no permite abordar su estudio completo en un simple artículo; ni siquiera el de un barrio individual. Mind the map! [¡Cuidado con el mapa!] (London Transport Museum, 2012) Por tanto, adaptándonos al formato, incluimos solamente algunos de los ejemplos más relevantes de Ciudad Jardín como apoyo al texto y como una imprecisa aproximación al estudio exhaustivo que se podrá llevar a cabo en el futuro: pretendemos advertir del sentido peyorativo y discriminatorio que estas representaciones adquieren en el imaginario colectivo, que incluso se ha con­ vencido de su inexistencia. La elección de dicho entorno responde asimismo a moti­ vos arbitrarios y asumimos la relatividad de las impresiones que se pudieran extraer: el mapa no refleja todo el Arte Urbano de Córdoba ni tampoco de la zona abarcada, sino aquello que más fácilmente puede ser identificado y aceptado por el ciudadano, al tiempo que describe unas ligeras pinceladas sobre la metodología a aplicar en el caso del estudio exhaustivo que se realizará a posteriori. 143 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García Figura 2. Mapa temático del barrio Ciudad Jardín, en el que se disponen algunas de las piezas más repre­ sentativas. (Elaboración propia sobre el plano de Córdoba cedido por la Gerencia Municipal de Figura 2. Mapa temático del barrio Ciudad Jardín, en el que se disponen algunas de las piezas más repre­ sentativas. (Elaboración propia sobre el plano de Córdoba cedido por la Gerencia Municipal de Figura 2. Mapa temático del barrio Ciudad Jardín, en el que se disponen algunas de las piezas más repre­ sentativas. (Elaboración propia sobre el plano de Córdoba cedido por la Gerencia Municipal de Figura 2. Mapa temático del barrio Ciudad Jardín, en el que se disponen algunas de las piezas más repre­ sentativas. (Elaboración propia sobre el plano de Córdoba cedido por la Gerencia Municipal de Arte, Individuo y Sociedad 2014, 26(1), 137-151 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 These walls don’t lie… [Esos muros no mienten] (Promoe, 2004) Cuando el mapa completo de Córdoba –macroestructura– esté confeccionado, po­ drá reflexionarse sobre aspectos como la diferencia de estilos entre barrios, la distinta profusión según zonas, el grado de actividad de cada una de ellas… Entretanto, no podemos adelantar más que unas palabras sobre el barrio elegido –microestructura–: Ciudad Jardín está infestada e infectada de grafitis de todas las tipologías, si bien la proporción entre el artístico y el vandálico es vergonzosamente preocupante; la ma­ yoría de aquellos, por cierto, sirven como imagen a privados. No existe, en ninguno de ellos, una coincidencia estilística que apunte a un grupo o un artista determinado, como tampoco aparecen firmas que reivindiquen dichos trabajos; más bien, debemos hablar de manifestaciones individuales y anónimas, sin conciencia de autoría ni vo­ luntad de prestigio. A pesar de la aleatoriedad de su disposición, podemos distinguir 144 Arte, Individuo y Sociedad 2014, 26(1), 137-151 , y 2014, 26(1), 137-151 Pablo Allepuz-García El Street Art y la (in)cultura urbana: el ejemplo de Córdoba algunas zonas de mayor concentración, en un ejercicio de mimetismo bastante co­ mún. Como decimos, todo ello se contrastará más adelante con las partes restantes para obtener los resultados. Nuestro interés en este artículo no es la documentación exhaustiva de la zona ni la atribución a uno u otro artífice, que aún deben esperar un tiempo prudencial, sino demostrar que existen suficientes manifestaciones para iniciar una discusión sobre su consideración. Así pues, prescindimos de un catálogo descriptivo de todas ellas e integramos algunos ejemplos con el único propósito de ilustrar los argumentos del discurso. Figura 3. Piezas A, B, C y D de la figura 2. (Fotografías de elaboración propia) Figura 4. Piezas E, F, G y H de la figura 2 (Fotografías de elaboración propia) Figura 3. Piezas A, B, C y D de la figura 2. (Fotografías de elaboración propia) Figura 3. Piezas A, B, C y D de la figura 2. (Fotografías de elaboración propia) Figura 4. Piezas E, F, G y H de la figura 2 (Fotografías de elaboración propia) Figura 4. Piezas E, F, G y H de la figura 2 (Fotografías de elaboración propia) Figura 4. Piezas E, F, G y H de la figura 2 (Fotografías de elaboración propia) 145 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García Figura 5. These walls don’t lie… [Esos muros no mienten] (Promoe, 2004) Piezas I, J, K y L de la figura 2 (Fotografías de elaboración propia) Figura 5. Piezas I, J, K y L de la figura 2 (Fotografías de elaboración propia) Gracias a la figura del flâneur (Cfr. Benjamin, 2005: 421-458), y sobre todo a la cartografía, hemos dado el salto desde el Street Art como constructo vago y abstracto hasta la realidad material y palpable que representan los grafitis, con los que podemos conectar… Arte, Individuo y Sociedad 2014, 26(1), 137-151 3. Estética y Filosofía del Arte (urbano) Las obras de arte son provocaciones. Nosotros no las explicamos, sino que polemiza­ mos con ellas (Hauser, 1981: 9) Las obras de arte son provocaciones. Nosotros no las explicamos, sino que polemiza­ mos con ellas (Hauser, 1981: 9) El siglo XX ha acabado con el arte tradicional. Tras las vanguardias –última mani­ festación del academicismo–, el Pop Art marcó una nueva dirección, un nuevo orden en el que todo es arte; o, idem, nada es arte (Cfr. Jiménez, 2002). Éste ha sido sustitui­ do por la Filosofía del Arte, que se instala en nuestra relación con la obra, indagando en los porqués de nuestra conmoción emocional y la catarsis que de ella se deduce. Esto es, hablamos de Erótica y Hermenéutica (Cfr. Álvarez, 2007). Arte, Individuo y Sociedad 2014, 26(1), 137-151 146 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García 3.1. Erótica Tanto menos se goza de las obras de arte cuanto más se entiende de ellas (Adorno, 1983: 25) La Erótica es la primera fase de la experiencia estética. Sobre ella debe recaer la reflexión, pues es aquello que nos vincula emocionalmente con la obra y crea una verdadera pasión por el arte. Sin embargo, esta faceta se ha menospreciado en las enseñanzas humanísticas a favor de una explicación histórica en la que prima el con­ tenido y el contexto. Se ha desarrollado toda una manera de hablar, una jerga en toda regla de discrimi­ nación de lo inmediato, de lo visual. […] La crítica de la subjetividad en nombre de intersubjetividad, objetividad y demás había declarado la guerra al sujeto que percibe y conoce, que también padece y obra. […] Conceptodependencia, mono de concepto o necesidad de muletas conceptuales son otras tantas maneras de nombrar una déforma­ tion profesionelle muy extendida (Schlögel, 2007: 265:269). En los últimos años se han venido sustituyendo las deterministas teorías del re­ flejo, de ascensión marxista, por otras más centradas en la percepción subjetiva del receptor. En otras palabras, se llama ahora la atención sobre aquello que nuestros ojos no han sabido amar en su cotidiano vistazo: el arte necesita de recogimiento, un tiempo que nos regalamos a nosotros mismos para conversar con la obra y entablar la llamada transformación en construcción (Cfr. Gadamer, 1999: 154-166). Esta tesitu­ ra parece haber quedado relegada a los espacios museísticos, artísticos por extensión, que se revisten de una cierta “religión del arte” (Cfr. Alonso, 1999: 21). Ahora bien, la imperante obsolescencia del presente –véase el apartado 1– nos ha robado el placer de vivir la ciudad: bajo ningún concepto hemos de resignarnos ante su pérdida.i En este sentido, el graffiti es un puente tendido hacia dicho placer, que suscita potentes y encontrados sentimientos: por un lado, los amantes del Street Art perciben en sus valores plásticos una gran capacidad evocadora, quizá condicionada por lo ilegal de su presencia y su futuro incierto; por otro, los detractores de este fenómeno se comunican de igual modo con las obras, aunque generen sentimientos negativos. Sea como fuere, la Erótica es conditio sine qua non para el posterior proceso herme­ néutico: no podemos negárnosla. Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Si el arte ha muerto: ¿qué es el arte? (Román, 2007) Si el arte ha muerto: ¿qué es el arte? (Román, 2007) Dentro del binomio que hemos establecido, la Hermenéutica supone la culmina­ ción de la fase Erótica y, por ende, de la experiencia estética en sí misma. Entonces se 147 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Pablo Allepuz-García El Street Art y la (in)cultura urbana: el ejemplo de Córdoba produce realmente un crecimiento interior: consiste en utilizar el goce de los sentidos para proyectar la personalidad y descubrir las razones profundas de aquella emoción; desnudarnos de complejos para comprender nuestra esencia más íntima. p j p p Tan importante es lo que vemos como lo que no vemos, que se encuentra presente por exclusión. Acaso la reflexión, en este caso, no se centre en la relación con la obra per se, sino en nuestra propia actitud. Las teorías actuales apuntan hacia una reinven­ ción del concepto de arte en el papel activo del espectador. Según esta estética de la recepción, el arte existe en tanto que alguien lo contempla como tal e inicia un proce­ so de construcción, un diálogo continuo con la obra. Pero, ¿qué ocurre si la obra pasa desapercibida ante nuestros ojos? En un museo estamos (pre)dispuestos a buscar esa comunión con el objeto artístico que, en suma, no es otra cosa que una comunión con nosotros mismos: hemos decidido que queremos dedicarle un tiempo determinado de nuestra apretada agenda. Sin embargo, si lo encontramos acechando –literalmente– a la vuelta de la esquina, lo obviamos con el mayor de los desprecios. Aprender los mecanismos del arte tiene, por supuesto, sus factores positivos; pero educar nuestra percepción es también limitar nuestra experiencia a unos parámetros predetermina­ dos. Definitio est negatio, máxime en el campo que nos atañe. Arte, Individuo y Sociedad 2014, 26(1), 137-151 A lot of people never use their initiative because no-one told them to [Mucha gente no emplea nunca su propia iniciativa porque nadie le dijo que lo hiciera] (Banksy, 2001: 23) A lot of people never use their initiative because no-one told them to [Mucha gente no emplea nunca su propia iniciativa porque nadie le dijo que lo hiciera] (Banksy, 2001: 23) A tenor de lo expuesto, ¿cuáles son los horizontes de expectativas para tales ma­ nifestaciones artísticas? Las nuevas corrientes destinadas a la musealización incurren en una flagrante contradicción: por un lado, el suculento mercado del arte puede suponer la fama y una nueva consideración del fenómeno –si bien se firma un pacto con el diablo, pues tan pronto como cesen sus repercusiones será desechado y olvi­ dado–; por otra parte, el Street Art pretende ser un arte puro, al margen de cualquier exigencia tendenciosa (Cfr. Ruiz, 2009). Existe una alternativa a la simplista solución de encerrar las obras en un recinto museístico: mantener su esencia más profunda, aquella que le da nombre, dejándolas vivir en libertad (condicional). En este sentido, la propuesta de futuro pasa por la puesta en valor del Patrimonio –porque, sin duda, estamos hablando de Patrimonio– in situ, es decir, respetando sus características in­ herentes; aunque para ello tengamos que ser tolerantes y abrir la mente: podemos adoptar un modelo “ideal” –benchmarking–, o bien, dejar volar nuestra imaginación para crear un nuevo paradigma. En cualquier caso, necesitamos un cuerpo teórico –que, desde la modestia, hemos pretendido plasmar en este trabajo– sobre el cual sustentar la reflexión y del que se carecía hasta el momento. l y q En cuanto a la primera opción, si tomamos como ejemplo Londres –salvando las evidentes distancias– destacamos Street Art London Tours: una iniciativa priva­ da que, por un módico precio, muestra el panorama de Arte Urbano londinense en una serie de rutas guiadas; por las condiciones especiales del objeto protagonista, se renuevan para cada cita: no existen dos iguales. Estos recursos se imbrican en una cultura callejera con mayor tradición, dentro de una estructura de la que se carece en Córdoba. Algo más ambicioso y complejo sería aceptar el mobiliario urbano a modo de soporte artístico y concederlo como tal en el marco de un concurso de ideas. An act of thought is an act of art [Un acto mental es un acto artistico] (Partum) An act of thought is an act of art [Un acto mental es un acto artistico] (Partum) An act of thought is an act of art [Un acto mental es un acto artistico] (Partum) Podríamos añadir una tercera fase, independiente de las anteriores, a modo de síntesis hegeliana. En esta ocasión se centra el foco de atención en el propio proceso Erótico-Hermenéutico, ampliando así el espectro de nuestro trabajo: analizar la ex­ periencia estética de una (hipotética) tercera persona alzándose un nivel por encima de ella, siendo conscientes de los mecanismos que subyacen a aquella observación de primer grado, discreta e ingenua en su individualidad. La observación de segundo orden correspondería a una metaposición analítica (Moyinedo, 2010) donde conver­ gen todos los puntos de vista y desde la cual es posible emitir valoraciones de carácter universal. La genealogía de la obra de arte se desdobla si consideramos una segunda posi­ ción de observación. […] pierde su estabilidad ontológica, es decir que, escindiéndo­ se de su manifestación material, se muestra ahora como resultado de un proceso cuya historicidad compromete no sólo las determinaciones de su origen sino también las de su destino (Moyinedo, 2010). De esta manera, el espectador –o su defecto– se ve despojado de la neutralidad que garantizaba su anonimato; en resumen, interviene más o menos directamente en la creación, con un mayor o menor grado de autoría –ya innegable–, deviniendo responsable de su entorno. Dicha postura ofrece un enfoque global, que permite en­ juiciar nuestro compromiso con la ciudad que vivimos, el paisaje urbano que confor­ mamos a diario a través de nuestras actividades ya sean positivas o negativas. Y el ve­ redicto puede derivar, consecuentemente, del Street Art: como arte urbano, representa sólo uno más de los elementos que nos pasan desapercibidos. Hemos de trascender 148 Arte, Individuo y Sociedad 2014, 26(1), 137-151 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García las fronteras del arte y alcanzar las mentalidades –fin último de todo proyecto– para extraer conclusiones. Arte, Individuo y Sociedad 2014, 26 (1), 137-151 A lot of people never use their initiative because no-one told them to [Mucha gente no emplea nunca su propia iniciativa porque nadie le dijo que lo hiciera] (Banksy, 2001: 23) En ocasiones se critica a Córdoba por su aparente carácter rancio sin hacer referencia, precisamente, a su abolengo, sino a su omnipresente visión de color sepia: esta inicia­ tiva, propia de un curador de las calles, podría actualizar dichas concepciones con una explosión de colores, solucionando de paso un “problema” público y aportando un nuevo atractivo a la ciudad. Año tras año, las arcas municipales destinan una impor­ tante cantidad de dinero a la limpieza de los grafitis presentes en sus infraestructuras. La creación de un programa iconográfico, de un discurso a través del cual ofrecer una imagen determinada –la temática podría renovarse– ha de entenderse como un ahorro para la comunidad y, al mismo tiempo, como un escaparate para el exterior; máxime, cuando en España es una práctica tan joven que despierta la curiosidad y tiene gran repercusión. 149 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Arte, Individuo y Sociedad 2014, 26 (1), 137-151 Pablo Allepuz-García Pablo Allepuz-García El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Sea como fuere, las nuevas tecnologías habrán de jugar un papel principal en el orden que se establezca: debemos disponer y hacer disponible a aquel que lo requiera toda la información sobre la materia, dentro de un mapa temático de Street Art en Córdoba; y aquí cobraría importancia el estudio que se viene apuntando durante todo el artículo. Éste tendría que ser actualizado a menudo, pero contaría con una serie de elementos permanentes –aquellos dictaminados por el Ayuntamiento tras el concurso de ideas–. Entonces, ¿es el Street Art una entelequia? Como hemos visto, existen algunas manifestaciones, mas Córdoba aún lo mantiene en un plano de inferioridad. A pesar de la contradictio in terminis, quizá haya que tender un puente a estos artistas hacia la legalidad y el reconocimiento mediante esta suerte de iniciativas, que pondrían en valor una dimensión de la ciudad apenas estudiada y que no puede continuar ocultán­ dose. En definitiva, se trata de adaptar nuestros modelos al dinamismo y la plurali­ dad; de ampliar la oferta cultural de Córdoba hacia sectores y franjas de edad que hoy están cambiando el mundo; de hacer de la ciudad un monumento integral, por fin… Referencias bibliográficas AA.VV. (2005). Granada Graffiti. 2005-1989. Armilla: El Lunes Adorno, T. (1983). Teoría estética. Barcelona: Orbis AA.VV. (2005). Granada Graffiti. 2005-1989. Armilla: Adorno, T. (1983). Teoría estética. Barcelona: Orbis AA.VV. (2005). Granada Graffiti. 2005-1989. Armilla Adorno, T. (1983). Teoría estética. Barcelona: Orbis Álvaréz, L. (2007). La aporía del arte: ‘hipertrofia’ del entendimiento y ‘represión’ de la sensibilidad. En Fedro. Revista de Estética y Teoría de las Artes, 5, pp. 50-75 y pp Banksy (2001). Banging Your Head Against a Brick Wall. United Kingdom: Weapons of Mass Distraction Banksy (2001). Banging Your Head Against a Brick Wall. United Kingdom: Weapons of Mass Distraction Benjamin, W. (2005). Libro de los pasajes. Madrid: Akali Benjamin, W. (2005). Libro de los pasajes. Madrid: Akali j ( ) p j Cambil Hernández, M. E. (2012). Los graffitis y el espacio urbano: el ‘niño de las pinturas’. Quiroga, 2, pp. 10-29 Cambil Hernández, M. E. (2012). Los graffitis y el espacio urbano: el ‘niño de las pinturas’. Quiroga, 2, pp. 10-29 Cook, N. (2001). De Madonna al canto gregoriano. Una muy breve introducción a la música. Madrid: Alianzai Cook, N. (2001). De Madonna al canto gregoriano. Una muy breve introducción a la música. Madrid: Alianzai De Diego, J. (2000). Graffiti, la palabra y la imagen: un estudio de la expresión en las culturas urbanas en el fin del siglo XX. Barcelona: Los libros de la fronterai De Diego, J. (2000). Graffiti, la palabra y la imagen: un estudio de la expresión en las culturas urbanas en el fin del siglo XX. Barcelona: Los libros de la fronterai Gadamer, H-G. (1999). Verdad y Método. Fundamentos de una hermenéutica filosó­ fica, Volumen I. Salamanca: Sígueme Gadamer, H-G. (1999). Verdad y Método. Fundamentos de una hermenéutica filosó­ fica, Volumen I. Salamanca: Sígueme fi g Hauser, A. (1981). Teorías del arte. Barcelona: Guadarrama fi g Hauser, A. (1981). Teorías del arte. Barcelona: Guadarrama Hessel, F. (1984). Ein Flaneur in Berlin. Berlin Jiménez, J. (2002). Teoría del arte. Madrid: Tecnos-Alianza Lefebvre, H. (1974). Le droit à la ville suivi de Espace et politique. Paris: Anthropos López Jiménez, Á. (1998). El arte de la calle. Reis: Revista española de investigacio­ nes sociológicas, 84, pp. 173-194 , ( ) p p q p López Jiménez, Á. (1998). El arte de la calle. Reis: Revista española de investigacio­ nes sociológicas, 84, pp. 173-194 ( ) p p q p López Jiménez, Á. (1998). Referencias electrónicas Abarca Sanchís, F. J. (2010). El postgraffiti, su escenario y sus raíces: graffiti, punk, skate y contrapublicidad, (Tesis de doctorado, Universidad Complutense de Madrid). Recuperado de http://eprints.ucm.es/11419/1/T32410.pdfi Arjona, A.R. (2012, 14 de octubre). Callejero pirata: ‘Por fin te he encontrado’. Diario Córdoba. Recuperado de http://www.diariocordoba.com/noticias/cordo­ balocal/callejero-pirata-por-fin-te-he-encontrado-_752624.html i Figueroa Saavedra, F. (1999). El ‘graffiti movement’ en Vallecas: historia, esté­ tica y sociología de una subcultura urbana, (1980-1996). (Tesis de doctorado, Universidad Complutense de Madrid). Recuperado de http://biblioteca.ucm.es/ tesis/19972000/H/0/H0041601.pdf p Lima Barbosa, L. R. (2011, 8 de noviembre). Conferencia inaugural de la celebración de los 80 años de Bellas Artes. Universidad de Caldas. Recuperado de http:// www.ucaldas.edu.co/index.php?option=com_content&view=article&catid=419 %3Auniversidad-al-dia&id=6419%3A-una-sociedad-sin-arte-es-una-sociedad- sin-vida-luis-roberto-lima-barbosa&Itemid=1039 London Transport Museum (2012, 18 de mayo): Mind the map. Inspiring art, design and cartography. Recuperado de http://www.ltmuseum.co.uk/whats-on/exhibi­ tions/past-exhibitions/395-exhibition-mind-the-map p p Moyinedo, S. (2010): La obra de la crítica. Formulaciones metodológicas para una metacrítica. Figuraciones. Teoría y crítica de artes, 7. Recuperado de http://www. revistafiguraciones.com.ar/numeroactual/articulo.php?ida=154&idn=7&arch=1 Partum, E. (2001, 16 de abril): Ewa Partum. Retrospektive 1965-2000. Badischer Kunstverein. Recuperado de http://www.badischer-kunstverein.de/index.php?Dir ection=Programm&list=Ausstellungen&Jahr=2001&Detail=260i Pérez, Y (2012, 26 de julio). Algo más que graffitis. Diario Córdoba. Recuperado de http://www.diariocordoba.com/noticias/contraportada/algo-mas-que-graffi­ tis_732818.html Referencias bibliográficas El arte de la calle. Reis: Revista española de investigacio­ nes sociológicas, 84, pp. 173-194 Román, R. (2007). Del arte con fronteras a las obras nómadas. Si el arte ha muerto: ¿qué es el arte? Fedro, Revista de Estética y Teoría de las Artes, 6, pp. 4-18 Román, R. (2007). Del arte con fronteras a las obras nómadas. Si el arte ha muerto: ¿qué es el arte? Fedro, Revista de Estética y Teoría de las Artes, 6, pp. 4-18 150 Arte, Individuo y Sociedad 2014, 26(1), 137-151 El Street Art y la (in)cultura urbana: el ejemplo de Córdoba Pablo Allepuz-García Ruiz, M. (2009). Disquisiciones graffiteras. Fedro. Revista de Estética y Teoría de las Artes, 8, pp. 39-50 pp Schlögel, K. (2007). En el espacio leemos el tiempo. Sobre Historia de la civilización y Geopolítica. Madrid: Siruela y p Thiebaut, C. (1999). La mal llamada postmodernidad (o las contradanzas de lo mo­ derno). En BOZAL, V. (ed.). Historia de las ideas estéticas y de las teorías artís­ ticas contemporáneas, Volumen II (377-393). Madrid: Visor Promoe (2004): These walls don’t lie. The Long Distance Runner. Burning Heart Records Black Star (1998): Respiration. Mos Def & Talib Kweli are Black Star. Rawkus Records Referencias musicales Black Star (1998): Respiration. Mos Def & Talib Kweli are Black Star. Rawkus Records Promoe (2004): These walls don’t lie. The Long Distance Runner. Burning Heart Records 151 Arte, Individuo y Sociedad 2014, 26 (1), 137-151

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How Should Crypto Lending Be Regulated Under EU Law?

European business organization law review

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How Should Crypto Lending Be Regulated Under EU Law? Accepted: 12 June 2023 / Published online: 31 July 2023 © The Author(s) 2023 Extended author information available on the last page of the article Keywords  Crypto lending · Prudential regulation · Financial stability · EU law · DeFi · Fintech · Crypto assets European Business Organization Law Review (2023) 24:421–438 https://doi.org/10.1007/s40804-023-00293-3 European Business Organization Law Review (2023) 24:421–438 https://doi.org/10.1007/s40804-023-00293-3 ARTICLE ARTICLE 1  Beganski (2022); Hetzner (2022). 2  The overdependence of investors on credit ratings and the flawed business model of credit rating agen- cies fueled the subprime mortgage bubble and are considered to be among the causes of the financial crisis. See generally Partnoy (2017). For the shortcomings of the credit rating agency industry, which contributed to the Eurozone debt crisis, see Gaillard 2013. In the aftermath of the financial crisis the EU adopted the so-called CRA Regulation, which provided for the mandatory registration and supervision of credit rating agencies. The Regulation was amended in 2011 and 2013. For an overview of regulatory reform both in Europe and the US, see generally Coffee Jr (2010). 3 Abstract The collapse of Genesis is the latest in a cascade of failures of crypto lenders. The last year has seen numerous major crypto lenders, such as Celsius, Voyager and BlockFi, going out of business in domino-like fashion. The failures have revealed the vulnerabilities of crypto-market lenders’ business model, most notably the liquidity and maturity mismatches in their loan portfolios, and their markedly weak corporate governance. The present article explores avenues to regulate crypto lend- ing within the framework of EU financial services regulation. It argues that crypto lenders should be taken as falling within the definition of credit institutions under EU law, and thus, as a result, should be subject to the stringent licensing and pruden- tial requirements introduced by the Capital Requirements Directive and Regulation. Prudential regulation is one of the ways that have been suggested for the regulation of crypto-market operators, alongside the investor protection framework. Taking into account that crypto lenders easily operate on a cross-border basis and that prudential regulation is fully harmonized in the EU, we take an EU-wide perspective and focus our analysis on EU law, rather than member state laws. In addition, prudential regu- lation can deal with any systemic risk issues with which investor protection regula- tion cannot deal. However, in order to avoid moral hazard and not give investors the false impression that crypto lenders are safe too-big-to fail institutions, we suggest that crypto lenders should not enjoy the full protection of prudential regulations. In particular, they should not be offered lender of last resort support and they should not be allowed to subscribe into a deposit insurance scheme. Even though it is often said that crypto markets pose no risk to the regulated sector due to limited intercon- nectedness, it should be noted that due to the high leverage of crypto investors, the real risk to the regulated sector comes from the possibility of crypto investors mas- sively liquidating their positions in other asset markets. Keywords  Crypto lending · Prudential regulation · Financial stability · EU law · DeFi · Fintech · Crypto assets Keywords  Crypto lending · Prudential regulation · Financial stability · EU law · DeFi · Fintech · Crypto assets Keywords  Crypto lending · Prudential regulation · Financial stability · EU law · DeFi · Fintech · Crypto assets Vol.:(0123456789) 123 Vol.:(0123456789) 123 422 E. Avgouleas, A. Seretakis 4  Fletcher and Oliver (2022); Findlay et al. (2023). More than $900 million in customer funds rema frozen in Genesis’s bankruptcy. See Sweet (2023). 3  Shimron (2020). 1  Beganski (2022); Hetzner (2022). 2 1  Beganski (2022); Hetzner (2022). 2  The overdependence of investors on credit ratings and the flawed business model of credit rating agen- cies fueled the subprime mortgage bubble and are considered to be among the causes of the financial crisis. See generally Partnoy (2017). For the shortcomings of the credit rating agency industry, which contributed to the Eurozone debt crisis, see Gaillard 2013. In the aftermath of the financial crisis the EU adopted the so-called CRA Regulation, which provided for the mandatory registration and supervision of credit rating agencies. The Regulation was amended in 2011 and 2013. For an overview of regulatory reform both in Europe and the US, see generally Coffee Jr (2010). 3  Shimron (2020). 4  Fletcher and Oliver (2022); Findlay et al. (2023). More than $900 million in customer funds remain frozen in Genesis’s bankruptcy. See Sweet (2023). 1  Introduction Furthermore, as the recent Celsius debacle demonstrates, the procyclical nature of 123 How Should Crypto Lending Be Regulated Under EU Law? 423 crypto-lending activities, fire sales of investor holdings in other asset classes, high leverage employed, and the risk of depositor runs may give rise to systemic risk.5 Prudential regulation will make crypto-lending institutions safer and more stable. For example, prudential regulation would have prevented crypto lenders’ exposure to a single asset class and would have cured their vulnerability to liquidity risks (e.g., user runs). It would also have limited the ability of crypto lenders to be highly leveraged. In this way crypto lenders would have become more stable and resilient, and the string of recent failures would have been averted. It is plausible to argue that if crypto lenders were subject to prudential regulation, recent crypto-lender failures and attendant investor losses6 would have been prevented, e.g., in the US only banks and similar regulated depositary institutions are allowed to take deposits.7 Unregulated crypto lenders have not been subject to any conduct of business or other rules for the protection of investors or users, making it easy for crypto lend- ers to misrepresent their status and conceal the risks of their products from market users. The present article will treat crypto lending as an activity distinct from other crypto-asset market activities, such as crypto-currency trading or taking custody of crypto assets, which are activities dealt with by the draft EU Regulation on Markets in Crypto-Assets, also known as ‘MiCA’.8 1  Introduction One of the biggest challenges facing policymakers with respect to crypto markets is the treatment of crypto lending. ECB President Lagarde recently stated that crypto lending should be regulated.1 According to the ECB President, the growing inci- dences of fraud, criminal dealings and dubious valuation practices in the crypto- lending space pose severe risks to consumers. One question flowing from her state- ment is: how should crypto lending be regulated? The present article will explore avenues to regulate crypto lending within the framework of EU financial services regulation. It will argue that crypto lenders fall within the definition of credit insti- tutions under EU law. As a result, they should be subject to the stringent licensing and prudential requirements provided by the Capital Requirements Directive and Regulation. It should be noted that crypto lenders are predominantly operating in the US, with their presence in Europe still limited. However, the considerable growth of crypto lending in Europe may lead crypto-lending firms to expand their operations in Europe, thus necessitating a regulatory response from European policymakers. A similar pattern could be observed with regard to the regulation of credit rating agen- cies. The Big Three credit rating agencies were all based in the US. Nevertheless, their expansion in Europe and their role in aggravating and/or causing the financial and sovereign debt crisis forced European policymakers to adopt a comprehensive regulatory and supervisory framework.2 The last few years have seen the exponential growth of crypto lending, with lend- ers such as Celsius, BlockFi and DeFi protocols, such as MakerDAO and Com- pound, dominating the space.3 Nonetheless, the failure of Celsius Network and Voyager has alarmed policymakers to the importance of crypto lenders for crypto markets and the fragility of their business model. Moreover, the spectacular col- lapse of FTX created contagion across the industry and had a spillover effect on crypto lenders, with major firms such as Genesis and BlockFi suspending withdraw- als of customer funds and filing for bankruptcy.4 As the present article will argue, the activities of crypto lenders, which involve the taking of deposits in crypto assets and the granting of crypto loans, resemble banking activities. However, lack of regu- lation creates a competitive advantage for crypto lenders vis-à-vis licensed banks. Unregulated crypto lenders are able to produce returns by taking on excessive risk. 5  Ponnezhath and Wilson (2022). See also IMF (2021). 6  Inductively, the total of investor losses in the Celsius saga was in the vicinity of $4.7 billion. This sum shows how important it is to protect investors and markets from the egregious practices of crypto lenders. Sigalos (2022). 7  Alexander et al. (2014). See 26 U.S.C Sect. 581. The term ‘bank’ means a bank or trust company incorporated and doing business under the laws of the United States or of any State, a substantial part of the business of which consists of receiving deposits and making loans and discounts, or of exercising fiduciary powers similar to those permitted to national banks under authority of the Comptroller of the Currency, and which is subject by law to supervision and examination by State or Federal authority hav- ing supervision over banking institutions. 8  MiCA introduces a regulatory framework for the issuance and trading of crypto assets. It covers crypto assets that are not classified as financial instruments under MiFID II, such as utility tokens and stablecoins. Furthermore, MiCA introduces rules for crypto-asset service providers, which are required to be authorized in order to operate within the EU. Council of the EU (2022). 9  The Ethereum platform is the most popular choice for DeFi financial services and products. The Ethereum blockchain allows the design and employment of highly programmable smart contracts with automated execution. Buterin defined smart contracts as systems which automatically move digital assets according to arbitrary pre-specified rules. See Buterin (2014), p 1. Moreover, Ethereum’s composable software stack ensures that DeFi applications (dapps) are built to integrate and complement one another. See Avgouleas and Seretakis (2022), p 17. 2.1  DeFi The combination of blockchain technology and smart contracts has given rise to a new financial ecosystem known as decentralized finance or DeFi.9 The total value 5  Ponnezhath and Wilson (2022). See also IMF (2021). 7  Alexander et al. (2014). See 26 U.S.C Sect. 581. The term ‘bank’ means a bank or trust company incorporated and doing business under the laws of the United States or of any State, a substantial part of the business of which consists of receiving deposits and making loans and discounts, or of exercising fiduciary powers similar to those permitted to national banks under authority of the Comptroller of the Currency, and which is subject by law to supervision and examination by State or Federal authority hav- ing supervision over banking institutions. 8  MiCA introduces a regulatory framework for the issuance and trading of crypto assets. It covers crypto assets that are not classified as financial instruments under MiFID II, such as utility tokens and stablecoins. Furthermore, MiCA introduces rules for crypto-asset service providers, which are required to be authorized in order to operate within the EU. Council of the EU (2022). 9i 5  Ponnezhath and Wilson (2022). See also IMF (2021). 5  Ponnezhath and Wilson (2022). See also IMF (2021). 6  Inductively, the total of investor losses in the Celsius saga was in the vicinity of $4.7 billion. This sum shows how important it is to protect investors and markets from the egregious practices of crypto lenders. Sigalos (2022). 7  Alexander et al. (2014). See 26 U.S.C Sect. 581. The term ‘bank’ means a bank or trust company incorporated and doing business under the laws of the United States or of any State, a substantial part of the business of which consists of receiving deposits and making loans and discounts, or of exercising fiduciary powers similar to those permitted to national banks under authority of the Comptroller of the Currency, and which is subject by law to supervision and examination by State or Federal authority hav- ing supervision over banking institutions. 8  MiCA introduces a regulatory framework for the issuance and trading of crypto assets. It covers crypto assets that are not classified as financial instruments under MiFID II, such as utility tokens and stablecoins. Furthermore, MiCA introduces rules for crypto-asset service providers, which are required to be authorized in order to operate within the EU. Council of the EU (2022).i 9  The Ethereum platform is the most popular choice for DeFi financial services and products. The Ethereum blockchain allows the design and employment of highly programmable smart contracts with automated execution. Buterin defined smart contracts as systems which automatically move digital assets according to arbitrary pre-specified rules. See Buterin (2014), p 1. Moreover, Ethereum’s composable software stack ensures that DeFi applications (dapps) are built to integrate and complement one another. See Avgouleas and Seretakis (2022), p 17. 123 123 123 424 E. Avgouleas, A. Seretakis locked in DeFi reached an all-time high of $253 billion in December 2021.10 DeFi is a term used to describe an ecosystem comprising financial applications built on top of blockchain networks which do not rely on traditional financial intermedi- aries such as brokerages, exchanges, or banks.11 DeFi aims at replicating existing financial services without the involvement of centralized intermediaries.12 In a DeFi environment the users can maintain full control over their assets and interact with this ecosystem through peer-to-peer (P2P), decentralized applications (dapps). DeFi applications do not need any intermediaries or arbitrators. Pre-set software code specifies the resolution of disputes that can be predicted in advance. 10  Minter (2021). The collapse of the crypto market severely impacted the DeFi sector, with the sector partly recovering during the summer of 2022. 11  Avgouleas and Seretakis (2022), p 13. 12  Ibid., at pp 16–17. 13  See generally De Filippi and Wright (2018) and Dimitropoulos (2020). 14  Schar (2021). 15  The largest decentralized exchange is Uniswap, whose total trading volume exceeds $1 trillion. Quarmby (2022). 16  Wharton Blockchain and Digital Asset Project (2021), p 14. 17  Ibid., p 15. 18  Ibid., p 16. 5  Ponnezhath and Wilson (2022). See also IMF (2021). Essentially, the Code is law among users and thus, in the context of blockchain platforms, it has been given the name ‘Lex Cryptographia’.13 Among the alleged advantages of DeFi is the bypassing of rent-seeking interme- diaries in financial services and the cultivation of an environment where technologi- cal innovation can thrive and offer more consumer choice when it comes to pay- ments and lower transaction costs. According to DeFi proponents, the removal of centralized intermediaries will lead to a more open, transparent and resilient finan- cial ecosystem.14 DeFi infrastructures provide flexibility and transparency in con- tract design as well as a high level of record security. DeFi platforms enable the creation of new financial instruments and digital assets by allowing developers to build on top of existing protocols, customize interfaces, and integrate third-party applications. As a result, they are often compared with lego pieces and referred to as money legos. DeFi operations include decentralized exchanges, decentralized derivatives, insurance, asset management and crypto lending. Decentralized exchanges allow the trading of digital assets without taking custody of user assets, which allows users to re-deploy their assets in other investment activities.15 Decentralized derivatives are tokens that derive their value from an underlying asset or the outcome of an event.16 DeFi insurance services are mostly used for insuring against the risks posed by smart contract failures and hacks of DeFi protocols.17 Claims are paid out with digi- tal assets after the vote of claim assessors. As far as asset management is concerned, DeFi investment funds invest in crypto assets, which are locked up in a smart con- tract.18 The lending market is another fast-growing sector of the DeFi ecosystem. DeFi seeks to bypass the traditional intermediaries in borrowing and lending, most notably banks. DeFi lending and borrowing are governed by smart contracts, and 123 123 How Should Crypto Lending Be Regulated Under EU Law? 425 loans are often overcollateralized, with borrowers depositing collateral in crypto coins. It should be noted that DeFi creates major challenges, especially relating to fraud and market instability and volatility. Indeed, crypto lending has been identified as a potential source of risk to the financial system. Also, crypto markets have recently been implicated in alleged money-laundering schemes and efforts to bypass recent Western sanctions on Russia.19 19  Flitter and Yaffe-Bellany (2022). 20  IMF (2021), p 39. 21  Brainard (2022), p 2. 22  de Hernandez (2022), pp 4–5. 23  Ibid. 24  Arguably, the best way to approach crypto lending is as a form of shadow banking. It should be noted that a run on collateral in the shadow banking sector was held to be one of the main causes of the global financial crisis. See the classic paper by Gorton and Metrick (2012). Other scholars also understand decentralized finance (DeFi) as a form of shadow banking, see, e.g., Allen (2022). 25  Drakopoulos (2021). 26 Aramonte et al (2022) p 2 26  Aramonte et al. (2022), p 2. i 25  Drakopoulos (2021). 2.2  The Nature of DeFi Markets According to the IMF, the tremendous growth and expansion of crypto markets pre- sents risks for financial stability.20 The recent turbulence in crypto markets, includ- ing DeFi markets, has exposed structural vulnerabilities in the ecosystem. In par- ticular, the mayhem in crypto markets has exposed crypto assets’ volatility, with the crypto market witnessing wild price swings.21 Crypto assets exhibit extreme fluc- tuations which are greater than those of other financial assets.22 Moreover, despite claims to the contrary, the correlation between the changes in the price of crypto assets and riskier assets, such as equities, has been increasing over the past few years.23 Another major source of vulnerability is the ability of investors to estab- lish highly leveraged positions, which exacerbate procyclicality and volatility and create, like other forms of shadow banking,24 invisible links of interconnectedness. Dapps, such as trading and lending platforms, facilitate the build-up of leveraged positions. For instance, the maximum permitted margin in decentralized exchanges is higher than in regulated exchanges. Moreover, collateralized lending allows the recycling of collateral, enabling investors to build large exposures using the same crypto assets. Leveraged positions are the first to be unwound when there is down- ward price pressure in crypto markets. Finally, the pseudonymous nature of the DeFi ecosystem and crypto markets more in general may facilitate money laundering, terrorist financing and market manipulation. Furthermore, regulators are unable to have a complete view of the market and monitor financial stability risks.25 What is more, as the Bank of International Settlements notes, anonymity and dependence on collateral undermine DeFi’s goal to promote financial inclusion.26 Especially in the context of DeFi lending, reliance on collateral benefits the owners of assets. 123 123 426 E. Avgouleas, A. Seretakis Most of today’s DeFi activity is outside the regulatory perimeter, but this is a situation that is no longer tenable. Thus, the European Commission has recently proposed a digital finance package aimed at fostering Europe’s competitiveness and innovation in the financial sector. The package includes a Digital Finance Strategy, a Retail Payments Strategy, and legislative proposals on crypto assets and digital operational resilience and a plot regime for market infrastructures powered by dis- tributed ledger technology. But the Digital Package that is still under consideration is only the beginning. 27  In re: CELSIUS NETWORK LLC, et al., Declaration of Alex Mashinsky, Chief Executive Officer of Celsius Network LLC, in Support of Chap. 11 Petitions and First Day Motions, p 2. 28  Ibid. 29  Ibid. 30  Tobias and Ashcraft (2012). 31  FSB (2011). 2.2  The Nature of DeFi Markets EU financial services regulation will soon require a wholesale overhaul in order to keep pace with the digital transformation of the financial value chain both within the EU and globally. 2.3  The Particular Case of Crypto Lending The sudden collapse of Celsius and Voyager has turned the attention of policymak- ers to the fragility of the business model of crypto lenders and their contribution to systemic risk. Crypto lenders, such as Celsius and Voyager, sought to provide a solution to two distinct problems facing crypto holders: lack of liquidity and lack of market purchasing power.27 Crypto holders face a liquidity problem since crypto currencies are not widely accepted as a medium of exchange. As a result, holders of crypto who want to monetize their holdings can convert them into fiat currency.28 Moreover, it offers them the opportunity to earn handsome returns on their crypto holdings, through staking, which is only available to holders of big portfolios.29 Spe- cific crypto lenders engage in secured lending, which allows holders to deposit their assets and borrow fiat currency or other digital assets using their crypto holdings as collateral. Furthermore, users can also earn rewards on these assets at rates that are more favorable than those offered by traditional intermediaries or other crypto platforms. Crypto lenders are in essence performing credit intermediation outside the regular banking system. As a result, they should be understood as a form of shadow bank- ing. For example, Adrian and Ashcraft define shadow banking as ‘a web of special- ized financial institutions that channel funding from savers to investors through a range of securitization and secured funding techniques’,30 while the Financial Stabil- ity Board defines it as ‘credit intermediation involving entities and activities outside the regular banking system’.31 It should be noted that Celsius was one of the biggest crypto platforms in the world. Headquartered in New Jersey, USA, Celsius had, in May 2022, around $12 billion of assets under management and had issued loans in excess of $8 billion. According to its chief executive Alex Mashinsky, the Celsius business model was 123 123 123 How Should Crypto Lending Be Regulated Under EU Law? 427 centered on deploying digital assets to generate income for Celsius’ operations and growth.32 Celsius offered the so-called ‘Earn’ program that enabled users to deposit their digital assets with Celsius, which was then allowed to use these assets in order to generate yield. 32  In re: CELSIUS NETWORK LLC, et al., Declaration of Alex Mashinsky, Chief Executive Officer of Celsius Network LLC, in Support of Chap. 11 Petitions and First Day Motions, p 5. 33  Ibid. 34  Ibid. 35  Ibid., pp 22–23. 36  Oliver (2022). 37  In re: VOYAGER DIGITAL HOLDINGS INC., Declaration of Stephen Ehrlich Chief Executive Office of the Debtors, in Support of Chap. 11 Petitions and First Day Motion, pp 11–12. 38  Singapore-based Three Arrows was one of the best known crypto hedge funds, making large lever- aged bets on rising crypto prices. The collapse of crypto token Luna inflicted heavy losses on Three Arrows, which had made significant investments in the token. Chipolina and Samson (2022). 39  In re: VOYAGER DIGITAL HOLDINGS INC., Declaration of Stephen Ehrlich Chief Executive Office of the Debtors, in Support of Chap. 11 Petitions and First Day Motion, pp 12–24. 40  Oliver et al. (2022). 41  Huang (2022). 32  In re: CELSIUS NETWORK LLC, et al., Declaration of Alex Mashinsky, Chief Executive Officer of Celsius Network LLC, in Support of Chap. 11 Petitions and First Day Motions, p 5. 33  Ibid. 37  In re: VOYAGER DIGITAL HOLDINGS INC., Declaration of Stephen Ehrlich Chief Executiv Office of the Debtors, in Support of Chap. 11 Petitions and First Day Motion, pp 11–12. 35  Ibid., pp 22–23. i 39  In re: VOYAGER DIGITAL HOLDINGS INC., Declaration of Stephen Ehrlich Chief Executiv Office of the Debtors, in Support of Chap. 11 Petitions and First Day Motion, pp 12–24. 36  Oliver (2022). 41  Huang (2022). fi 38  Singapore-based Three Arrows was one of the best known crypto hedge funds, making large leve aged bets on rising crypto prices. The collapse of crypto token Luna inflicted heavy losses on Thr Arrows, which had made significant investments in the token. Chipolina and Samson (2022). fi 40  Oliver et al. (2022). 2.3  The Particular Case of Crypto Lending Users earned rewards on their assets in the form of payment in kind interest or Celsius tokens, with the annual percentage yield reaching 17% on certain assets.33 The company generated the yield through various activities, includ- ing lending services, and also provided borrowing services to retail and institutional clients. Furthermore, the company had extended loans to its clients secured by digi- tal assets, which it was allowed to rehypothecate.34 Moreover, it engaged in staking and deployed digital assets into automated market maker or lending protocols, for a fee.35 Losses suffered on certain illiquid investments and the collapse of the crypto market led to massive withdrawals by depositors, destabilizing the company, which was forced to impose a ban on withdrawals to stem the depositor run.i Voyager was the next major crypto lender to file for bankruptcy following the turbulence in the crypto market and the default of one of its borrowers.36 Voyager operated a crypto-currency platform that enabled its users to trade and store crypto currency. Customers were able to deposit their crypto holdings and earn interest on them.37 Voyager was able to pay interest on deposits by lending crypto currency deposited on its platform to third parties at a pre-negotiated interest rate. The wide- spread panic in crypto-currency markets, the announcement by Celsius Network that it was suspending all account withdrawals and transfers and the collapse of Three Arrows, a crypto fund,38 which had borrowed more than $670 million, led to a run by Voyager’s customers.39 The company was forced to suspend withdrawals and trading activity on its platform and file for bankruptcy. i Finally, crypto lenders were severely hit by the sudden collapse of crypto exchange FTX. The FTX empire, founded by fallen crypto mogul Sam Bankman- Fried, included the FTX crypto exchange and Alameda Research, a quantitative crypto hedge fund speculating in digital assets.40 Following the announcement of Binance, a rival exchange, that it would liquidate its holdings in FTT, FTX’s native token, FTX suffered an effective run on the bank, with customers’ withdrawal requests amounting to an estimated $6 billion over 3 years.41 FTX, which was using 123 123 428 E. Avgouleas, A. Seretakis customer funds in order to finance the risky and illiquid bets by its affiliated trading firm Alameda Research, was unable to fulfil the requests.42 The resulting liquidity crunch forced FTX to file for bankruptcy. 43  John Ray, the new chief executive and chief restructuring officer of FTX, stated: ‘Never in my career have I seen such a complete failure of corporate controls and such a complete absence of trustworthy financial information as occurred here.’ See In re: FTX TRADING LTD et al., Debtors, Declaration of John J. Ray III in Support of Chap. 11 Petitions and First Day Pleadings, p 2. According to bankruptcy lawyers, Sam Bankman-Fried ran FTX as a personal fiefdom with a substantial amount of money being used to buy vacation homes in the Bahamas. Kinder (2022). 44 2.3  The Particular Case of Crypto Lending The bankruptcy proceedings have revealed aggressive risk-taking, an utter lack of corporate controls and risk management, absence of transparency and trustworthy financial information, and self-dealing.43 In particular, the case exposed the poor corporate governance standards and lack of accountability permeating the crypto industry. Headquartered in Nassau, the Baha- mas, FTX had a three-person board, including its founder Sam Bankman-Fried and a lawyer in Antigua.44 Indeed, some companies of the FTX Group never even held a board meeting. FTX’s collapse had a wider impact, leading to widespread conta- gion in crypto markets and a market-wide run on crypto lenders, which were forced to halt redemptions and loan originations.45 Crypto lenders’ difficulties revealed the inherent vulnerability of their business model caused by the liquidity and duration mismatch of their loan portfolio. customer funds in order to finance the risky and illiquid bets by its affiliated trading firm Alameda Research, was unable to fulfil the requests.42 The resulting liquidity crunch forced FTX to file for bankruptcy. The bankruptcy proceedings have revealed aggressive risk-taking, an utter lack of corporate controls and risk management, absence of transparency and trustworthy financial information, and self-dealing.43 42  Michaels et al. (2022). 42  Michaels et al. (2022). 43  John Ray, the new chief executive and chief restructuring officer of FTX, stated: ‘Never in my career have I seen such a complete failure of corporate controls and such a complete absence of trustworthy financial information as occurred here.’ See In re: FTX TRADING LTD et al., Debtors, Declaration of John J. Ray III in Support of Chap. 11 Petitions and First Day Pleadings, p 2. According to bankruptcy lawyers, Sam Bankman-Fried ran FTX as a personal fiefdom with a substantial amount of money being used to buy vacation homes in the Bahamas. Kinder (2022). 44  CBS News (2022). 45  Sigalos and Capoot (2022). 46  According to Makavor and Schoar (2022), p 26, NFTs are ‘a unique piece of data stored on a block- chain. The data can be associated with a particular digital or physical asset or a license to use the asset for a specified purpose.’ 47  Collateral made up of crypto assets can be very volatile and can quickly lose value. For instance, Sam Bankman-Fried argued in a letter to staff that the value of collateral held by FTX dropped from $60 bil- lion to $9 billion. De (2022). 45  Sigalos and Capoot (2022). 44  CBS News (2022). 46  According to Makavor and Schoar (2022), p 26, NFTs are ‘a unique piece of data stored on a block- chain. The data can be associated with a particular digital or physical asset or a license to use the asset for a specified purpose.’ 3.1  What Are the Risks? The key financial stability threat of crypto lending comes from the excessive volatil- ity of crypto-currency markets and the fact that lots of crypto assets, such as non- fungible tokens (NFTs),46 are very complex and very difficult to value, making it very difficult to obtain adequate collateral to secure the loan.47 So, as a result, user leverage within the system remains uncontrolled. This practice exposes crypto lend- ers to suspicions and rumors about their financial health, thus causing market panic, manifested as depositor runs, which expose the well-concealed liquidity imbalances within crypto lenders, leading crypto lenders and crypto-exchange platforms to face the risk of illiquidity. An indicative example is the FTX debacle where market con- fidence in FTX evaporated shortly after the release of a report by crypto-currency news platform CoinDesk, which on this occasion revealed the close ties between 46  According to Makavor and Schoar (2022), p 26, NFTs are ‘a unique piece of data stored on a block- chain. The data can be associated with a particular digital or physical asset or a license to use the asset for a specified purpose.’ i 47  Collateral made up of crypto assets can be very volatile and can quickly lose value. For instance, Sam Bankman-Fried argued in a letter to staff that the value of collateral held by FTX dropped from $60 bil- lion to $9 billion. De (2022). 123 used to buy vacation homes in the Bahamas. Kinder (2022). 44  CBS News (2022). 45  Sigalos and Capoot (2022). 46  According to Makavor and Schoar (2022), p 26, NFTs are ‘a unique piece of data stored on a block- chain. The data can be associated with a particular digital or physical asset or a license to use the asset for a specified purpose.’ 47 Collateral made up of crypto assets can be very volatile and can quickly lose value For instance Sam 123 How Should Crypto Lending Be Regulated Under EU Law? 429 Alameda and FTX.48 In this way, instability can spread to other institutions or mar- ket segments (contagion), resulting in a generalized confidence crisis.49 Even though the interconnectedness between crypto lenders and mainstream financial institutions is limited, a market panic, including a flight to save assets, is a behavioral phenom- enon and is very hard to contain ex ante.50 A valid concern here is whether investor runs from the crypto markets can evolve into a generalized confidence crisis, despite the fact that the links between crypto lenders and regulated financial institutions appear to be limited. Moreover, the number of retail investors with exposures to crypto-currency mar- kets is ever increasing. The proportion of Bitcoin supply held by retail investors has reached an all-time high at 17%.51 The volatility of crypto currencies and crypto markets, and their boom and bust cycles, can leave investors exposed to significant losses and amplify market instability through the aforementioned collateral channel. For example, a recent paper by the Bank for International Settlements found that the overwhelming majority of retail investors in Bitcoin, around 73–81%, lost money on their initial investment.52 Investors that lose money on their crypto investments may be forced to sell assets that they hold in other markets, thus putting a downward pressure on prices. As a result, contagion can spread to unrelated markets. It should be noted here that leverage is an inherent characteristic of crypto mar- kets because crypto exchanges allow investors to take highly leveraged positions and borrow heavily. Leveraged positions are the first to be unwound and additional sell- ing activity adds further downward price pressure,53 further diminishing the value of crypto assets as collateral. In addition, given the liquidity problems facing crypto markets, namely the imbalances between supply and demand, users of crypto lend- ing also face a marked settlement risk, i.e., the risk that their trade will not settle, and their position will remain open. What is more, widespread incidents of fraud have been observed in the crypto lending markets. The opaque and complex nature of crypto lending provides fertile ground for fraudsters. 53  See Avgouleas (2010) and Shleifer and Vishny (2011). 48  Nelson and Schickler (2022). 49  See Elliott et al. (2014). 50  According to the FSB, this is also the main risk that non-bank financial intermediation poses to the regulated sector. Even though the interconnectedness between NBFI operators and institutions in the reg- ulated sector is very limited, liquidation of positions in asset markets (fire sales) that regulated financial institutions carry on their balance sheets presents stability and solvency issues for regulated institutions as well. This effect is the main systemic risk concern emanating from the crypto sector. See FSB (2022a). 51 Thouvalas (2022) 52  Auer et al. (2022). 48  Nelson and Schickler (2022). f 51  Thouvalas (2022). 49  See Elliott et al. (2014). 48  Nelson and Schickler (2022). 49  See Elliott et al. (2014). 50  According to the FSB, this is also the main risk that non-bank financial intermediation poses to the regulated sector. Even though the interconnectedness between NBFI operators and institutions in the reg- ulated sector is very limited, liquidation of positions in asset markets (fire sales) that regulated financial institutions carry on their balance sheets presents stability and solvency issues for regulated institutions as well. This effect is the main systemic risk concern emanating from the crypto sector. See FSB (2022a). 51  Thouvalas (2022). 52  Auer et al. (2022). 53  See Avgouleas (2010) and Shleifer and Vishny (2011). 123 For example, in July 2022, the Securities and Exchange Com- mission (SEC) issued a cease and desist order against US crypto lender Voyager for falsely presenting itself as being covered by the US Federal Deposit Insurance Corporation (hereinafter ‘FDIC’), misleading users into believing that their deposits were insured by the FDIC and the FDIC would insure them against the failure of 123 430 E. Avgouleas, A. Seretakis Voyager.54 Finally, concerns have been raised regarding the potential use of crypto lending as a vehicle for money laundering, tax evasion and terrorist financing. Voyager.54 Finally, concerns have been raised regarding the potential use of crypto lending as a vehicle for money laundering, tax evasion and terrorist financing. 57  The rules, e.g., impose limits on the number of directorships, mandate the separation of the positions of chairman and CEO, and provide for training and induction for new board members and periodic self- evaluation exercises. Furthermore, significant institutions are required to establish a remuneration com- mittee, a nomination committee composed of non-executive members and a risk committee composed of non-executive board members. Institutions must have a risk management function independent of the operational function. The risk management function must be actively involved in elaborating the institu- tion’s risk strategy. See Clarke (2020).f 54  Federal Deposit Insurance Corporation and Board of Governors of the Federal Reserve System (2022), p 1. 55  Art. 4(1)(1) of Regulation (EU) No 575/2013 of the European Parliament and the Council of 26 June 2013 on prudential requirements for credit institutions and investment firms and amending Regulation (EU) No 648/2012. 56  The CRD package implemented the Basel III agreement adopted in the aftermath of the financial crisis. 57  The rules, e.g., impose limits on the number of directorships, mandate the separation of the positions of chairman and CEO, and provide for training and induction for new board members and periodic self- evaluation exercises. Furthermore, significant institutions are required to establish a remuneration com- mittee, a nomination committee composed of non-executive members and a risk committee composed of non-executive board members. Institutions must have a risk management function independent of the operational function. The risk management function must be actively involved in elaborating the institu- tion’s risk strategy. See Clarke (2020). 58  Remuneration policies should be consistent with and promote sound and effective risk management, policies should be in line with the firm’s business strategy, objectives, values and long-term interests of the firm, and the implementation of the remuneration policy should be subject to central and inde- pendent internal review by the firm’s management body at least annually. The remuneration rules impose stringent limits regarding the structure of remuneration, including a bonus cap, capping variable remu- neration at 100% of the fixed component for material risk-takers. The bonus can be raised to 200% of the fixed remuneration with shareholder approval. See EBA (2015). 54  Federal Deposit Insurance Corporation and Board of Governors of the Federal Reserve Syste (2022), p 1. 55 55  Art. 4(1)(1) of Regulation (EU) No 575/2013 of the European Parliament and the Council of 26 Jun 2013 on prudential requirements for credit institutions and investment firms and amending Regulatio (EU) No 648/2012.i p p y (2022), p 1. 55  Art. 4(1)(1) of Regulation (EU) No 575/2013 of the European Parliament and the Council of 26 June 2013 on prudential requirements for credit institutions and investment firms and amending Regulation (EU) No 648/2012. 56  The CRD package implemented the Basel III agreement adopted in the aftermath of the financial crisis. ( ) 56  The CRD package implemented the Basel III agreement adopted in the aftermath of the financial crisis. 3.2  Proposed Regulatory Response: A Prudential Regime for Crypto Lenders The activities of crypto lenders, which involve the taking of deposits in crypto assets and the granting of crypto loans, resemble the activities of credit institutions. Pursu- ant to the Capital Requirements Regulation (CRR), a credit institution is defined as an ‘undertaking the business of which is to take deposits or other repayable funds from the public and to grant credit for its own account’.55 Credit institutions are sub- ject to a strict licensing regime built upon the prudential rules introduced by the Capital Requirements Directive (CRD) and the Capital Requirements Regulation.56 The rules apply to banks and investment firms and include stringent capital require- ments and liquidity requirements. Moreover, prudential oversight under the Regu- lation extends to corporate governance provisions which seek to ensure the inde- pendence and diversity of the board of directors and strengthen risk management.57 Systems and controls requirements would make sure that crypto lenders remain safe from the risk of cyber attacks. Furthermore, the prudential rules impose remu- neration restrictions, whose goal is to promote prudent risk-taking and ensure that remuneration policies are aligned with the long-term interests of the institutions.58 Given that crypto lenders satisfy the definition of credit institutions under EU law, EU bank prudential regulation should be extended to crypto lenders as an effective remedy against the risks created by crypto lending. As a result, crypto lenders would need to be licensed and follow the complex web of prudential rules imposed by the CRD and CRR. The application of a national regulatory regime is another possibil- ity. Nevertheless, taking into account that the activities of crypto lenders resemble The activities of crypto lenders, which involve the taking of deposits in crypto assets and the granting of crypto loans, resemble the activities of credit institutions. Pursu- ant to the Capital Requirements Regulation (CRR), a credit institution is defined as an ‘undertaking the business of which is to take deposits or other repayable funds from the public and to grant credit for its own account’.55 Credit institutions are sub- ject to a strict licensing regime built upon the prudential rules introduced by the Capital Requirements Directive (CRD) and the Capital Requirements Regulation.56 i Given that crypto lenders satisfy the definition of credit institutions under EU law, EU bank prudential regulation should be extended to crypto lenders as an effective remedy against the risks created by crypto lending. 3.2  Proposed Regulatory Response: A Prudential Regime for Crypto Lenders As a result, crypto lenders would need to be licensed and follow the complex web of prudential rules imposed by the CRD and CRR. The application of a national regulatory regime is another possibil- ity. Nevertheless, taking into account that the activities of crypto lenders resemble 57  The rules, e.g., impose limits on the number of directorships, mandate the separation of the positions of chairman and CEO, and provide for training and induction for new board members and periodic self- evaluation exercises. Furthermore, significant institutions are required to establish a remuneration com- mittee, a nomination committee composed of non-executive members and a risk committee composed of non-executive board members. Institutions must have a risk management function independent of the operational function. The risk management function must be actively involved in elaborating the institu- tion’s risk strategy. See Clarke (2020).f 58  Remuneration policies should be consistent with and promote sound and effective risk management, policies should be in line with the firm’s business strategy, objectives, values and long-term interests of the firm, and the implementation of the remuneration policy should be subject to central and inde- pendent internal review by the firm’s management body at least annually. The remuneration rules impose stringent limits regarding the structure of remuneration, including a bonus cap, capping variable remu- neration at 100% of the fixed component for material risk-takers. The bonus can be raised to 200% of the fixed remuneration with shareholder approval. See EBA (2015). 123 123 How Should Crypto Lending Be Regulated Under EU Law? 431 the activities of banks, which are regulated at EU level, and that crypto lenders sat- isfy the definition of a credit institution under the CRR, the application of the EU’s prudential bank regulatory regime is the appropriate solution. the activities of banks, which are regulated at EU level, and that crypto lenders sat- isfy the definition of a credit institution under the CRR, the application of the EU’s prudential bank regulatory regime is the appropriate solution. Crypto lenders are currently not captured by banking regulation. In the US, numerous state regulators and the SEC have taken the view that the interest-bearing accounts offered by crypto lenders are unregistered securities. 59  In the Matter of BlockFi Lending LLC, SEC Order. 60  Pursuant to the Howey Test ‘an investment contract for purposes of the Securities Act means a con- tract, transaction or scheme whereby a person invests his money in a common enterprise and is led to expect profits solely from the efforts of the promoter or a third party.’ Securities and Exchange Commis- sion v. W. J. Howey Co., 328 U.S. 293 (1946). 61  Mahoney (2021), Mahoney (1995) and Coffee Jr (1984). 62  Avgouleas (2009). 63  Laux and Leuz (2010), pp 93–118. 64  Cranston et al. (2018), p 31. 63  Laux and Leuz (2010), pp 93–118. 9  In the Matter of BlockFi Lending LLC, SEC Orde 64  Cranston et al. (2018), p 31. 3.2  Proposed Regulatory Response: A Prudential Regime for Crypto Lenders For instance, in Feb- ruary 2022,f the SEC charged BlockFi, a major crypto lender, with failing to register the offers and sales of its retail crypto-lending product.59 BlockFi offered so-called Interest Accounts (‘BIAs’) to investors, through which the latter lent crypto assets to BlockFi in exchange for BlockFi’s promise to provide a variable monthly interest payment. BlockFi generated the yield paid out to investors by making loans of crypto assets, lending dollars and investing in equities and futures. The SEC determined that the products offered by BlockFi were investment contracts pursuant to the Howey test.60 In particular, the SEC held that investors in BIAs had a reasonable expectation that BlockFi would use the invested crypto assets in BlockFi’s lending and principal investing activity and that they would obtain a future profit in the form of inter- est payments, resulting from BlockFi’s efforts. As a result, the SEC considered that BIAs were securities, which were required to be registered with the SEC. BlockFi violated the Securities Act of 1933 by offering and selling securities without filing a registration statement. As a result, US regulators seek to regulate crypto lenders and protect the pub- lic against their risks via securities law. Nevertheless, securities regulation is not suitable for tackling the risks posed by crypto lending. Instead, it may exaggerate financial instability. Securities regulation is based on disclosure.61 In the event of a market panic, market players do not act rationally and it is unlikely that they will stop ‘running’ when faced with more information. On the contrary, the disclosure of more, usually negative information, will accelerate the run.62 For instance, accord- ing to numerous commentators, fair-value disclosures contributed to the financial crisis of 2008–2009 by increasing leverage during boom times and accelerating write-downs during the bust.63i Cranston et al. define prudential regulation as the thick and complex web of rules employed to (a) keep financial institutions safe and a going concern, and, failing that, (b) to assist their resolution and/or restructuring, and (c) to augment the resil- ience of financial systems to withstand shocks.64 Even though crypto lending is a form of narrow banking and the usual rationales for prudential regulation, namely 64  Cranston et al. (2018), p 31. 123 432 E. Avgouleas, A. Seretakis fractional reserve and depositor protection, may not apply, the risks created by the crypto-lending industry are important enough to justify the full panoply of pruden- tial regulation. 65  In its proposed framework for the regulation of crypto-asset activities the Financial Stability Board states that where crypto assets and intermediaries perform an equivalent economic function to one per- formed by instruments and intermediaries in the traditional financial system, they should be subject to regulations in line with the principle of ‘same activity, same risk, same regulation’. See FSB (2022b), p 1. As a result, the FSB argues in favor of extending prudential rules on capital and liquidity to crypto- asset companies when undertaking similar functions to banks. See FSB (2022c), p 6 and Annex I. 66  ECB (2022). 68  Liquidity requirements are composed of the Liquidity Coverage Ratio and the Net Stable Funding Ratio. The Liquidity Coverage Ratio seeks to ensure that institutions have enough liquid assets to with- stand a 30-day stress period. The Net Stable Funding Ratio forces institutions to finance long-term assets with long-term liabilities. See Bonner and Hilbers (2015). 69 67  It should be noted that the exact requirements for own funds that banks should set aside for exposure to the crypto-market risk are not known until the BIS finalizes its prudential standard for credit institu- tions’ exposure to the crypto markets. BIS (2022). 65  In its proposed framework for the regulation of crypto-asset activities the Financial Stability Board states that where crypto assets and intermediaries perform an equivalent economic function to one per- formed by instruments and intermediaries in the traditional financial system, they should be subject to regulations in line with the principle of ‘same activity, same risk, same regulation’. See FSB (2022b), p 1. As a result, the FSB argues in favor of extending prudential rules on capital and liquidity to crypto- asset companies when undertaking similar functions to banks. See FSB (2022c), p 6 and Annex I. 66  ECB (2022). 67  It should be noted that the exact requirements for own funds that banks should set aside for exposure to the crypto-market risk are not known until the BIS finalizes its prudential standard for credit institu- tions’ exposure to the crypto markets. BIS (2022). 68  Liquidity requirements are composed of the Liquidity Coverage Ratio and the Net Stable Funding Ratio. The Liquidity Coverage Ratio seeks to ensure that institutions have enough liquid assets to with- stand a 30-day stress period. The Net Stable Funding Ratio forces institutions to finance long-term assets with long-term liabilities. See Bonner and Hilbers (2015). 69  Armour et al. (2016), p 279. 69  Armour et al. (2016), p 279. 69  Armour et al. (2016), p 279. 3.2  Proposed Regulatory Response: A Prudential Regime for Crypto Lenders As the Celsius and Voyager debacles demonstrated, crypto lenders face the risk of investor runs, which can lead to their demise, triggering a cascade of failures in crypto markets. Turbulence in crypto markets can quickly spread to the mainstream financial system, posing a threat to global financial stability. What is more, crypto lending is a very important segment of open finance markets. How- ever, paradoxically, crypto lending is introducing a new form of intermediation in the open finance market, with the operations of crypto lenders resembling those of banks. Consequently, taking a functional approach, regulation should not distinguish between the two types of intermediaries, i.e., the mainstream lending institutions and crypto lenders.65i Crypto lenders satisfying the definition of a credit institution would need to be licensed in accordance with the Capital Requirements Directive and the criteria it imposes for the assessment of licensing requests. The ECB has stated that when assessing licensing requests covering crypto-asset activities and services, the ECB and the national competent authorities must examine how the proposed activity matches the overall activity and risk profile of the institution, whether the institu- tion’s policies and procedures are adequate to identify and tackle the risks unique to crypto assets and whether senior managers and board members have knowledge and experience in IT and crypto markets.66 The application of these licensing crite- ria would ensure that only crypto lenders with sound business models and internal governance and competent senior management would be able to obtain a license as credit institutions. Prudential regulatory tools include capital requirements,67 liquidity require- ments,68 corporate governance and remuneration rules, lender of last resort facilities and deposit insurance.69 The application of prudential rules, excluding lender of last resort and deposit insurance arrangements in order not to heighten moral hazard, would have averted the recent collapses of Voyager and Celsius. Adequate capital reserves would have ensured the stability of crypto-lending operators and reduced the risk of bankruptcy. The balance sheet hole would have been covered. Prudential 69  Armour et al. (2016), p 279. 123 123 How Should Crypto Lending Be Regulated Under EU Law? 433 regulation would also have prevented concentration of the balance sheet on a single asset class. Moreover, liquidity requirements would have required crypto lenders to hold some of their assets in liquid form, thus ensuring that they had enough funds to repay users and avert the run. 70  On how deposit insurance creates moral hazard, see Calomiris (1990) and Fischer (1999). 71  ‘Robust segregation and separation between traditional business and crypto business are desirable, although group-wide and step-in risk would also need to be considered even when crypto businesses are located in a separate entity’. IMF (2019). See, inter alia, IMF blog available at https://​blogs.​imf.​org/​2021/​ 10/​01/​crypto-​boom-​poses-​new-​chall​enges-​to-​finan​cial-​stabi​lity (accessed 03 Mar 2023). 72  For an overview of the MiFID product governance regime see Avgouleas and Seretakis (2022), pp 27–28, and Colaert (2019). 70  On how deposit insurance creates moral hazard, see Calomiris (1990) and Fischer (1999). 4  Conclusion This article has examined the mechanics of a key segment of crypto markets. It has also suggested that crypto lenders should be licensed and regulated as credited insti- tutions under EU law in order to boost the stability of the crypto-lending sector and create a level playing field with mainstream lenders such as banks. A careful exami- nation of recent failures has shown that the sector is very unstable and ripe for dras- tic regulation, which will stabilize the sector, limit the risk of contagion triggered by depositor runs and prevent future bankruptcies. It should be noted that Awrey and Macey also suggest a licensing regime for data aggregators in the case of open banking.73 But the authors’ suggestion refers to controlling market power, not boost- ing financial stability like the present proposal. A plausible alternative to licensing would be to systematically curb the promotion of crypto-lending schemes by con- sumer protection regulators. Nevertheless, the regulation of crypto-lending schemes from a consumer protection perspective may not be sufficient to tackle the financial stability risks emanating from their activities. Arguably, a licensing regime for crypto lenders may herald the end of DeFi as an unregulated market segment. But it should be noted here that other parts of the crypto markets, such as trading, will remain unaffected. The application of pruden- tial regulation to crypto lenders will certainly increase the compliance burden and costs, eroding crypto lenders’ profits. However, the recent FTX debacle has revealed that the business model and profits of many crypto firms are the result of regula- tory arbitrage, weak corporate governance, excessive risk-taking and outright fraud. Moreover, the unregulated nature of crypto lending offers crypto lenders an unfair advantage over regulated financial institutions such as banks, which are subject to stringent prudential and conduct of business rules. While there is no evidence of any concrete benefits brought about by crypto lending, the level and kind of risks (market failures) associated with this activity fully justify invasive regulation, and prudential regulation is the most effective tool to control this activity. Acknowledgements  We would like to thank the participants in the CBFL Banking, Finance, Technology Conference organized by the National University of Singapore, School of Law, on 18–19 May 2023, and in the International Conference on Cryptocurrency and Central Bank Digital Currency organized by the Chinese University of Hong Kong, School of Law on 2–3 December 2022, for helpful comments. 73  According to the authors, data aggregators are technological platforms that develop and manage application programming interfaces designed to access the customer data held by incumbent financial institutions and to share it with fintech disruptors. The authors argue that a small handful of data aggre- gators erect substantial barriers to entry and exert monopoly power, thus becoming a new breed of too- big-to fail institutions. Awrey and Macey (2022), p 22. 3.2  Proposed Regulatory Response: A Prudential Regime for Crypto Lenders Corporate governance standards and remuneration rules would have guaranteed effective risk management and prevented excessive risk-taking. For instance, Celsius’s collapse can in part be attributed to the losses suffered from erroneous and risky asset deployment decisions, such as investments in long-term and illiquid assets. To avoid giving false assurances to crypto-lending users, we do not suggest here that crypto lenders should benefit from deposit insurance schemes or lender of last resort facilities. The application of deposit insurance and lender of last resort facili- ties to crypto lenders could create moral hazard and lead to implicit government guarantees being extended to crypto lenders.70 This would prevent crypto lenders from turning into yet another category of too-big-to fail institutions. In the absence of the safety net provided by deposit insurance and lender of last resort facilities, liquidity requirements within prudential regulation are the only way to alleviate the liquidity risks that crypto lenders face. Finally, a licensing regime would also facili- tate the segregation of crypto-asset holdings within the organization, which would boost crypto lenders’ stability71 and offer protection against any designs by crypto operators to misappropriate client holdings. Apart from boosting the stability of individual crypto lenders, prudential regulation would also enhance regulatory scru- tiny and market discipline.i The additional benefit of a licensing regime for crypto lenders is that licensed institutions would also be subject to the MiFID II product governance regime,72 and thus they would have to disclose to users the historical volatility and default rates of their products, thus minimizing any attempts to mislead the investors about the true risks of the product and maximizing user/consumer protection. The product govern- ance requirements introduced by MiFID II have proved to be among the most impor- tant elements of the MiFID II investor protection framework, aimed at ensuring that firms act in their clients’ best interests during all stages of the investment product’s life cycle and preventing mis-selling. As part of the product governance require- ments, a target market of end clients must be identified and periodically reviewed for each product, as must a distribution strategy that should be consistent with the identified target market. Furthermore, assuming that crypto lending could be used for money-laundering activities, authorization would resolve this concern by default because authorized institutions would impose Know Your Customer (‘KYC’) safe- guard requirements on their customers. 123 123 434 E. Avgouleas, A. Seretakis 4  Conclusion Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. 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References Accessed 03 Mar 2023 f Thouvalas A (2022) Portion of Bitcoin supply held by retail investors reaches all-time high: Glassnode. Decrypt, 20 December 2022. https://​decry​pt.​co/​117685/​porti​on-​bitco​in-​supply-​held-​retail-​reach​es-​ all-​time-​high-​glass​node. Accessed 03 Mar 2023 123 123 438 E. Avgouleas, A. Seretakis f Wharton Blockchain and Digital Asset Project (2021) DeFi beyond the hype, the emerging world of decentralized finance. https://​wifpr.​whart​on.​upenn.​edu/​wp-​conte​nt/​uploa​ds/​2021/​05/​DeFi-​Beyond-​ the-​Hype.​pdf. Accessed 03 Mar 2021 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. * Emilios Avgouleas [email protected] Alexandros Seretakis [email protected] Tobias A, Ashcraft A (2012) The regulation of the shadow banking system. Federal Reserve Board of New York Staff Report No. 559. http://ssrn.com/abstract=2043153. 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https://openalex.org/W2188760800

https://jurnal.ugm.ac.id/ijccs/article/download/7541/5864

Indonesian

Analisis Kualitas VoIP pada SCTP Menggunakan ECN dan AQM

Indonesian Journal of Computing and Cybernetics Systems

cc-by-sa

IJCCS, Vol.9, No.2, July 2015, pp. 121~132 ISSN: 1978-1520 IJCCS, Vol.9, No.2, July 2015, pp. 121~132 ISSN: 1978-1520  121 121  Analisis Kualitas VoIP pada SCTP Menggunakan ECN dan AQM La Surimi*1, MHD. Reza M.I. Pulungan2 1Program Studi S2/S3 Ilmu Komputer, FMIPA UGM, Yogyakarta 2Jurusan Ilmu Komputer dan Elektronika, FMIPA UGM, Yogyakarta email: *[email protected],[email protected] Abstrak VoIP merupakan aplikasi real time yang kualitasnya sangat tergantung pada delay dan jitter, yang mana hal ini sulit dipenuhi oleh protokol yang bersifat reliable dan memiliki congestion control seperti TCP. Di sisi lain penggunaan UDP yang tidak memiliki congestion control menyebabkan peluang terjadinya congestion pada jaringan sangat besar. Penggunaan SCTP sebagai protokol alternatif juga belum mampu mengakomodasi kekurangan TCP dan UDP. Beberapa hasil penelitian menunjukkan perlu adanya perbaikan ataupun modifikasi pada mekanisme congestion control yang dimiliki oleh SCTP. g y g Penggunaan mekanisme ECN dan AQM pada beberapa penelitian menunjukkan bahwa kedua mekanisme ini dapat menurunkan delay dan jitter. Penelitian ini melakukan pengujian terhadap kualitas VoIP di atas SCTP yang menggunakan ECN dan AVQ pada network simulator NS2. Hasil simulasi menunjukkan bahwa penggunaan mekanisme ECN dan AVQ pada protokol SCTP menghasilkan kualitas VoIP yang lebih baik pada kondisi jaringan yang tidak ideal (high Latency low Bandwidth dan low Latency low Bandwidth dari pada penggunaan protokol SCTP tanpa menggunakan mekanisme ECN dan AVQ. Penelitian ini juga melakukan perbandingan nilai MOS panggilan VoIP SCTP yang menggunakan ECN dan AVQ dengan nilai MOS panggilan VoIP yang menggunakan protokol TCP dan UDP. Hasilnya SCTP dengan ECN dan AVQ mengungguli TCP namun belum dapat mengungguli UDP. Keywords— VoIP, SCTP, ECN, AQM,AVQ. Received August 28th,2014; Revised January 8th, 2015; Accepted July 10th, 2015 Abstract VoIP is the real time applications that are highly dependent on the quality of delay and jitter, which it is difficult to be met by protocol that has reliable data transfer feature and congestion control such as TCP. On the other hand the use of UDP that has no congestion control make chance of causing congestion in the network is very large. The use of SCTP as an alternative protocol was also not able to accommodate the weaknesses of TCP and UDP. Some research shows that repairs or modifications to the SCTP congestion control mechanism is needed. The Use of ECN and AQM in some studies show that these two mechanisms can reduce delay and jitter. This study tested the quality of VoIP over SCTP with ECN and AVQ, in NS2. Simulations carried out by independent replication technique, and the results showed that ECN and AVQ can increase the value of MOS VoIP calls significantly in non ideal network scenarios. This study also did comparison of SCTP MOS that uses ECN and AVQ with MOS values VoIP using TCP and UDP. The result showed that SCTP with ECN and AVQ outperform TCP but can not surpass UDP yet. Keywords— VoIP, SCTP, ECN, AQM,AVQ. Keywords— VoIP, SCTP, ECN, AQM,AVQ. Received August 28th,2014; Revised January 8th, 2015; Accepted July 10th, 2015  122 122 ISSN: 1978-1520 1. PENDAHULUAN 1. PENDAHULUAN ransmission Control Protocol (TCP) dan User Datagram Protocol (UDP) merupakan dua buah transport layer yang paling banyak digunakan di internet saat ini. TCP menyediakan layanan reliable data transfer dan mekanisme congestion control TCP banyak digunakan oleh aplikasi yang menuntut reliable data transfer seperti aplikasi berbasis web dan aplikasi file transfer. Berbeda dengan TCP, UDP merupakan transport layer yang bersifat unreliable dan tidak menyediakan mekanisme congestion control. Namun karena kedua sifat itulah UDP banyak dipakai oleh aplikasi yang bersifat real time seperti aplikasi VoIP. Aplikasi real time lebih mementingkan timing guarantees dibanding reliable dan ordered data transfer. Aplikasi VoIP merupakan aplikasi real time yang sangat tergantung pada delay dan jitter, sehingga UDP merupakan pilihan terbaik sebagai transport layer.Tidak adanya congestion control pada UDP menyebabkan peluang terjadinya congestion pada jaringan sangat besar. Selain itu UDP dengan mudahakan memonopoli bandwidth pada suatu link karena sending rate Di lain pihak TCP menjadi tidak cocok dengan aplikasi yang membutuhkan end to end delay yang seminimal mungkin [1]. 2. METODE PENELITIAN Berikut adalah uraian dari tahapan dan metode yang digunakan dalam penelitian : 2.1 Studi Kepustakaan Pengumpulan bahan referensi, seperti jurnal penelitian, prosiding, tesis, buku-buku teori dan sumber-sumber lain termasuk informasi yang diperoleh melalui internet. Pengumpulan bahan referensi, seperti jurnal penelitian, prosiding, tesis, buku-buku teori dan sumber-sumber lain termasuk informasi yang diperoleh melalui internet. Abstract Beberapa tahun belakangan ini, para peneliti di bidang jaringan menawarkan beberapa transport layer baru, yang diharapkan mampu mengakomodasi kelebihan dan meminimalkan kekurangan dari TCP dan UDP. Beberapa transport layer tersebut sudah mendapatkan standarisasi dari Internet Enginering Task Force (IETF), salah satunya adalah Streaming Control Protocol (SCTP). T T Beberapa penelitian sebelumnya memfokuskan pada pertanyaan apakah SCTP dapat menggantikan UDP sebagai transport layer paket VoIP. Beberapa penelitian seperti [2], [3], dan [4] menunjukkan hasil yang mengejutkan, beberapa penelitian tersebut menunjukkan bahwa tanpa adanya modifikasi pada SCTP, maka protokol ini memberikan perfoma yang tidak baik dengan delay yang cukup tinggi dalam mentransportasikan paket VoIP. Beberapa tahun belakangan ini penelitian mengenai Explicit Congestion Notification (ECN) dan Active Queue Management (AQM) sangat populer di kalangan peneliti jaringan. Berbeda dengan congestion control umumnya yang mekanismenya mengharuskan sender mengurangi congestion windownya setelah terjadinya congestion, mekanisme ECN dan AQM mampu mengirimkan feedback ke sender tentang peluang akan terjadinya congestion agar sender mengurangi congestion windownya sebelum congestion terjadi. Kemampuan ECN dan AQM mampu mengurangi drop packet rate sehingga mengurangi jitter dan delay [5]. Penggunaan ECN dan AQM pada penelitian [6] dan [7] mampu meningkatkan performa SCTP. Namun pada kedua penelitian tersebut simulasi tidak dilakukan pada aliran paket VoIP. Penelitian [5] menganalisa efek penggunaan ECN dan AQM pada kualitas aliran paket VoIP di atas protokol UDP. Hasil penelitian tersebut menunjukkan peningkatan signifikan pada kualitas panggilan aplikasi VoIP. Penelitian Reguera et al. [5] menggunakan protokol UDP sebagai transport layer aplikasi VoIP, namun sebagaimana disebutkan di atas, protokol UDP memiliki beberapa drawback pada internet. Penelitian ini akan melakukan analisa pengaruh mekanisme ECN dan AQM pada performa protokol SCTP sebagai transport layer aplikasi VoIP. 2.2 Analisis dan Perancangan Sistem Proses yang dilakukan dalam analisa dan perancangan sistem terbagi dalam beberapa bentuk diantaranya adalah sebagai berikut: IJCCS Vol. 9, No. 2, July 2015 : 121 – 132 123  IJCCS ISSN: 1978-1520 2.2.1 Analisis Sistem Aplikasi jaringan yang bersifat real time seperti VoIP adalah aplikasi yang membutuhkan jitter dan delay yang rendah. Pada saat ini penyedia layanan VoIP di internet lebih memilih protokol UDP ketimbang protokol TCP sebagai protokol transport. Hal ini terutama dikarenakan oleh tidak adanya congestion control pada UDP. Tidak adanya congestion control pada UDP menyebabkan rate sending UDP tidak terbatasi oleh congestion control, yang akhirnya menyebabkan delay dan jiiter pada receiver sangatlah kecil. Namun seperti telah disebutkan sebelumnya hal ini menimbulkan masalah pada jaringan. SCTP merupakan salah satu jenis protocol transport pada jaringan yang sampai sekarang masih terus dikembangkan. Desain awal SCTP ditujukan untuk membawa sinyal telepon SS7 [8] dalam jaringan IP. Kenyataan tersebut memunculkan beberapa penelitian yang mengangkat SCTP sebagai transport layer paket VoIP. Beberapa penelitian seperti [2], [3] dan [4] menunjukkan menunjukkan bahwa tanpa adanya modifikasi pada SCTP, maka protokol ini memberikan perfoma yang tidak baik dalam hal delay dan jitter yang cukup tinggi dalam mentransportasikan paket VoIP. Salah satu alasannya adalah karena sebagian besar mekanisme SCTP meniru mekanisme pada TCP, termasuk mekanisme congestion control. Penelitan- penelitian tersebut memberikan kesimpulan bahwa harus ada modifikasi pada congestion control SCTP. ECN dan AQM dapat berperan sebagai tambahan mekanisme pada congestion control SCTP. ECN dan AQM adalah dua mekanisme yang pada beberapa penelitian menujukkan performa yang cukup baik terutama dalam hal meminimalkan delay dan jitter. Berbeda dengan congestion control umumnya yang mekanismenya mengharuskan sender mengurangi congestion window (cwnd) setelah terjadinya congestion, mekanisme ECN dan AQM mampu mengirimkan feedback ke sender tentang peluang akan terjadinya congestion agar sender mengurangi cwnd sebelum congestion terjadi. Kemampuan ECN dan AQM yang mampu mendeteksi peluang terjadinya congestion dapat mengurangi drop packet rate sehingga mengurangi jitter dan delay [5]. Oleh karena itu pada penelitian ini akan dilakukan simulasi untuk mengukur sejauh mana ECN dan AQM mempengaruhi kualitas VoIP yang berjalan di atas protokol SCTP. 2.2.2 Perancangan Sistem Gambar 1 secara umum menunjukkan rancangan sistem yang akan dijalankan dalam simulasi. Rancangan sistem tersebut terdiri dari NS2VoIP++ sebagai generator traffic VoIP, SCTP agent sebagai transport layer, mekanisme ECN sebagai tambahan mekanisme congestion control pada SCTP, dan mekanisme AVQ sebagai mekanisme queue. Analisis Kualitas VOIP pada SCTP Menggunakan ECN dan AQM (La Surimi) Node (AVQ ) SCTP Agent +ECN Network Stat send() process_data() Stat::put() Node (AVQ ) NS2VoIP++ pada receiver recv() voip_decoder NS2VoIP++ (Trafffic Voip Generator) voip_bedirectional voip_source handle() talksspurt() recv() recvPayLoad() sendmsg() NS2VoIP++ pada sender voip_encoder voip_aggregate voip_header SCTP Agent +ECN Gambar 1 Rancangan Sistem Stat::put() Network Gambar 1 Rancangan Sistem Analisis Kualitas VOIP pada SCTP Menggunakan ECN dan AQM (La Surimi) Analisis Kualitas VOIP pada SCTP Menggunakan ECN dan AQM (La Surimi)  124 ISSN: 1978-1520 1. NS2VoIP++ Traffic Voip Generator) Pada penelitian ini traffic VoIP dihasilkan oleh modul NS2VoIP++. NS2VoIP++ adalah modul yang mensimulasikan traffic VoIP pada NS2NS2VoIP++ dirancang berdasarkan model user behaviour dari aplikasi VoIP. Modul ini dapat didownload dalam bentuk patch NS2 pada NS2Voip++ pada dasarnya dirancang untuk berjalan di atas protokol UDP, sehingga dalam penelitian ini harus dilakukan modifikasi pada modul NS2VoIP++ agar dapat berkomunikasi dengan SCTP agent. g p g g 2. SCTP Agent NS2 mulai pada versi 2.29 telah menyediakan modul untuk SCTP agent. Rancangan modul SCTP agent pada ns2 dirancang oleh Protocol Engineering Lab, Universitas Delaware. Penelitian ini menggunakan modul versi SCTP 3.5 yang terdapat pada NS2 2.34 dengan beberapa modifikasi. Modifikasi dilakukan untuk mengakomodir penambahan mekanisme ECN dan VoIP Traffic Generator NS2VoIP++. 2. SCTP Agent NS2 mulai pada versi 2.29 telah menyediakan modul untuk SCTP agent. Rancangan modul SCTP agent pada ns2 dirancang oleh Protocol Engineering Lab, Universitas Delaware. Penelitian ini menggunakan modul versi SCTP 3.5 yang terdapat pada NS2 2.34 dengan beberapa modifikasi. Modifikasi dilakukan untuk mengakomodir penambahan mekanisme ECN dan VoIP Traffic Generator NS2VoIP++. ECN Echo TSN < ECN_ECHO_TSN? Reduce CWND Inisialisasi koneksi Buat variabel ECN_ECHO_LAST=InitialTsn-1 ECN_ECHO_TSN=InitialTsn-1 ECNE packet? ECN_ECHO_LAST=ECN Echo TSN ECN_ECHO_TSN =TSN_data_Chunk yang_paling terakhir dikirim Generate paket CWR dengan TSN Number=ECN_ECHO_LAST Send Paket CWR start end sepakat ecn? Receive packet dari receiver? YES NO YES YES NO NO YES pernah mereduce cwnd untuk current_window_data ? NO Set ect=1 pada header paket data Send paket data YES Receive Data dari Upper layer/ Applikasi? NO YES NO Gambar 2 Mekanisme ECN pada Sender Send paket data Gambar 2 Mekanisme ECN pada Sender IJCCS Vol. 9, No. 2.2.2 Perancangan Sistem 2, July 2015 : 121 – 132  125 125 IJCCS IJCCS ISSN: 1978-1520 3. Mekanisme ECN pada SCTP SCTP modul versi 3.5 belum menyediakan dukungan untuk mekanisme ECN. Oleh karena itu, dilakukan modifikasi terhadap modul SCTP agent untuk menambahkan mekanisme ECN. Mekanisme ECN yang ditambahkan mengikuti prosedur dari IETF yang termuat dalam draft-stewart-tsvwg-sctpecn-05 [9].Mekanisme ECN bekerja pada sisi sender dan sisi receiver. 3. Mekanisme ECN pada SCTP SCTP modul versi 3.5 belum menyediakan dukungan untuk mekanisme ECN. Oleh karena itu, dilakukan modifikasi terhadap modul SCTP agent untuk menambahkan mekanisme ECN. Mekanisme ECN yang ditambahkan mengikuti prosedur dari IETF yang termuat dalam draft-stewart-tsvwg-sctpecn-05 [9].Mekanisme ECN bekerja pada sisi sender dan sisi receiver. Inisialisasi koneksi start end sepakat ecn? YES NO recv DATA PACKET? recv CWR PACKET? ect=1 && ec =1 Create ECNE paket TSN number =tsn pada chunk terakhir DATA PACKET Send ECNE paket CWR TSN number >= TSN number ECNE Chunk NO YES NO YES NO NO YES YES Gambar 3 Mekanisme ECN pada receiver CWR TSN number >= TSN number ECNE Chunk recv CWR PACKET? ect=1 && ec =1 Gambar 3 Mekanisme ECN pada receiver Gambar 2 menunjukkan diagram alir mekanisme ECN pada sender. Setelah tahap inisialisasi selesai dilakukan dan penggunaan mekanisme ECN disetujui oleh sender dan receiver maka untuk keperluan mekanisme ECN nantinya, sender akan membuat dua buah variabel, yaitu ECN_ECHO_LAST dan ECN_ECHO_TSN untuk setiap destination. Nilai inisial untuk kedua variabel ini adalah nilai InitialTSN-1. Setiap saat sender menerima data dari upper layer atau aplikasi, sebelum mengirim data yang telah dienkapsulasi menjadi paket ke receiver, mekanisme ECN mengharuskan sender untuk menset ECT header flag pada paket data dengan nilai 1. ECT header flag tersebut akan memberi tanda kepada jaringan bahwa paket tersebut mendukung mekanisme ECN. Jika sender menerima paket berupa ECNE chunk dari receiver, maka hal ini menandakan bahwa jaringan mulai mendeteksi akan terjadinya congestion. Oleh karena itu sender harus melakukan penurunan cwnd. Penurunan cwnd oleh sender harus dilakukan sekali dalam satu window data. Jika penurunan cwnd belum pernah dilakukan maka sender akan melakukan penurunan cwnd. Setelah melakukan penurunan cwnd, maka nilai ECN_ECHO_TSN akan diset dengan nilai TSN pada DATA chunk terakhir yang telah dikirim sedangkan Nilai ECN_ECHO_LAST diset dengan nilai TSN yang ada pada ECNE chunk. Setelah mengeset kedua variable tersebut sender akan menggenerate CWR chunk dan mengirimkannya ke receiver sebagai tanda sender telah menurunkan cwnd. 2.2.2 Perancangan Sistem TSN Analisis Kualitas VOIP pada SCTP Menggunakan ECN dan AQM (La Surimi)  126 126  ISSN: 1978-1520 number pada CWR chunk diset dengan nilai ECN_ECHO_LAST. Jika pengecekan pertama dan kedua tidak mengizinkan penurunan cwnd maka sender akan tetap menggenerate CWR chunk dan kemudian mengirimkanya ke receiver. Gambar 3 menunjukkan diagram alir mekanisme ECN pada receiver. Setelah tahap inisialisasi selesai dilakukan dan penggunaan mekanisme ECN disepakati oleh sender dan receiver. Setiap kali receiver menerima paket bertipe DATA, receiver akan mengecek ECT flag header dan EC flag header pada paket. ECT flag header bernilai 1 dan EC flag header bernilai 1 mengindikasikan jaringan memiliki peluang besar mengalami congestion. Receiver kemudian akan menggenerate ECNE chunk dengan nilai TSN number diset dengan nilai TSN chunk terakhir dalam paket DATA yang diterima. Kemudian receiver akan mengirimkan ECNE chunk tersebut dalam bentuk paket ke sender. Receiver akan terus mengirimkan ECNE chunk ke sender hingga receiver menerima CWR chunk dari sender yang nilai TSN number pada CWR chunk tersebut bernilai lebih besar sama dengan TSN number ECNE chunk yang terakhir dikirim. 4. Adaptive Virtual Queue Pada [10] menawarkan mekanisme Adaptive Virtual Queue (AVQ) berbasis AQM. AVQ telah diimplementasikan dalam NS2 dengan nama modul vq queue 5. Lingkungan Simulasi Rancangan topologi untuk lingkungan simulasi mengikuti rancangan simulasi yang diajukan oleh [3] ditunjukkan pada Gambar 4. Node 1 dan node 2 merupakan node router dengan panjang buffer yang terbatas. Node-node yang lain merupakan end node. Node 0 dan node 3 merupakan node VoIP dengan NS2VoIP++ sebagai pembangkit aliran VoIP. Node 4 dan node 5 digunakan untuk mensimulasikan traffic FTP dengan TCP sebagai transport agent. Node 6 dan node 7 digunakan untuk mensimulasikan HTTP traffic menggunakan modul pack mime HTTP dengan TCP sebagai transport agent. Link 1-2 digunakan secara share oleh semua aliran (aliran VoIP, FTP dan HTTP). Simulasi dilakukan menggunakan keempat skenario (hLhB, hLlB, lLhB dan lLlB) dengan mengubah-ubah ukuran bandwidth dan delay pada link 1-2. 5. Lingkungan Simulasi Rancangan topologi untuk lingkungan simulasi mengikuti rancangan simulasi yang diajukan oleh [3] ditunjukkan pada Gambar 4. Node 1 dan node 2 merupakan node router dengan panjang buffer yang terbatas. Node-node yang lain merupakan end node. Node 0 dan node 3 merupakan node VoIP dengan NS2VoIP++ sebagai pembangkit aliran VoIP. Node 4 dan node 5 digunakan untuk mensimulasikan traffic FTP dengan TCP sebagai transport agent. 2.2.2 Perancangan Sistem Penarikan kesimpulan Penarikan kesimpulan, apakah penggunaan ECN dan AVQ pada protokol SCTP mempengaruhi kualitas VoIP yang dihasilkan, dilakukan menggunakan uji statistik Wilcoxon Signed Rank. 8. Penarikan kesimpulan Penarikan kesimpulan, apakah penggunaan ECN dan AVQ pada protokol SCTP mempengaruhi kualitas VoIP yang dihasilkan, dilakukan menggunakan uji statistik Wilcoxon Signed Rank. 2.2.2 Perancangan Sistem Node 6 dan node 7 digunakan untuk mensimulasikan HTTP traffic menggunakan modul pack mime HTTP dengan TCP sebagai transport agent. Link 1-2 digunakan secara share oleh semua aliran (aliran VoIP, FTP dan HTTP). Simulasi dilakukan menggunakan keempat skenario (hLhB, hLlB, lLhB dan lLlB) dengan mengubah-ubah ukuran bandwidth dan delay pada link 1-2. Router 2 Router 1 . . . FTP/TCP Source HTTP Source VoIP A Bottle Neck 30 ms 0.3 Mb/s HTTP Sink FTP/TCP Sink VoIP B 30 ms 0.3 Mb/s 30 ms 0.3 Mb/s 25 ms 0.5 Mb/s 30 ms 0.3 Mb/s 30 ms 0.3 Mb/s Network Characteristic Bandwith Delay hLhB 2 Mbps 140 ms hLlB 0,6 Mbps 140 ms lLHB 2 Mbps 70 ms 4 5 1 2 6 7 0 3 lLlB 0.6 Mbps 70 ms Gambar 4 Lingkungan Simulasi 5 Router 2 HTTP Source 3 VoIP A Gambar 4 Lingkungan Simulasi IJCCS Vol. 9, No. 2, July 2015 : 121 – 132 IJCCS Vol. 9, No. 2, July 2015 : 121 – 132 127 IJCCS IJCCS  ISSN: 1978-1520 6. Rancangan Pengujian Terdapat empat pengujian, pengujian pertama akan dilakukan terhadap kualitas VoIP yang dihasilkan oleh SCTP tanpa menggunakan ECN dan AVQ, pengujian kedua dilakukan untuk menguji kualitas VoIP yang dihasilkan oleh SCTP yang menggunakan ECN dan AVQ. Pengujian tiga dilakukan untuk menguji kualitas VoIP di atas protokol TCP. Pengujian empat dilakukan untuk menguji kualitas VoIP di atas protokol UDP. Pada masing-masing pengujian diberlakukan empat buah skenario jaringan hLhB, hLlB, lLhB, dan lLlB. Simulasi dilakukan dengan teknik independent replication, dengan jumlah replikasi maksimal sebanyak 10 kali, persentase error yang diperbolehkan sebesar 5%, dan setiap simulasi berdurasi 300 s. 7. Pengambilan Data kualitas VoIP Ukuran kualitas VoIP yang diukur berupa MOS.Ukuran kualitas MOS diukur dalam bentuk average dalam satu simulasi.Nilai MOS dikonversi dari nilai R-Model persamaan (1). 7. Pengambilan Data kualitas VoIP Ukuran kualitas VoIP yang diukur berupa MOS.Ukuran kualitas MOS diukur dalam bentuk average dalam satu simulasi.Nilai MOS dikonversi dari nilai R-Model persamaan (1). (1) (1) Dimana adalah kondisi sinyal asli tanpa noise, dengan nilai default 93,2 [11], adalah gangguan yang disebabkan oleh delay, adalah gangguan yang disebabkan low bit rate codec dan packet lost, dan A adalah nilai kompensasi terhadap gangguan- gangguan di atas. Konversi dilakukan dengan persamaan (2). MOS = (2) 1 4,5 ( )( ) 8. 3.1.3 Low Latency high Bandwidth (delay 70 ms dan bandwidth 2 Mbps) y g ( y p ) Tabel 3 menunjukkan perbedaan antara nilai MOS SCTP tanpa ECN+AVQ dan nilai MOS SCTP dengan ECN+AVQ, tidaklah signifikan. Sama halnya dengan skenario pertama, pada skenario ini bandwidth yang cukup besar mengurangi kinerja AVQ+ECN, ditambah lagi oleh rendahnya latency membuat queue yang menuju ke router lain (router 1 ke router 2 dan sebaliknya) tidak terlalu terbebani oleh paket yang akan dipropagasikan. Pada kondisi seperti ini peluang terjadinya congestion sangat kecil, kualitas teknik queue DropTail menghampiri kualitas yang diberikan oleh AVQ bahkan lebih baik. 3.1.2 high Latency low Bandwidth (delay 140 ms dan bandwidth 0.6 Mbps) high Latency low Bandwidth (delay 140 ms dan bandwidth 0.6 Mbps) Uji Wilcoxon Signed Rank, seperti yang ditunjukkan pada Tabel 2 memberikan kesimpulan bahwa terdapat perbedaan yang signifikan antara nilai MOS protokol SCTP yang menggunakan ECN+AVQ dengan nilai MOS protokol SCTP yang tidak menggunakan ECN+AVQ. Pada skenario kedua ini, skenario yang paling sering terjadi pada jaringan, kinerja ECN+AVQ lebih baik ketimbang skenario pertama. Bandwidth yang kecil ditambah dengan latency yang cukup besar menyebabkan paket bertumpuk di queue. AVQ melakukan mark paket lebih sering, mekanisme ECN menurunkan congestion window pada sender sehingga SCTP sender dapat menghindari kemacetan yang berujung pada rendahnya jitter dan paket loss dari SCTP yang menggunakan ECN+AVQ. 3. HASIL DAN PEMBAHASAN 3.1 Perbandingan Kualitas VoIP SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ 3.1.1 Skenario 1, high Latency high Bandwidth (delay 140 ms dan bandwidth 2 Mbps) 3.1.1 Skenario 1, high Latency high Bandwidth (delay 140 ms dan bandwidth 2 Mbps) 3.1.1 Skenario 1, high Latency high Bandwidth (delay 140 ms dan bandwidth 2 Mbps) Tabel 1 memperlihatkan hasil uji statistik Wilcoxon Signed Rank terhadap nilai MOS SCTP yang tidak menggunakan ECN+AVQ dan nilai MOS protokol SCTP yang menggunakan ECN+AVQ. Terlihat bahwa nilai MOS SCTP yang menggunakan ECN+AVQ lebih besar dibandingkan dengan nilai MOS SCTP yang tidak menggunakan ECN+AVQ namun uji statistik Wilcoxon Signed Rank memberikan kesimpulan perbedaan yang dihasilkan tidak signifikan. Hal ini disebabkan oleh bandwidth antara router 1 dan router 2 cukup besar hingga menyebabkan kinerja ECN+AVQ pada queue antara router 1 dan router 2 tidak maksimal. Setiap paket yang telah diproses pada router dan akan dipropagasikan ke router berikutnya tidak membebani queue terlalu berat karena bandwidth untuk propagasi cukup besar, walupun terdapat delay 140 ms. Hal ini membuat kinerja AVQ menghampiri DropTail, sehingga nila MOS yang diperoleh tidak jauh berbeda dengan nilai MOS pada SCTP yang menggunakan DropTail. Analisis Kualitas VOIP pada SCTP Menggunakan ECN dan AQM (La Surimi)  128 ISSN: 1978-1520 128 3.1.4 Low Latency low Bandwidth (delay 70 ms dan bandwidth 0.6 Mbps) Tabel 4 menunjukkan perbedaan yang signifikan antara protokol SCTP yang tidak menggunakan ECN+AVQ dan protokol SCTP yang menggunakan ECN+AVQ Pada skenario ini, kinerja ECN+AVQ lebih baik ketimbang skenario pertama dan ketiga. Bandwidth yang kecil menyebabkan paket bertumpuk di queue. AVQ melakukan mark paket lebih sering, mekanisme ECN menurunkan congestion window pada sender sehingga SCTP sender dapat menghindari kemacetan yang berujung pada rendahnya jitter dan paket loss dari SCTP yang menggunakan ECN+AVQ. Tabel 1 Hasil uji Wilcoxon Signed Rank terhadap nilai MOS SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ pada skenario 1 Replik- asi Kualitas MOS SCTP SCTP (ECN+AVQ) Difference (D) |D| Rank |D| Signed Rank of |D| SQR(∑Signed Rank of |D|) 1 2,1126 2,15254 -0,03996 0,04 4,0 -4,0 16 2 2,1104 2,16429 -0,05387 0,0539 5,0 -5,0 25 3 2,1811 2,26402 -0,08289 0,0829 8,0 -8,0 64 4 2,1493 2,13428 0,01501 0,015 2,0 2,0 4 5 2,1937 2,10656 0,0871 0,0871 9,0 9,0 81 6 2,0797 2,1979 -0,11814 0,1181 10,0 -10,0 100 7 2,1564 2,1814 -0,025 0,025 3,0 -3,0 9 8 2,1564 2,2129 -0,05649 0,0565 7,0 -7,0 49 9 2,1354 2,1495 -0,0141 0,0141 1,0 -1,0 1 10 2,1276 2,1824 -0,05478 0,0548 6,0 -6,0 36 |W| 1,68 Kesimpulan: |W| < c c 1,96 Tidak Terdapat perbedaan yang signifikan antara SCTP dan SCTP (ECN+AVQ) Tabel 1 Hasil uji Wilcoxon Signed Rank terhadap nilai MOS SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ pada skenario 1 IJCCS Vol. 9, No. 3.2 Perbandingan Kualitas VoIP SCTP dengan ECN+AVQ, SCTP tanpa ECN+AVQ, TCP dan UDP Perbandingan dilakukan terhadap nilai MOS SCTP dengan ECN+AVQ, SCTP tanpa ECN+AVQ, TCP dan UDP. Hasil simulasi menunjukkan untuk semua skenario SCTP dengan ECN+AVQ memiliki kualitas MOS yang lebih baik dari TCP namun tidak lebih baik dari UDP belum dapat melebihi UDP. Perbedaan nilai MOS yang diberikan oleh TCP dan SCTP terletak pada kelebihan feature SCTP berupa kemampuan multystreaming dan teknik pempaketan chunk data. SCTP dapat mengirimkan satu paket yang berisikan beberapa chunk data dalam beberapa stream dalam satu satuan waktu namun TCP mengirimkan hanya satu chunk data dalam satu paket melalui satu stream pada satu satuan waktu. Pada skenario ini tercatat SCTP tanpa ECN+AVQ maupun SCTP yang menggunakan ECN+AVQ mengirimkan paket rata-rata sebanyak 17695 buah paket, bandingkan dengan TCP yang hanya mengirimkan 4757 buah paket. Kualitas UDP tidak terpengaruh oleh high latency maupun low bandwidth,, hal ini disebabkan UDP tidak memiliki congestion control. Ketika ketiga protokol lainya menurunkan sending rate akibat congestion dan harus melakukan proses retransmisi akibat packet loss, UDP yang tidak memiliki mekanisme congestion control dan tidak bersifat reliable data transfer, tetap mengirimkan paket dengan sending rate yang konstan. 3.2 Perbandingan Kualitas VoIP SCTP dengan ECN+AVQ, SCTP tanpa ECN+AVQ, TCP dan UDP 3.2 Perbandingan Kualitas VoIP SCTP dengan ECN+AVQ, SCTP tanpa ECN+AVQ, TCP dan UDP 3.1.4 Low Latency low Bandwidth (delay 70 ms dan bandwidth 0.6 Mbps) 2, July 2015 : 121 – 132 ISSN: 1978-1520 129 IJCCS  IJCCS ISSN: 1978-1520  129 Tabel 2 Hasil uji Wilcoxon Signed Rank terhadap nilai MOS SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ pada skenario 2 Replikasi Kualitas MOS SCTP SCTP (ECN+AVQ) Difference (D) |D| Rank |D| Signed Rank of |D| SQR(∑Signed Rank of |D|) 1 1,64344 1,88078 -0,23734 0,2373 1,0 -1,0 1 2 1,54468 1,92765 -0,38297 0,383 5,0 -5,0 25 3 1,58658 1,89037 -0,30379 0,3038 3,0 -3,0 9 4 1,63305 1,92832 -0,29527 0,2953 2,0 -2,0 4 5 1,61161 1,92098 -0,30937 0,3094 4,0 -4,0 16 6 1,61872 1,89888 -0,28016 0,2802 5,0 -5,0 25 7 1,69339 1,80013 -0,10674 0,1067 2,0 -2,0 4 8 1,60587 1,89361 -0,28774 0,2877 6,0 -6,0 36 9 1,58276 1,8378 -0,25504 0,255 4,0 -4,0 16 10 1,74973 1,83469 -0,08496 0,085 1,0 -1,0 1 ∑Signed Rank of |D| -33,0 Kesimpulan: |W| > c √(SQR(∑Signed Rank of |D|)) 11,7 Terdapat perbedaan yang signifikan antara SCTP dan SCTP(ECN+AVQ) |W| 2,82 c 1,96 Tabel 3 Hasil uji Wilcoxon Signed Rank terhadap nilai MOS SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ pada skenario 3 Replikasi Kualitas MOS SCTP SCTP (ECN+AVQ) Difference (D) |D| Rank |D| Signed Rank of |D| SQR (∑Signed Rank of |D|) 1 2,44611 2,39984 0,04627 0,0463 7,0 7,0 49 2 2,39272 2,38548 0,00724 0,0072 1,0 1,0 1 3 2,39621 2,43698 -0,04077 0,0408 6,0 -6,0 36 4 2,36672 2,4481 -0,08138 0,0814 10,0 -10,0 100 5 2,44881 2,43535 0,01346 0,0135 2,0 2,0 4 6 2,44164 2,38992 0,05172 0,0517 8,0 8,0 64 7 2,42493 2,44578 -0,02085 0,0209 3,0 -3,0 9 8 2,44105 2,4066 0,03445 0,0345 5,0 5,0 25 9 2,40752 2,37731 0,03021 0,0302 4,0 4,0 16 10 2,32755 2,40017 -0,07262 0,0726 9,0 -9,0 81 ∑Signed Rank of |D| -1,0 Kesimpulan: |W| < c √(SQR(∑Signed Rank of |D|)) 19,6 Tidak Terdapat perbedaan yang signifikan antara SCTP dan SCTP(ECN+AVQ) |W| 0,05 c 1,96 Tabel 2 Hasil uji Wilcoxon Signed Rank terhadap nilai MOS SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ pada skenario 2 Replikasi Kualitas MOS SCTP SCTP (ECN+AVQ) Difference (D) |D| Rank |D| Signed Rank of |D| SQR(∑Signed Rank of |D|) 1 1,64344 1,88078 -0,23734 0,2373 1,0 -1,0 1 2 1,54468 1,92765 -0,38297 0,383 5,0 -5,0 25 3 1,58658 1,89037 -0,30379 0,3038 3,0 -3,0 9 4 1,63305 1,92832 -0,29527 0,2953 2,0 -2,0 4 5 1,61161 1,92098 -0,30937 0,3094 4,0 -4,0 16 6 1,61872 1,89888 -0,28016 0,2802 5,0 -5,0 25 7 1,69339 1,80013 -0,10674 0,1067 2,0 -2,0 4 8 1,60587 1,89361 -0,28774 0,2877 6,0 -6,0 36 9 1,58276 1,8378 -0,25504 0,255 4,0 -4,0 16 10 1,74973 1,83469 -0,08496 0,085 1,0 -1,0 1 ∑Signed Rank of |D| -33,0 Kesimpulan: |W| > c √(SQR(∑Signed Rank of |D|)) 11,7 Terdapat perbedaan yang signifikan antara SCTP dan SCTP(ECN+AVQ) |W| 2,82 c 1,96 Tabel 3 Hasil uji Wilcoxon Signed Rank terhadap nilai MOS SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ pada skenario 3 Replikasi Kualitas MOS SCTP SCTP (ECN+AVQ) Difference (D) |D| Rank |D| Signed Rank of |D| SQR (∑Signed Rank of |D|) 1 2,44611 2,39984 0,04627 0,0463 7,0 7,0 49 2 2,39272 2,38548 0,00724 0,0072 1,0 1,0 1 3 2,39621 2,43698 -0,04077 0,0408 6,0 -6,0 36 4 2,36672 2,4481 -0,08138 0,0814 10,0 -10,0 100 5 2,44881 2,43535 0,01346 0,0135 2,0 2,0 4 6 2,44164 2,38992 0,05172 0,0517 8,0 8,0 64 7 2,42493 2,44578 -0,02085 0,0209 3,0 -3,0 9 8 2,44105 2,4066 0,03445 0,0345 5,0 5,0 25 9 2,40752 2,37731 0,03021 0,0302 4,0 4,0 16 10 2,32755 2,40017 -0,07262 0,0726 9,0 -9,0 81 ∑Signed Rank of |D| -1,0 Kesimpulan: |W| < c √(SQR(∑Signed Rank of |D|)) 19,6 Tidak Terdapat perbedaan yang signifikan antara SCTP dan SCTP(ECN+AVQ) |W| 0,05 c 1,96 Analisis Kualitas VOIP pada SCTP Menggunakan ECN dan AQM (La Surimi) ISSN: 1978-1520  130 130 Tabel 4 Hasil uji Wilcoxon Signed Rank terhadap nilai MOS SCTP dengan ECN+AVQ dan SCTP tanpa ECN+AVQ pada skenario 4 Replikasi Kualitas MOS SCTP SCTP (ECN+AVQ) Difference (D) |D| Rank |D| Signed Rank of |D| SQR (∑Signed Rank of |D|) 1 2,08404 2,14286 -0,05882 0,0588 6,0 -6,0 36 2 2,1155 2,18749 -0,07199 0,072 7,0 -7,0 49 3 2,06395 2,13967 -0,07572 0,0757 8,0 -8,0 64 4 2,02747 2,08433 -0,05686 0,0569 5,0 -5,0 25 5 2,14319 2,13505 0,00814 0,0081 2,0 2,0 4 6 2,00593 2,24014 -0,23421 0,2342 10,0 -10,0 100 7 2,01159 2,0344 -0,02281 0,0228 3,0 -3,0 9 8 2,13087 2,18048 -0,04961 0,0496 4,0 -4,0 16 9 2,09219 2,09215 4E-05 4E-05 1,0 1,0 1 10 1,96009 2,12986 -0,16977 0,1698 9,0 -9,0 81 ∑Signed Rank of |D| -49,0 Kesimpulan: |W| > c √(SQR(∑Signed Rank of |D|)) 19,6 Terdapat perbedaan yang signifikan antara SCTP dan SCTP(ECN+AVQ) |W| 2,50 c 1,96 5. SARAN Untuk pengembangan penelitian lebih lanjut, diberikan saran sebagai berikut: 1. Penelitian berikutnya dapat melakukan pengujian kualitas VoIP untuk jenis Active Queue Management (AQM) lain. Selain AVQ ada beberapa jenis AQM lain, misalnya ARED, PI, SFB, RRED dan REM. 2. Penelitian berikutnya dapat mengimplementasikan ECN pada SCTP dan melakukan pengujian pada sistem nyata. Pengujian pada sistem nyata dapat menutupi asumsi-asumsi yang dilakukan pada pengujian simulasi. protokol SCTP tanpa menggunakan mekanisme ECN dan AVQ p p gg 2. SCTP dengan ECN dan AVQ mengungguli TCP namun belum dapat mengungguli UDP p p gg 2. SCTP dengan ECN dan AVQ mengungguli TCP namun belum dapat mengungguli UDP. 4. KESIMPULAN Berdasarkan dari hasil penelitian dan pembahasan yang dilakukan maka diperoleh kesimpulan sebagai berikut: 1. Penggunaan protokol SCTP yang menggunakan mekanisme ECN dan AVQ sebagai transport layer VoIP menunjukkan performa yang lebih baik daripada penggunaan 1. Penggunaan protokol SCTP yang menggunakan mekanisme ECN dan AVQ sebag transport layer VoIP menunjukkan performa yang lebih baik daripada penggunaa IJCCS Vol. 9, No. 2, July 2015 : 121 – 132 131 IJCCS ISSN: 1978-1520  protokol SCTP tanpa menggunakan mekanisme ECN dan AVQ [9] Stewart, R., Tuexen, M. dan Dong, X., 2014, ECN for Stream Control Transmission Protocol (SCTP), http://tools.ietf.org/id/draft-stewart-tsvwg-sctpecn-00.txt, diakses 1 Januari 2014 [10] Kunniyur, S.S. dan Srikant, R., 2004, An Adaptive Virtual Queue (AVQ) Algorithm for Active Queue Management, The IEEE/ACM Transactions on Networking, 2, 21, 286-299 [11] ITU-T, 2009, ITU-T Recommendation G.107, The E-Model: A Computational model for Use in Transmission Planning. IJCCS Vol. 9, No. 2, July 2015 : 121 – 132 DAFTAR PUSTAKA [1] Rakocevic,V., 2004, Congestion Control for Multimedia Applications in the Wireless Internet, International Journal of Communication Systems, 17,723–734. [2] Lim, P.H., Myungchul, K. dan Jeong-Seon, K., 2007, Evaluation of Stream Control Transmission Protocol as a Transport for VoIP over WLAN, International Conference on Advanced Communications Technology 2007, Paris. [3] Asodi, S., Ganesh, S.V., Seshadri, E. dan Singh, P.K., 2009, Evaluation of Transport layer Protocols for Voice Transmission in Various Network Scenarios, International Conference on the Applications of Digital Information and Web Technologies 2009, London, 4-6 Agustus 2009. [4] Gangurde, P., Waware, S. dan Sarwade, N., 2012, Simulation of TCP, UDP and SCTP with Constant Traffic for VOIP Services, International Journal of Engineering Research and Applications (IJERA), ISSN: 2248-9622, 2, 1245-1248. [5] Reguera, V.A., Paliza, F.Á., Fernandez, E.M.G. dan Godoy, W., 2008, On the Impact of Active Queue Management on VoIP Quality of Service, Computer Communications, 1, 31, 73-87, http://www.sciencedirect.com/science/article/pii/S0140366407004148. [6] Ye, G., Saadawi, T.N. dan Lee, M., 2003, Using Explicit Congestion Notification in Stream Control Transmission Protocol in Lossy Networks, 23rd International Conference on Distributed Computing Systems Workshops, Macau, 19-22 Mei 2003. [7 Tahir, H.M., Abas, M.S., Elhalabi, M. J.M., Puteh, N., Othman, A., Zain, N.M., Dahalin, Z.M., Ismail, M.H., Zaini, K.M. dan Hussin, M.Z., 2011, Improving Network Performance by Enabling Explicit Congestion Notification (ECN) in SCTP Control Chunks, Snasel, V., Platos, J., dan Eyas El-Qawasme, E., Digital Information Processing and Communications, 188, Springer, Heidelberg. [8] Stewart, R., 2007, Stream Control Transmission Protocol, http://tools.ietf.org/html/rfc4960, diakses 03 Januari 2013. Analisis Kualitas VOIP pada SCTP Menggunakan ECN dan AQM (La Surimi)  132 ISSN: 1978-1520 ISSN: 1978-1520 IJCCS Vol. 9, No. 2, July 2015 : 121 – 132

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Ethical potentialities on physical education as a vehicle for ethical education through sports

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Abstract Sports occupy an interesting ethical space from a pedagogic point of view, being in- cluded in physical education curricula in most Western countries. The approach of physical education to sports as vehicle for ethical education is too limited when it is restricted to their minimal functional, constitutive and regulatory goals. This essay’s aim is to argue the extent to which the ethical potential of physical education can embrace more than func- tional purposes, or whether that will be neglected in terms of limited educational aspira- tions. We present data from nineteen exploratory interviews with experienced philosophy, sports and physical education researchers and teachers, from six different nations, con- cerning the ethical potentiality of physical education. We highlight five ethical themes: (i) the regulatory and normative structure of sports; (ii) the spirit of sports and its internal values; (iii) the right playing/doing of sports; (iv) the overcoming in sports; and (v) sports as an opportunity for a supererogatory ethics as fertile ground for future operationalization of the potential of physical education for ethical education through sports. Keywords: sport, ethics, physical education. RECERCA, REVISTA DE PENSAMENT I ANÀLISI, NÚM. 18. 2016. ISSN: 1130-6149 – pp. 29-48 doi: http://dx.doi.org/10.6035/Recerca.2016.18.3 RECERCA, REVISTA DE PENSAMENT I ANÀLISI, NÚM. 18. 2016. ISSN: 1130-6149 – pp. 29-48 doi: http://dx.doi.org/10.6035/Recerca.2016.18.3 Una nueva concepción del potencial ético de la educación física LUÍSA ÁVILA DA COSTA*; MICHAEL MCNAMEE**; TERESA LACERDA*** * University of Porto and Member of the Centre of Research, Education, Innovation and Intervention in Sport (Portugal). ** Swansea University (Wales, United Kingdom). *** University of Porto and Member of the Centre of Research, Education, Innovation and Intervention in Sport (Portugal) Artículo recibido: 18 septiembre 2014 Solicitud de revisión: 15 marzo 2015 Artículo aceptado: 7 julio 2015 Resumen El deporte ocupa un interesante espacio ético desde un punto de vista pedagógico, integrándose en los curricula de educación física en la mayoría de países occidentales. El planteamiento de la educación física como vehículo de la educación ética es limitado cuando restringido a sus objetivos mínimos funcionales, constitutivos y regulatorios. El objetivo de este estudio es discutir si el potencial ético de la educación física puede ir más allá de los propósitos funcionales, que considerados aisladamente constituyen un desper- dicio de la experiencia pedagógica del deporte. Para conseguirlo, presentamos datos de diecinueve entrevistas exploratorias con experimentados investigadores y profesores de filosofía, deporte y educación física relacionadas con las potencialidades éticas de la edu- cación física. La muestra incluye individuos de seis diferentes nacionalidades, cuyo trabajo RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 30 demuestra preocupaciones con el tema. La argumentación resulta de cinco temáticas prin- cipales: (i) Estructura normativa y regulativa del deporte; (ii) El espírito del deporte y sus valores internos; (iii) El bien hacer deportivo; (iv) La superación deportiva; (v) El deporte como oportunidad de una ética supererogatoria como contexto fértil para la concretiza- ción del potencial ético de una educación deportiva. Palabras clave: deporte, ética, educación física. Palabras clave: deporte, ética, educación física. INTRODUCTION Ethics is a contested terrain in general, and specifically in the contexts of sports. Not uncommonly, and often in the case of the interviews con- ducted in this study, «ethics» is referred to as a discipline or field of phi- losophy that concerns the study and reasoning of normative appraisal of values and practices that drive human actions towards the common good. Many scholars, from various modern and postmodern traditions, have al- ready defined ethics as the quest for the good life with and for the good of others (Ricoeur, 1990). Thus, the consideration of the ethics of physical education and sports exists within the frame of human coexistence ori- ented towards the good, individual and collective, that requires a human experience that is lived in a free, responsible, and fair way, exhibiting suf- ficient degrees of solidarity or communal living. This is the sense outlined long ago by Aristotle in his account of living well (Aristotle, 2009). Modern scholarship in ethics is rooted in three key areas, namely the ethics of virtues whose focus is the personal quality of individuals and how they should be aimed towards the good (MacIntyre, 2007); the ethics of duty (deontology) that is related to the criteria and rules frameworks, more or less universal and paradigmatic, that should guide individuals in acting according rightly; and the consequential-practical (typically utilitar- ian) mode through which individuals exercise their reason to discern the optimal way of acting before ethical problems with a precise, specific, contextual and localized storyline (oss3, iss2, iss6. pet2). The specific ap- proach of sports in the light of these three main family of ethical theories (utilitarian or consequentialism; duty or deontological; and virtue-ethical), resulted in several works produced by sports philosophers that debated issues such as justice, integrity, responsibility and respect between players, the rules and norms for a healthy coexistence in sports, the problem of cheating, doping and medical intervention with the intent of artificially improving the performance, violence, racism, exclusion, inequality, and so on. (McNamee, 2007) In this sense, sports ethics has, in recent decades, 31 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical proved to be an area of strong scholarly growth, mastering most part of the works dedicated to sports philosophy (McNamee and Parry, 1998; Mc- Namee, 2010, McNamee and Morgan, 2015; Torres, 2014). 1 See for example: Boxill, J. (ed) (2002) Ethics and Sport, Oxford: Blackwell; Galasso, P.J. (Ed.) (1988) Philosophy of Sport and Physical Activity Issues and Concepts, Toronto: Canadian Scholars Press; Loland, S. (2002) Fair Play in Sport: A Moral Norm System, London: Routledge; Morgan, W.J. (2000) Ethics in Sport, Illinois: Human Kinetics; Simon, R.L. (1991) Fair Play: Sports, Values, and Society, Colorado: Westview Press; McNamee, M. J. & Parry, S. J. (Eds.) (1998) Ethics and Sport, London, Routledge; McNamee, M. J. & Parry, S. J. (Eds.) (1998) Ethics and Sport, London, Routledge. INTRODUCTION Considering that there are many relevant works that set the foundation of sports ethics1, this study arises not with the intention to exhaust the subject, but in order to get together specific arguments on sport’s ethics which underwrite the ethic potential of physical education. Considering this paper as a part of a broader study in the aesthetic-ethics relations within physical education (Ávila da Costa, McNamee and Lacerda, 2015a), we focus here only on the ethical elements of this relation. The purpose of the present study, within that framework and based on our research group’s aims, concerns the identification of some ethical subjects of sports, beyond their regulatory, constitutive and functional aspects, that may have relevance for a broader ethical consideration of physical education. To this end we identify and discuss these subjects in 19 semi-structured and exploratory interviews that enabled the data collection, analysis and discussion of viewpoints of representative subjects among those that are the main players in aesthetic education through sports, namely, experi- enced teachers and researchers in the context of ethics, philosophy, sports science and physical education, from six different nationalities in Western countries. We conducted a hermeneutic analysis on some of the main as- pects as they enable the understanding of physical education as a vehicle for ethical education through sports. The 19 interviews were conducted with three different groups of indi- viduals that, considering their relationship with ethics, with sport and with physical education, can make different contributions and complement the problem under study. These were: a) «Outside Sport Sciences»: teachers/ researchers from the areas of ethical education outside sports sciences referred to as oss; b) «Inside Sport Sciences»: teacher/researchers inside sports sciences whose work reveals ethical concerns in the context of pedagogy and education through sports, referred to as IIS: c) «Physical Edu- cation Teachers», physical education teachers who provided a more focused and practical look on how these dimensions are implemented in physical education lessons, referred to as pet. In order to guarantee the 32 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 anonymity of their discourses, the quotes included throughout the text are identified with theses acronyms, in order to recognise the group from which they come and with a random numerical order. 1. PHYSICAL EDUCATION AND ETHICAL EDUCATION THROUGH SPORTS In the course of the study we tried to understand, together with our interviewees, the role that ethics has in sports and the importance of eth- ics in understanding sports and physical education. Since the Greek educational model, the essential substantiation of sports is deeply ethical, in the sense that it leads Man to search for the aret, understood as human excellence or perfection. Pestalozzi (2009) advocat- ed the pedagogical importance of exercising the will, coordinating the in- tellectual and moral education of subjects for which sports can greatly contribute. iss5 supports these tendencies, arguing that sports is a vehicle for the education of will, against a contemporary logic of a hedonistic and painless ethic (Lipovetsky, 2010) saying: I think there is no other justification for sports. Teaching sports or physical education is only justified in two ways. The first is that (...), the reference that human beings are artistic, that become human as they acquire that art, the arete, from which they are born naked, deprived, as they are born without doing, due to the neoteny in the body, feelings, values, etc... (...) The second justification, for me, is still the education of the will, explained by the substantiation of Pestalozzi’s corporal exercises that aim the moral. (...) At a time of painless ethics, (...) sports is clearly a pedagogy of will since it leads us to do things that make us sweat, it is necessary to train and practice to acquire competence, to learn what we don’t know (iss5, p.4). In our interviews, however, oss5, iss4, iss5 and pett add that ethics is not only the ground to consider these goals. In sport, ethical considerations on the one hand, lead to the practical configuration of normative, constitutive and regulatory structures that make sports practicable: «For instance, if suddenly football had no rules it would not be football and it would be a bit more difficult...what are they doing? Where are they going?» (pett, p.18). On the other hand ethics is constitutive of the identity or essence and sense of any sport. Moreover, oss6 and iss1 agree with what had already been stated by Mor- gan, that the awareness of the ethical nature of sports and how central it is, requires from the subject a deep knowledge and involvement with sports (Morgan, 2007). INTRODUCTION 33 SA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical INTRODUCTION It is not the purpose of this work to include or exhaust every possible relevant issue for an ethics of sports in general in an educational point of view which, indeed would be impossible. More specifically, our purpose was to debate some specific ethical potentialities of physical education based on the narrative of our interviewees, were they have stressed what are particularly important and relevant ideas for physical education that might enrich this quest for well-living in sports and that can, thus, propose ways or means of living well. Besides the permanent feeling of difficulty in handling ethical ambigui- ties, and also the need for coherence and completeness that are normally associated with normative theories such as ethics embodies, this subject seems not prove an obstacle to dialogue among either common citizens nor the participants. Everyone seems to have a view on ethical matters even if only a few are capable of theorizing or even systemically evaluating them. In contrast to what happens with aesthetics (Ávila da Costa, Mc- Namee and Lacerda, 2015b), these ethically focused interviewees discussed the subject in a fearless, fluid and spontaneous way: «Ethics....that part is probably easier to debate than aesthetics. At least for me!» (pett, p.16). This is because, for oss2, even though it is a subject that not all of us study is one that we all face daily. And, thus, the approach to ethics proposed in this study, taking into consideration the academic background of most of the subjects in the study group, as well as that of the researchers involved assumes a more functional, hermeneutic and interpretative nature than theoretical, descriptive, normative or analytic. This leads us to generate perceptions that may not be generalizable in their content. That is to say, based on what has been widely included in literature, we aim to under- stand what is nowadays considered relevant for daily life ethics in the quotidian contexts of physical education. Thus, our framework draws on many of the elements of sports ethics in the context of physical education, as a subject with ethical potentialities that can and should be used in the pedagogical sense: the regulatory and normative structure of sports; the spirit of sports and its internal values; the right playing/doing of sports; the overcoming in sports; sports as an opportunity for a supererogatory ethics. 1. PHYSICAL EDUCATION AND ETHICAL EDUCATION THROUGH SPORTS It would be difficult, according to these interview- ees, that someone deeply involved in sports is not immersed in its ethical nature, even if in an unconscious way: «You can ignore it if you haven’t thought about it, but it is a bit like aesthetics. The more you look and the more you learn about it, the more you will see the aesthetic values. The RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 34 same happens with ethics» (oss6, p.7); «(…) I think that people are not aware of that but they act according to some values such as not hurting others and so one, respecting the rules, playing in a fair way, etc... (…) I think that even those persons that cheat this aspect, that try to cause dam- age, that play in a violent way, that use other means...even those persons are aware that they are contravening, that they are ignoring what would be correct.... what they are expected to do» (iss1, p.13). In this way, when they are asked about the possibility of understanding sports ignoring the ethical dimension, our interviewees were unanimous in stating that for any quest in the understanding of sports, ignoring the ethical dimension may be possible, but nevertheless represents an artificial way of approaching sports, impoverished, limited and lacking what is es- sential in, or partly constitutive of, its identity. Nevertheless, if one aims at a specific ethical pedagogy of sport, one must be aware that it is permanently conditioned by the social reality that pedagogues and learners are dealing with: «You can’t understand ethics in the abstract. (…) And so, situating ourselves in a meaningful storyline is a fundamental step for the understanding of the right, that is, behavioural ethics» (iss6, p.5). Thus, for example, an ethics of sports has boundaries and critical aspects that are different from art ethics. If, in sports, the aspiration of an ethical experience in its different levels is apparently common and foreseeable, the same does not happen with art which frequently claims for the independency and the transgression of any axiological framework; this kind of autonomy also supports claims for its being amoral (osst). 2 An example of this openness of the art world to works with a highly debatable and ethical content that is open to criticism is the exhibition by Guillermo Vargas Jiménez, entitled «Exposición nº1», in Nicaragua where, for a long period of time, he tied up and displayed a starving dog. 1. PHYSICAL EDUCATION AND ETHICAL EDUCATION THROUGH SPORTS That is to say, art is not intended to be moralised and its frequently transgressive nature is also revealed in the domain of an ethics of transgression, shock, rejection and rupture with values, independently from their positive or negative, universal or particular nature (osst). With this we do not mean to say that art does not have either more or less ethical reference, but only that it is different from sports in a special way, with a permanent questioning and confrontation with the axiological benchmarks of each era and their ethical criteria often iterating between universality and particularity. Nowadays, art is characterised by personal values that can naturally trigger critical and conflicting reactions that are sometimes ethical in character, but this does not constitute any threat to the development of its space and place in our world.2 Such ethical trans- 35 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical gression – as an artistic or aesthetical value – is open to question and al- ways debatable, rejected and accepted, by different interviewees (osst, oss1). This does not happen in sports, at least not this way, where the norma- tive and regulatory structure present stricter and more tightly. The formal or constitutive rules (Reddiford, 1993), are defined and the ethical para- digm seems to require minimal universality criteria, that are reproduced in the practices of physical education. This means that, even if ethical trans- gression is frequent and relevant in sports world, it is not accepted in such a ready way as in art. Thus, in sports, ethical particularism, sometimes even relativism, is generally considered as a problem to overcome or solve (osst, oss1, oss3). For oss3 and oss6, grounding the debate on ethics in the context of a polarity between universality and relativism embodies a too simplistic di- chotomisation of ethics: «You can have a bit of both sides. (...) There is an adequate answer that changes according to time, situations and people, in particular. (...) You cannot simply apply the rules from top to bottom and say that this answers everything. It is always necessary to interpret the situation, the motivations, the consequences and so on. (oss3, p.8); (…) It is not an entirely subjective experience, but it is not simply objectivism. If there were no human being there perceiving the world, I don’t think there would be ethical values. 1. PHYSICAL EDUCATION AND ETHICAL EDUCATION THROUGH SPORTS It is a mutual manifestation of the object of ethical evaluation and human perceiver» (oss6, p.6). For oss2, the ethical patterns and the concepts of right and goodness, depends on the internal characteristics of the reality we experience. Thus, «In music, I think that the ethics of each style is different. There are great difference in the ethics behind jazz, for example, and classic or popular music. One person plays guitar in a totally different way depending on the music styles. The way he plays, how he holds the guitar, the way he ap- proaches the music is totally different. In classical music we are much more formal and this determines many things, not only how we dress on stage (...), but also how we approach the written music. In popular music or jazz there is much more freedom of interpretation. In classical music there are also requirements related to a certain ethics that we must respect to a certain degree and that defines the shades. (...) The way entertaining music faces a musical score would be considered wrong, for us, classics» (oss2, pp.3,4). This means that the ethical consideration of reality is not abstract or blind, it requires a deep understanding of the nature and internal structure 36 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 of the object under consideration. The same happens in physical education when we define the set of sports contents (knowledge, skill, rules, etc) that will be taught. The ethical criteria of a basketball game are, obviously, dif- ferent from those of a rugby game. Considering the aim of this study that was to investigate the ethical nature or aspects of sports as a pedagogical tool in physical education, we propose a more specific approach to the most relevant ethical elements and criteria that characterise sports. If, for instance in a sport such as bas- ketball, defensive actions forbid, both from the cultural and regulatory point of view, great physical contact with the opponent, in rugby, the tackle is a compulsory technical gesture and, thus, the ethical legitimacy of that technical gesture that can, somehow, physically attack those involved, is highly different in both realities (pett). 1. PHYSICAL EDUCATION AND ETHICAL EDUCATION THROUGH SPORTS In the quest for a more specific ethics applied to sports, more specifi- cally to physical education, we discussed what we considered to be the key elements for an ethical debate on physical education, that is to say, the main discussion of the threads of identity within pedagogical sports. De- spite the broad boundaries of this subject, we attempt to map the contours of ethical concern in sports, as a key element in terms of: a) the regulatory and normative structure of sports; b) the spirit of sports and its internal values; c) the right playing/doing at sports; d) the overcoming in sports; and e) sports as an opportunity for a supererogatory ethics (i.e. one over above compliance with ethical duties). A) THE REGULATORY AND NORMATIVE STRUCTURE OF SPORTS Sports represent a highly regulated social reality. Each sport has a set of constituent and regulatory norms that characterises it and provide its iden- tity (Torres, 2011). Normally this structure corresponds to one of the first contents that are provided when we wish to teach any sports in physical education lessons. Thus, it is an artificial reality, consisting of artificial cri- teria and norms that create unnecessary obstacles, deliberately invented and handled by man, to answer his desire to meet that challenge or take a test (Suits, 2005). «While Suits says that sports creates artificial problems, for me sports itself «is» a great artificial problem that we have created to make life inter- esting» (oss3, pp. 8-9). The creation of a symbolic conflict that becomes a practical conflict requires that the human relationship assumes itself as an 37 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical ethical relationship (iss2). For this reason, the participation in a sports ac- tivity requires the previous acceptance of the entry into an ethical uni- verse: «And the reason we face these unnecessary obstacles is so that sport can be played, and so if you are not going to obey the rules it is almost as you are opting to be out of sport» (oss6, p.7). The setting of rules in sports is mainly related to the type of challenge that man wishes to face and, also, to the way he wants to answer it. What is the challenge? How ought we to overcome it? Which criteria are used to provide answers to that challenge? Do we wish to challenge ourselves in- dividually or in group? Do we wish to compare our answer with that of our counterparts? The answers to these questions will then result in the type of sports activity in which, for example in a physical education lesson, we decide to take part, as well as to create an opportunity of making sports a place of concrete evidences of our virtue (iss6). In the type of education of sports that is mainly functional, namely in the context of a physical education lesson, these are, however, questions answered and provided to students. Sport activities are selected and pre- determined in (e.g.) the national curricula and presented to students along with its most frequent norms, regulations and techniques and skills. B) THE SPIRIT OF SPORTS AND ITS INTERNAL VALUES But if the creation and regulation of sports arise only from rules that are explicitly described, then the ethical debate would be much more straight- forward, simple and objective than it seems. There is something endlessly debatable in the ethical dimension of sports that in turn leads the quest for the good in this field to become prominent and often without definitive answers, in the reflection and discussion by its main social players. This ethical element that goes beyond explicit regulatory and normative crite- ria, that generates further complexity in our understanding of the ethical nature of sports is, entitled «the spirit of sports and its internal values» (Si- mon, 2000) and emerges with the intention of searching for a better and more enriching way of living sports, with a better interpretation and not only considering the minimum criteria that make it possible (iss4, iss5). Without contemplating this spirit that is mainly ethical, there is a negli- gence of sport itself and of aspects of its nature that are essential (oss3, oss6, iss1, iss2, iss4, iss5, iss6, pett, pet1, pet2, pet3, pet4, pet5). As an example, oss3 refers that even within the explicit set of regulatory and constituent norms, some are more central than others and must be respected in order not to deprive that sport from its characteristics: «Foot- ball rules have changed a lot throughout the years, for example the offside. It is still football and rules continue to change. Some rules are more basic and central. If you decide that in football you cannot use the feet anymore, unless you are the goalkeeper, then you are totally changing the nature of the game. You can keep calling it football, but it will be a different version of football. (...) you must be aware that, even though you use the same name, it is not the same activity» (oss3, p.10). Nevertheless, it is possible to identify ethical aspects that are common across sports and that go far beyond its explicitly normative dimension. The notion of fair play is a good example and this subject constantly arises in physical education lessons (iss1, iss2, iss4, iss6). For instance, for iss1, «the idea that we can live collectively, even if we have different views (...) and we can share the same world» (iss1, p.19) is crucial to the spirit of sports, especially when considered as a vehicle for an ethical education. A) THE REGULATORY AND NORMATIVE STRUCTURE OF SPORTS In contrast to this didactic, iss1 in line with Meakin (1986; 1990) and McNamee, (1992) suggests the importance of creating a space in physical education lessons for raising questions of this nature with students in or- der to promote a greater awareness and participation in the ethical activi- ties in which they take part. This way, regulations are not something that is only externally imposed, they can be internally incorporated and become the result of a choice. Thus, for instance, if the student chooses an activity whose challenge entails the impossibility of individually carrying the ball, he knows and accepts that he is not going to play football or basketball but that he can choose volleyball, for example. The same way that if part of the challenge corresponds to including physical contact with the opponent, the tolerance of the student for accepting a one-to-one battle will be higher in sports such as handball or rugby. According to the interviewees, when we ask students to think about these questions, we are necessarily promoting a more deliberate and involved attitude with the ethical con- tent in classroom activities, thus making greater advances in terms of ethi- cal education. 38 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 B) THE SPIRIT OF SPORTS AND ITS INTERNAL VALUES This supra-regulatory understanding, and independ- ent from the different roles and point of views that we have, seems to be part of an internal spirit of the verbally inexplicit sport, and it can then set the basis for extremely rich learning situations during physical education lessons: «I think that for us, in the field of sports, the ideal would be that 39 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical one day we could play without a referee, isn’t it?» (iss1, p.19); «(…) [In a game], if we could ensure that everybody raised their hand when there is a foul, we would contribute for justice and for fairplay» (iss5, p.7). This is why physical education lessons, in contrast to what happens in more strictly regulated competition contexts, where regulatory aspects are strict- er, are a valuable space for the promotion of this spirit that, in a certain way, results from the legal and regulatory understanding of sports. In recreational sports, from which we can learn lessons for educational contexts, there is even a tendency to break some regulations in order to promote the internal values of sports. For instance, in handicapping con- testants, or when we create teams with different numbers of elements, contrary to the normal regulations, we artificially create balance in the confrontation and dispute so that it is real and has potential for growth through challenge of sufficiently similar capabilities: «The fundamental idea is that the sports relationship requires treating people with equality and trying to ensure that it is a relationship of equals. Equals does not mean that they are equal, it means they have the same dignity, the same credit and thus they can have an equal treatment» (iss2, p.8). In this sense, sports ethics in physical education is a highly relational concept and pro- vides references and norms on how we relate to our counterparts, creating what we can call a social ethics, where the displacement of ourselves and otherness, that is to say, the sensitivity and availability in relation to the place/role of the other, are crucial (oss3, oss5, iss1, iss3). This equitably-conditioned environment calls for another internal and common value of sports, the idea of mutual commitment (iss3). B) THE SPIRIT OF SPORTS AND ITS INTERNAL VALUES The idea of a mutual search for excellence via competition (Simon, Torres and Hager, 2015) is, for our interviewees, a non– or supra-regulatory ethical require- ment of sports that requires specific pedagogical commitment: an engagement where teachers and learners deploy all their skills, strengths and energy to their maximum capacity. There is something deeply ethical in this full dedication to sports challenge that human beings can make and think about and that, besides that, reflects the consideration of the other (op- ponent, teammate) as someone that deserves that mutuality of commit- ment and dedication (pett). Hence, there is a mutual logic in the ethical requirement of commitment, without which, even if we comply with all the regulations, we can disrespect the other or the sport itself in which we engage. One interviewee captures this mutuality with particular insight: «(...) since when we try to do better, we also enable the others to do their RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 40 best. We create room so that the other can offer his best and vice versa» (iss3, p.15). In competitive sports the levels of commitment are normally associated to the competitive needs of that moment. This means than the maximum commitment may not be necessary when the aim of winning does not re- quire that effort. In physical education, where we often realise that stu- dents’ performance is very weak due to their limited sports literacy, the mutual value of this commitment of showing the best performance of each one and, mainly, the best group performance, is pedagogically priceless. C) THE RIGHT PLAYING/DOING OF SPORTS When we think about ethics, especially in common sense conversa- tions, we often run the risk of finding moral perspectives on the notion of good in sports. Sports goodness includes, but is not limited to, fair play, justice, and the kindness of players’ actions and character. There is an es- sential aspect in sports goodness that is related to more technical, tactical and/or pragmatic aspects of sports performance that lead to an adjustment of the gesture to the requirements of each moment, which we call the right playing/doing of sports (iss2): «For me, when I am watching [sports], of whatever kind, it is important that gestures are well performed» (issT, p. 15). The right performing of sports gesture is not only related to the techni- cal criteria, it also includes the ethical dimension that should be pedagogi- cally analysed more in depth in physical education. In this sense, for iss5, the right playing/doing is an essential aspect of sports ethics since nor- mally the sportsmen that most break rules and do not respect the sports’ spirit are usually the technically less skilled professionals, which have a shorter range of legitimate tools to reach their objectives: «The improve- ment of the gesture is important because of ethics, for example. The best we teach the gesture, the less players need to cheat or use violence to reach their ends, since they acquire tools that enable them to reach them in a legal way» (iss5, p. 14). Normally, the good playing of sports is thought to require the correct performance of the technical movements, the correct use of sport materi- als, their functionality, a concern for efficiency and effectiveness, and are based on standardised criteria even those criteria can be altered from standard competitive forms to those more apt to the teachers’ pedagogical goals (osst). 41 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical Thus, the technical domain is the support and the basis of any right (i.e. rule-observing) playing/doing, whether it is sportive, artistic, technological or mechanical (osst). In this sense, we can find here a link between ethics and technique that can be relevant for an ethical interpretation of sports teaching through physical education, since technical competences enable the sportsman to overcome the challenges created by sports. C) THE RIGHT PLAYING/DOING OF SPORTS It would be too simplistic, therefore, to say that for an ethical concern in physical education it is only necessary to respect its normative structure and its internal structure, since the respect for the rules and the maximum commitment and good will of students is not enough. For our interviewees it is essential that, besides the incorporation of normative criteria and a committed mutuality, there is also the serious work of learning technical and tactical knowledge that are specific of each sport and without which not only the technical aspect would be jeopardised, but also the ethical considerations. D) OVERCOMING IN SPORTS So it is the same thing as you swimming against an historical record and if you finish one minute shorter time you can say «I won, I beat», because you beat the record. But who did you beat? Because the person who did that record didn’t have the chance to adjust a strategy or to know that you are a little bit ahead of them» (iss6, p.4). – cannot be compared to that of non-simultaneous competitions, where one performs alone or against our previous results, since: «(…) my historical self, the person who performed yesterday and run in two hours and twenty two minutes does not have the chance to try harder against myself today. So it is the same thing as you swimming against an historical record and if you finish one minute shorter time you can say «I won, I beat», because you beat the record. But who did you beat? Because the person who did that record didn’t have the chance to adjust a strategy or to know that you are a little bit ahead of them» (iss6, p.4). These aspects raise relevant questions related to the fairness of sports challenge, the merit of the overcoming process and its didactic utility. Thus, competition constitutes an important part of the ethical dimen- sion of sports. For pet2, the commitment to ethics becomes increasingly more difficult the higher the competitive level is, and the higher the number of other aspects that are considered beyond sports entertainment and the mere aspects of winning or losing. The dominance of the com- petitive aspect of sports often compromises its ethical experience, accord- ing to oss5, pet4 and pet5, since it is also necessary to learn to compete, including the value of the fight for victory and success in its correspond- ent axiological hierarchical place. Equally, oss5 and pet5 argue that sports must be a place of inclusion and that, often, particularly at high level, it becomes just the opposite, a place of exclusion: «It is essential that people respect each other’s differences. As it is also important to respect our skills, doing our best» (pet5, p.6). Also for pet4 the selection process of athletes in school-age (children and young people) contributes to the marginalisation of those that are less skilled for the benefit of the absolute value of performance. D) OVERCOMING IN SPORTS For oss3, sports is an arena for «human betterment» (Hämäläinen, 2014) at different levels. When they submit themselves to a sports challenge, sportspersons voluntarily embark upon a path of personal and/or collec- tive improvement, challenging themselves, the others, or a result/record: «The sportsman (sic) has an interesting problem – no matter if they are opponents, or a very difficult wave, for a surfer – and he managed with his skills and right-doing to overcome himself, to achieve something unex- pected» (oss3, p.12). This overcoming notion is not only a practical one, but it has a symbolic meaning too. When overcoming a sports challenge, indi- viduals (more or less self-consciously) wander a path of personal growth and overcoming (Lacerda and Mumford, 2010). Yet for issT and iss6, this dimension of sports overcoming is not always straightforward or easy to judge, and the ethical nature of the sports chal- lenge changes considerably according to both practical and formal criteria of that specific challenge. For instance, the overcoming capacity and the capacity of performing at the best of his ability for a student in gymnastics, as it is an individual activity and not performed simultaneously with op- ponents, is totally different of that of the student that takes part in a relay race and that can permanently compare and adjust his performance, in real time, according to the performance of his opponents. In this case, the stu- 42 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 42 dent can decide not to do his best, in case something below is enough for succeeding. This raises ethical questions related to each one’s duty of per- forming his skills at their highest level and, at the same time, the right that each one has to manage their own efforts (isst). Moreover iss6 reports that the ethical value of competition, simultaneous with the performance of others – parallel and shared tests (Kretchmar, 1975) Moreover iss6 reports that the ethical value of competition, simultaneous with the performance of others – parallel and shared tests (Kretchmar, 1975) – cannot be compared to that of non-simultaneous competitions, where one performs alone or against our previous results, since: «(…) my historical self, the person who performed yesterday and run in two hours and twenty two minutes does not have the chance to try harder against myself today. 3 See another interesting example in this field, when the athlete Iván Fernández Anaya refused to take advantage from the runner that was ahead of him when this one stopped before the finish- ing line thinking he had already crossed it: http://elpais.com/elpais/2012/12/19/inenglish/­ 1355928581_856388.html D) OVERCOMING IN SPORTS When we speak of physical education, these questions should not be raised in this linear way, since it should be a place from all and for each individual, where in this aspect we can make the difference. pet5 adds that the non-acceptance of the weakness of others’ perform- ances, mainly in an educational context, is more serious in ethical terms than accidentally breaking some of the strictly regulated rules: «(…) [the 43 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical ethical attitude in sports] also depends on understanding that the others fail independently of complying or not with the rules. (...) The misunder- standing of others’ fails is a lack of respect in ethical terms, because as human beings we all fail» (pet5, p, 5). Thus, physical education, as a space for the teaching of sports that is loosened from the shackles of competition-dominated or supremacy-per- formance, is then an excellent place for learning situations and different performance criteria that increasingly value the ethical content. Here the pedagogues strives more for the inclusion of all than for a blind achieve- ment of numerical results, as already stressed by Manuel Sérgio: «The trans­ cendence and overcoming (namely in group, team, community) of what we are, towards what we should be: this is the sense of sports!» (Sérgio, 2014, p.80). E) SPORT AS AN OPPORTUNITY FOR A SUPEREROGATORY ETHICS As argued so far, in sports as in life, ethical problems are not solved al- ways by the respect for and compliance with all regulations. «We cannot reduce sports ethics to rules. (...) Ethics includes the idea of supereroga- tory, when someone goes beyond his duties. And this is what we most ad- mire» (oss3, p.10). An important part of the ethical experience and sports spirit, is based on the experience of a supererogatory ethics, that is to say, an ethics that goes beyond formal requirements of right conduct (Feinberg, 1968; Feld- man, 1986). As pointed out by oss1: «(...) because the rule has a very limited scope of action. (...) It is the administrative aspect. I can follow the rules but, for example, be unpleasant to my opponent ...(...). Thus, ethics is not at all limited to the compliance with the rules nor to their existence» (oss1, p.15). It is broadly consensual in the totality of our interviewees that there is a fundamental ethics not limited to rules and regulations. This ethical space is unregulated not merely because it is difficult so to do, but rather because its values depend properly on the fact that they are not imposed:3 «It some- 44 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 44 times happens in cycling. Someone has a flat tyre and we can try to run away or wait while his tire is changed. We do not have to wait. And there isn’t any ethical norm that says that. And it would be impossible to define procedures for each situation. We have to interpret. And then there are things that ethically cannot be requested. They are beyond what we should do. But they have ethical value! They can be appreciated, we can say that it was morally and aesthetically beautiful. But I think it is dangerous to try to put that down in writing, because that is when we see an aesthetically more boring side of sports, in which we want to foresee all situations and control every element» (oss3, p.10). E) SPORT AS AN OPPORTUNITY FOR A SUPEREROGATORY ETHICS According to iss1, the strong regulatory nature of sports can promote something that can be considered to be very dangerous because its ethical dimension: the fulfilment of ethical criteria only because they are exter- nally imposed and not because they are internal and part of our convic- tions: «The higher the competition in terms of performance, the more rules it has up to the smallest details, with the aim of finding increasingly thor- ough assessment methods. There we find a relation between ethics and law. Law tries that sports remains in an ethical relation between partici- pants, that it has the regulatory aspect (which is not necessarily ethical) and it can even frequently lead to strategic behaviours that may be ques- tioned from the point of view of sports virtue. It is possible to take advan- tage or profit from a situation that, at first, had the aim of punishing but that was then taken as an advantage» (iss2, p.7). In its turn, rather than imposing a normative structure with well-de- fined, strictly applied rules enforcing only minimum limits, a supereroga- tory ethics is transformative because it makes us think, reflect, and inter- pret the world and ourselves in a more holistic way, promoting ways of being that are built and grown internally. Physical education lessons seem to be a privileged space for this experience (oss3). oss5 and issT reinforce this idea, adding that what is imposed by the law, that is just equitable and faire, even being good, is not enough for us. This is why there is something especially interesting and attractive in an ethics that extrapolates the mere duty and that sports promotes with the idea of fair play: «For instance, when one player is about to score a goal and offers that goal (...) to a team- mate (...) or to the player who plays less time or that is still in an integra- tion process in the team [or class] (...) I think this is a demonstration of an ethical value...» (pett, p.17). E) SPORT AS AN OPPORTUNITY FOR A SUPEREROGATORY ETHICS The notion of fair play includes the active participant in the ethical process, providing a great opportunity for exercising his freedom, his cons­ 45 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical ciousness and autonomy in the process of thinking/building the ethical universe of practice that just extrapolates the minimum requirements, the duty, the fair and the equitable (iss1, pet4, pet5). It is through this notion that we can understand the distinction between the rule and the spirit of the game (Simon, Torres and Hager, 2015): «We can abide the rules of the game by the limit and have tricky tactics, throw ourselves on the floor, kick the ball out of play...» (iss2, p.10); «In many occasions I can enter a game and respect all the rules but without respecting the other, because I don’t rec- ognise him as someone that can create challenges...» (iss3, p.15). An equally interesting aspect in applying the notion of the supereroga- tory ethics to sports and physical education is that, as it is not descriptive nor explicitly defined, it is tacitly created and negotiated between the par- ticipants: «There is always a negotiation in every game. Teams enter the game and start to analyse one another: how are we going to play this game? Will we play clean or dirty? This relationship is developed through a dialog and events reveal that» (iss2, p.11). Sports, and more specifically the physical education lesson, is then an arena of opportunities where man can exercise and communicate this su- pererogatory or meta-ethics, in which he is highly qualified, an ethics that leads to overcoming and transcendence, that extrapolates law and duty requirements, that overcomes justice and equity, that makes us think be- yond the minimum limits, and an ethics that is not ordinary: «Sports is also a place where man can transcend himself... I think that this attitude leads him to make a difference» (pett, p.20). FINAL CONSIDERATIONS «Sports should do good and in order to do good it (sic) has to be linked to the idea of good» (iss5, p.7). Ethics appears in sports when there is also a need of preservation, de- fence and mainly persecution of its essential nature and of places, func- tions or roles of its participants (iss6). This need is even more urgent when we consider a sports education through physical education that is based on the idea that sports is a fertile ground that contributes to a meaningful life (Feezell, 2013; McNamee, 2008; Reid, 2010). No matter how arguable, variable and apparently intangible the nature of sports may be, largely due to the huge diversity of forms it assumes (dif- ferent sports and practices) and the different contexts where it is per- 6 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2016.18.3 · ISSN: 1130-6149 - pp. 29-48 46 formed (competition/high-performance, entertainment, teaching, training) there is an idea of sports that constitutes its identity and before which we fell the need of a truthful relationship: «A key question in sports is the existence of a relation of truth, not in the sense of an absolute truth but a relationship that is genuine in terms of the respect for an idea of the sports practice. Virtue appears in sports because it always challenges and places people in competition, and there are two sides in a competition that try to obtain a favourable result. This result involves a conflict of interests. And in this relation of conflict of interest, in order to promote a truthful relationship, honesty and courage have to stand out (...), the respect for the opponent, the recognition of the opponent, that is to say, seeing the opponent as equal» (iss2, p.6). In the pedagogical context of physical education we can, thus, con- clude that an interesting part of the ethical potential of sports, as stated by our interviewees, is based on the didactic contemplation, treatment and use of the ethical vector presented here, namely, the regulatory and normative structure of sports; the spirit of sports and its internal values; the right playing/doing of sports; the overcoming in sports; and sports as an opportunity for a supererogatory ethics. FINAL CONSIDERATIONS These are not, as we have seen, external or optional elements to add to physical education classes, but intrinsic features of sports’ contents that can and should be treated and promoted in physical education classes by an ethical pedagogical lens. Notwithstanding that our purpose was to identify, according to the main concerns of our study group, a relevant start point for promoting sports ethics in a physical education lesson that goes beyond the legal, regulatory and functionalist boundaries of the teaching of sports in this class, based on the idea that «universal values, linked and associated to ef- fort and sweating, help to create different and unique persons and indi- viduals, in terms of body and soul, spirit and mind, ways of feeling and thinking, understanding and assessing» (Bento, 2010).We conclude, how- ever, that the ethical potential of sports is not limited to these technical aspects and that, naturally, some relevant aspects of sports ethics in ge­neral have yet to be systematically exploited. 47 LUÍSA ÁVILA DA COSTA; MICHAEL MCNAMEE; TERESA LACERDA Re-envisioning the ethical potential of physical Mcnamee, M. (2010): The ethics of sport. A reader. London: Routledge. References Aristotle. 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Conversasiones para disfrutar el deporte plenamente.(Goal from the middle field. Conversations to fully enjoy sport). Buenos Aires: Miño y D’ávila editors. Torres, C. (2014): The Bloomsbury Companion to the Philosophy of Sport. London: Bloomsbuty.

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Survival Analysis in The Presence of a Cure Fraction Using Data of Dialysis Patients: A Bayesian Approach

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Laleh Hassani Mother and Child Welfare Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Mohammad Reza Baneshi Modeling in Health Research Center, Institute for Futures Studies in Health, Kerman University of Medical Sciences, Kerman, Iran. Shideh Rafati Social Determinants in Health Promotion Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Survival Analysis in The Presence of a Cure Fraction Using Data of Dialysis Patients: A Bayesian Approach Shideh Rafati Research Article Keywords: Survival, Bayesian Approach, Cure Fraction, Dagum Distribution Posted Date: January 27th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-145627/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License TITLE PAGE Type of manuscript (Original Article) Type of manuscript (Original Article) Title: Survival Analysis in the presence of a Cure Fraction Using Data of Dialysis Patients: A Bayesian Approach Tel: +98(0)9131404512 Fax: +983431325127 Tel: +98(0)9131404512 Fax: +983431325127 Email: [email protected] [email protected] [email protected] Original Article Authors: Shideh Rafati1,2. PhD in Biostatistics. ORCID ID: 0000-0001-7108-414X 1 Social Determinants in Health Promotion Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. 2 Department of Biostatistics and Epidemiology, Kerman University of Medical Sciences, Kerman, Iran. Email: [email protected] Mohammad Reza Baneshi2,3. Professor of Biostatistics. ORCID ID: 0000-0002-6405-8688 2 Department of Biostatistics and Epidemiology, Kerman University of Medical Sciences, Kerman, Iran. Mohammad Reza Baneshi2,3. Professor of Biostatistics. ORCID ID: 0000-0002-6405-8688 2 Department of Biostatistics and Epidemiology, Kerman University of Medical Sciences, Kerman, Iran. 2 Department of Biostatistics and Epidemiology, Kerman University of Medical Sciences, Kerman, Iran. 3 Modeling in Health Research Center, Institute for Futures Studies in Health, Kerman University of Medical Sciences, Kerman, Iran. Email: [email protected] Laleh Hassani4. Assistant Professor of health education and health promotion. ORCID ID: 0000-0001-8446-0992 3 Modeling in Health Research Center, Institute for Futures Studies in Health, Kerman University of Medical Sciences, Kerman, Iran. Email: [email protected] Laleh Hassani4. Assistant Professor of health education and health promotion. ORCID ID: 0000-0001-8446-0992 4 Mother and Child Welfare Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. Email: [email protected] Corresponding author: Abbas Bahrampour2,3. Professor of Biostatistics. ORCID ID: 0000- 0002-6343-9243 2 Department of Biostatistics and Epidemiology, Kerman University of Medical Sciences, Kerman, Iran. 3 Modeling in Health Research Center, Institute for Futures Studies in Health, Kerman University of Medical Sciences, Kerman, Iran. 1 1 Original Article 2 2 Survival Analysis in the presence of a Cure Fraction Using Data of Dialysis Patients: A Bayesian Approach Bayesian Approach Abstract Abstract Background: The aim of this study was to evaluate the goodness of fit of Bayesian mixture and non-mixture cure models to find the factors affecting dialysis patient’s survival time where a significant proportion of the population has a long-term survival. Study Design: A retrospective cohort study. Study Design: A retrospective cohort study. Methods: The data of 252 dialysis patients were used among whom 35 cases died. Since in this study a part of the population had long-term survival, Bayesian cure models were used and evaluated using DIC index. The data were analyzed by R and Openbugs Softwares. Results: Of the 252 dialysis patients, 136(54%) were males and the mean (SD) age was 53.39 (18.09) years. The patient’s follow-up time mean (SD) was 10.93(7.82) years. The 10 and 20- year survival rate of these patients were 87% and 73%, respectively. The findings show that the best fitting belonged to the Bayesian Non-mixture Cure Model (BNCM) with Dagum distribution. The variables of age, Body Mass Index, dialysis duration, frequency of dialysis, age of onset of dialysis, and occupation affected patients' survival based on BNCM with Dagum distribution. Conclusions: The results demonstrated that the BNCM with Dagum distribution can be a good selection model to analyze survival data, where there is the possibility of a fraction of cure. Keywords: Survival; Bayesian Approach; Cure Fraction; Dagum Distribution Introduction Keywords: Survival; Bayesian Approach; Cure Fraction; Dagum Distribution Introduction Introduction 3 The advanced phase of chronic renal failure, in which life relies on transplantation or dialysis, is called end-stage renal disease (ESRD) [1]. Due to its chronic and disabling nature, this disorder may have a detrimental impact on patients' quality of life and contributes to decreased social interactions, depression, anger, decreased capacity of an individual to perform independent everyday life activities, and eventually increased mortality. It is necessary to determine the factors that influence the survival of these patients, with regard to the challenges and issues that dialysis patients face [2]. With significant advances in all areas of medical science, a significant proportion of patients with various types of diseases have long-term survival [3]. Thus, in many cases we have survival data that the number of patients experiencing death from a disease is much lower than that of censored cases. In such a situation, if all the events occurred at the beginning of the study and no event happened at the end of the Kaplan-Meier (K-M) curve, cure models can be used to provide a more accurate interpretation of survival data [4]. Cure models are specific types of survival models in which the population is a combination of susceptible cases (those who may experience the event, ie, patients with short- term survival) and cured/non-susceptible individuals who have long-term survival (those who never experience the event in the follow-up period) [5]. There are 2 main methods of mixture and non-mixture to model the survival data with a cure fraction. The existence of a long, stable flatness with high censoring rate at the tail of the K-M curve and the adequacy of follow-up time, indicate that the data are suitable for applying cure models [6]. To identify cured individuals, statistical tests can be used to detect patients with long-term survival. The null hypothesis of this test indicates that all people may experience death and there is no cure fraction. To reject or accept the stated hypothesis, critical values that have been set by Maller and Zhou can be used [7, 8]. 4 Cure models do not work well when the sample size is small, and the high censoring rate at the end of the K-M curve is one of the favorable features required to use cure models. Introduction However, in the secondary data used in the present study, all events occurred at the beginning of the follow-up, and the K-M curve tail was stable. Still, the number of samples at the end of the study was low (Censoring rate at the end of the survival curve was not high.); thus, Bayesian analysis of cure models was used instead of classical analysis. Bayesian inference of cure models were introduced by some authors, including Martine [9, 10], Swain [11], Chen [12], Ibrahim [13], and Castro [14]. However, to date, no study has compared the prediction performance of the Bayesian mixture and non-mixture cure models based on Dagum, Weibull, and log-logistic distributions. Therefore, in this study, the mentioned models were used and compared. The results of this study may be helpful for future studies and analysis of medical and health-related data, where there is the possibility of a fraction of cure. Methods Secondary Data Of the total patients admitted from 2010 to 2016, the data of 252 patients were recorded in the dialysis ward of Bandar Abbas Hospitals, Iran. Mortality was considered as the event of interest and censored cases included those who were alive at the end of the study, excluded cases and those treated with kidney transplant. The survival time of the patients was calculated by years from the onset of dialysis to the end of the study in 2016. Data were obtained from a given checklist including age, gender, body mass index (BMI), education, age of onset of dialysis, job, blood type, marital status, smoking, history of diabetes, hypertension, renal stones and obstruction, renal cysts and congenital diseases, dialysis 5 duration (hour per session), number of dialysis sessions per week, history of cardiac-respiratory diseases, history of anemia and familial history of chronic renal failure. In the end, 19 variables, including gender, job (five indicator variables), blood type (three indicator variables), marital status, history of smoking, diabetes and hypertension (all binary), age of censoring or death time, education, dialysis duration, number of dialysis sessions, BMI, age of diagnosis (all continuous), are used in Bayesian cure models with regard to the low frequency in some categories of independent variables. Ethical considerations This manuscript has been approved by the Ethics Committee of Kerman University of Medical Sciences at No. IR.KMU.REC.1397.599. Written informed consent was obtained from all the participants. 1. Mixture cure models The population is separated into two parts in a mixture cure model. Cured or long-term survivors and survivors that are uncured or short-term. Let the probability of being cured be p (0 < p < 1) and therefore (1 - p) is the probability of being susceptible to an individual[9]. The corresponding t-time survival function is as follows: S(t) = p + (1 − p)S0(t) S(t) = p + (1 − p)S0(t) Where S(t) is the total population's survival function. S0(t) is the baseline survival function for the susceptible individuals for which type I Dagum, Weibull and Log-logistic distributions are assumed in this study. The distributions described below have been introduced. 2. Non-mixture cure models 6 6 The survival function is defined in this case as The survival function is defined in this case as The survival function is defined in this case as S(t) = p𝐹0(𝑡) = exp⁡[(ln 𝑝)𝐹0(𝑡)] S(t) = p𝐹0(𝑡) = exp⁡[(ln 𝑝)𝐹0(𝑡)] Where 𝐹0 (t) = 1 – S0(t) is the baseline cumulative distribution function for the susceptible people. Where 𝐹0 (t) = 1 – S0(t) is the baseline cumulative distribution function for the susceptible people. To model the cure probability (p) under both mixture and non-mixture cure models, we applied the logistic function. 3. The type I Dagum distribution Suppose that survival time for the uncured individuals has the Dagum distribution with three parameters. The This distribution's cumulative distribution function is defined through (for t > 0) 𝐹0(𝑡) = [1 + 1 𝑎𝑡−𝑏] −𝑐 = (1 + 𝑎𝑡𝑏 𝑎𝑡𝑏 ) −𝑐 0) 𝐹0(𝑡) = [1 + 1 𝑎𝑡−𝑏] −𝑐 = (1 + 𝑎𝑡𝑏 𝑎𝑡𝑏 ) −𝑐 Where b and c are positive parameters of shape and the scale parameter is a (𝑎= ⁡exp⁡(𝑋𝑖 ′𝜃) the covariates can be included in the model through 𝑎). Notice that the c=1 case leads to the log- logistics distribution[10]. The third distribution is also assumed to be the Weibull distribution with two parameters ⁡𝛼, 𝛾> 0 (S0(𝑡) = 𝑒𝑥𝑝⁡(−𝛾𝑡𝛼)), which are the shape and scale parameters respectively. The third distribution is also assumed to be the Weibull distribution with two parameters ⁡𝛼, 𝛾> 0 (S0(𝑡) = 𝑒𝑥𝑝⁡(−𝛾𝑡𝛼)), which are the shape and scale parameters respectively. Priors Priors The normal prior distributions N(0, 100) were considered in the Bayesian analysis for the vector of the parameter 𝜃. Also, in Dagum, Weibull, and log-logistic distributions, the uniform prior distribution was assumed for the shape parameters. Prior independence of the parameters was presumed in the model for all scenarios. 7 7 Bayesian Inference Here, the Bayesian mixture and nonmixture cure models were used based on Dagum, Weibull and log-logistic distributions. By combining the joint prior distribution and the likelihood function, the joint posterior distribution was obtained for the parameters of the model. Posterior summaries of interest are obtained from simulated samples for the joint posterior distribution using standard Markov Chain Monte Carlo (MCMC) procedures. Also, 1,010,000 samples were generated for each parameter of interest. The first 10000 simulated samples were discarded as a burn-in period, which is usually used to minimize the effect of the initial values. The posterior summaries of interest were based on 20,000 samples, taking every 50 sample to have approximately uncorrelated values[9]. The Bayes estimates of the parameters were the mean of Gibbs samples, which were drawn from the joint posterior distribution. Convergence of the MCMC algorithm was monitored by history and autocorrelation plots for the simulated samples[9, 15]. Inferences were obtained using R and OpenBUGS Softwares. In addition to the history plot, the Monte Carlo error (MC_error) were used to evaluate the accuracy of posterior estimates for each parameter, which is an estimate of the difference between the actual posterior mean and the estimated posterior mean for each parameter. If the Monte Carlo error value for each parameter is less than 5% of its standard deviation, then, convergence is obtained for that parameter, indicating no need for further simulation samples. Model Selection Model Selection 8 8 Deviance Information Criteria (DIC), as a measure of the goodness-of-fit, used to compare between the mixture and non-mixture models, where a lower DIC value indicates a better model fit. Results Overall, 252 hemodialysis patients were studied, of them, 35 (13.9%) cases faced the event of death and 217 (86.1%) cases were censored. The follow-up time mean (SD) was 10.93 (7.82) years. The 10 and 20-year survival rate of these patients were 87% and 73%, respectively. Table1 shows the patients characteristics. Figure 1 displays a K-M plot for overall survival function, which shows "flatness" near 0.60 in the survival curve, and thus a cure model appears to be suitable for this data. Based on this diagram, flatness occurs after about 20 years (fig1). In table2, the DIC values of Bayesian cure models based on Weibull, Log-logistic and type I Dagum distributions and in the presence all of covariates have been reported. Based on the DIC index in Table 2, the goodness of fit of the non-mixture models is better than the mixture ones, and belongs to the dagum distributions. The worst performance is related to the Bayesian Weibull cure model (mixture and non-mixture) because the most DICs belong to these two models. The posterior summaries of parameters of non-mixture cure models with type I Dagum distribution have been presented in table 3. In section short term survival in table3, the 95% credible interval for age of death or censoring time, BMI, duration of each dialysis, frequency of dialysis per week, age of onset of dialysis, and occupation does not include zero. Therefore, these variables have a significant effect on the short-term survival. 9 9 Based on this table, by adjusting other factors, for one unit of increase in age, the failure odds is increased by 0.11 (OR=exp(0.105)). for one unit of increase in BMI and duration of each dialysis, the failure odds is reduced by 0.17 (OR=exp(-0.181)) and 0.31 (OR=exp(-0.364)), respectively. For one unit of increase in frequency of dialysis and age of onset of dialysis, the failure odds is increased by 0.28 (OR=exp(0.252)) and 0.09(OR=exp(0.084)), respectively. The death odds of housewives is 0.17 less than employees (OR=exp(-0.192)). Also, in section long term survival, the 95% credible interval for age of death or censoring time, BMI and age of onset of dialysis does not include zero suggesting that these variables have significant effect on the long-term survival. Discussion A total of 252 dialysis patients with 35 (13.9%) death events were used for this study. Due to the low-event data, use of classical methods, such as Cox, was not possible. Also, all 35 death events (target event) occurred at the beginning of the study, and all cases were censored at the end of the study. However, the number of samples at the end of the study was low (The censoring rate was not high at the tail of the survival curve). Thus, Bayesian cure models were used to analyze the data. Given the nature of the data of dialysis patients, this study aimed to evaluate and compare the performance of Bayesian cure models with Weibull, log-logistic, and Dagum distribution to analyze these data. Based on the findings of the present study, the variables of age, Body Mass Index, dialysis duration, frequency of dialysis, age of onset of dialysis, and occupation affected dialysis patients' survival. The findings of present study revealed that older age was related to higher mortality in dialysis patients. This result is consistent with that of the previous studies[16, 17]. Also, similar to the 10 present study, Tsur’s study confirmed that higher BMI, was significantly associated with longer survival[17], hence, high BMI is protective in these patients. Further, based on the present study, occupation is one of the most important factors affecting dialysis patients' survival that its effect is confirmed in past studies[18]. present study, Tsur’s study confirmed that higher BMI, was significantly associated with longer survival[17], hence, high BMI is protective in these patients. Further, based on the present study, occupation is one of the most important factors affecting dialysis patients' survival that its effect is confirmed in past studies[18]. Based on the present study, for one unit of increase in the duration of dialysis, the odds of death was decreased by 0.31; the significance of this variable was verified in other studies[19]. Also, increasing one unit in the number of dialysis sessions per week increases the odds of death that is inconsistent with some other studies[20]. Discussion The results of this study indicated that the Bayesian non-mixture cure models are superior in compared to the Bayesian mixture cure models, and the results of some previous studies are consistent with those of the present study [15, 21].However, The Swain study showed that the mixture cure model has a better fit than the nonmixture cure model based on the generalized Gompertz distribution [11]. Another study has compared mixture and non-mixture cure models based on the generalized modified Weibull distribution by Bayesian approach. Based on DIC values, it was found that the mixture and nonmixture cure models provide very close results [9]. Also, a study aimed to assess mixture and nonmixture cure models that revealed both classes fit the data well [6]. The results of Jafari Koushaki study showed the performance of the non-mixture cure model with log-logistic distribution is better than the Weibull non-mixture cure model, as the value of the DIC is lower for the log-logistic non-mixture cure model [22] that present study confirms it. it. the implementation of the Bayesian cure models with the less used distribution of Dagum is the strength of the present study and the lack of more important and more effective variables on 11 patient survival such as adequacy of dialysis, creatinine level, urea, albumin, and hemoglobin is its weakness. Abbreviations DIC: Deviance Information Criteria; MC_error: Monte Carlo error; BMI: Body Mass Index; K-M curve: Kaplan-Meier curve; SD: Standard Deviation; BNCM: Bayesian Non- mixture Cure Model; ESRD: end-stage renal disease Ethics approval and consent to participate This manuscript has been approved by the Ethics Committee of Kerman University of Medical Sciences at No. IR.KMU.REC.1397.599. Written informed consent was obtained from all the participants. In addition to this, all methods were performed in accordance with relevant guidelines and regulations. Conclusion The results of the present study demonstrated that the Bayesian non-mixture cure model with type I Dagum distribution can be a good choice for analyzing survival data, where there is the possibility of a fraction of cure. Consent for publication Not applicable: individual information has not been published. Availability of data and materials Availability of data and materials 12 The data sets used and/or analyzed during the current study are not publicly available due to confidentiality of data and subsequent research, but are available from the corresponding author on reasonable request. Funding No funding was received for the study. Authors' contributions SR proposed the study. MB and LH were involved in the design of study with the supervision of AB. LH collected the data. SR performed the statistical analysis. SR, MB and AB prepared the first draft of the manuscript and the authors read, revised and approved the final manuscript. Acknowledgements The authors thank staff of Dialysis Ward of Shahid Mohammadi and Persian Gulf Hospitals located on Bandar Abbas (Iran) for assisting and allowing for data collecting. Also, the authors wish to acknowledge Martinez and colleagues[9] for their study, as their method is the original source of described method in this study. Competing interests The authors declare that they have no competing interests. References 1. Rafati F, Mashayekhi F, Dastyar N. Caregiver Burden and Spiritual Well-being in Caregivers of Hemodialysis Patients. Journal of Religion and Health. 2019:1-13. 1. Rafati F, Mashayekhi F, Dastyar N. Caregiver Burden and Spiritual Well-being in Caregivers of Hemodialysis Patients. Journal of Religion and Health. 2019:1-13. 2. Rafati S, Baneshi MR, Hassani L, Bahrampour A. 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International journal of nephrology. 2018;2018. 14 17. Tsur N, Menashe I, Haviv YS. Risk Factors Before Dialysis Predominate as Mortality Predictors in Diabetic Maintenance Dialysis patients. Sci Rep. 2019;9(1):1-8. 18. Tao S, Zeng X, Liu J, Fu P. Socioeconomic status and mortality among dialysis patients: a systematic review and meta-analysis. Int Urol Nephrol. 2019;51(3):509-18. 19. Goh A, Vathsala A. Native renal cysts and dialysis duration are risk factors for renal cell carcinoma in renal transplant recipients. Am J Transplant. 2011;11(1):86-92. 20. Chandrashekar A, Ramakrishnan S, Rangarajan D. Survival analysis of patients on maintenance hemodialysis. Indian J Nephrol. 2014;24(4):206. 21. Achcar JA, Coelho-Barros EA, Mazucheli J. Cure fraction models using mixture and non-mixture models. Tatra Mountains Mathematical Publications. 2012;51(1):1-9. 22. Jafari-Koshki T, Mansourian M, Mokarian F. Exploring factors related to metastasis free survival in breast cancer patients using Bayesian cure models. Asian Pac J Cancer Prev. 2014;15(22):9673-8. Tables Table1-the patients characteristics Table1-the patients characteristics 15 15 Continuous Variables Died (n=35) Censored (n=217) Pvalue Mean SD Mean SD Age (year) 54.14 22.38 53.27 17.36 0.69 Age starting dialysis 42.77 19.38 42.90 16.72 0.04 Body Mass Index 21.51 3.61 23.10 4.30 0.76 dialysis duration (hours) 3.66 0.48 3.79 3.40 0.06 Categorical Variables Number Percent Number Percent Blood group O 16 45.7 92 42.4 0.71 Blood group A 5 14.3 57 26.3 0.12 Blood group B 14 40.0 58 26.7 0.10 Blood group AB 0 0.0 10 4.6 0.19 Employee 2 5.7 17 7.8 0.32 Jobless 10 28.6 36 16.6 0.08 Housewife 13 37.1 88 40.6 0.69 Farmer 2 5.7 6 2.8 0.36 Retired 4 11.4 38 17.5 0.36 Other occupation 4 11.4 32 14.7 0.60 Illiterate 16 45.7 70 32.3 0.21 Low literacy 15 42.9 102 47.0 0.65 Diploma 2 5.7 35 16.1 0.11 Collegiate 2 5.7 10 4.6 0.77 Males 18 51.4 118 54.4 0.74 Married 27 77.2 180 82.9 0.41 Tobacco use 15 42.9 76 35.0 0.36 Diabetes 24 68.6 110 50.7 0.04 Hypertension 18 51.4 134 61.8 0.24 Table2- DIC values of Bayesian cure models DIC Models Mean 280.2 Mixture type I Dagum 110.7 Non-mixture type I Dagum 280.6 Mixture Log-logistic Table2- DIC values of Bayesian cure models 16 249.3 Non-mixture Log-logistic 523.2 Mixture Weibull 490.9 Non-mixture Weibull Table 3: Posterior summaries of the non-mixture type I Dagum cure models. Parameter Posterior mean Posterior SD* MC_error 95%Credible Interval Odds Ratio Table 3: Posterior summaries of the non-mixture type I Dagum cure models. 17 Short-term survival age of death or censoring time 0.105 0.039 <0.001 (0.048, 0.169)** 1.110 body mass index -0.181 0.050 <0.001 (-0.292,- 0.085)** 0.834 dialysis duration -0.364 0.070 <0.001 (-0.825,- 0.097)** 0.694 frequency of dialysis per week 0.252 0.100 <0.001 (0.242, 0.700)** 1.286 age of onset of dialysis 0.084 0.041 <0.001 (0.044, 0.171)** 1.087 Occupation(Housewives) Reference: Employees -0.192 0.080 <0.001 (-0.721,- 0.337)** 0.825 Long-term survival age of death or censoring time -0.357 0.009 <0.001 (-1.889,- 0.831)** 0.699 body mass index -0.666 0.007 <0.001 (-1.253,- 0.072)** 0.513 age of onset of dialysis -0.620 0.067 <0.001 (-1.629,- 0.500)** 0.537 * Standard Deviation ** significant at 95% level Figure Figure 18 18 Fig1. Kaplan–Meier estimate of the overall survival function for the dialysis patient data Fig1. Kaplan–Meier estimate of the overall survival function for the dialysis patient data Fig1. Kaplan–Meier estimate of the overall survival function for the dialysis patient dat 19 19 Figures Figure 1 Kaplan–Meier estimate of the overall survival function for the dialysis patient data Figure 1 Kaplan–Meier estimate of the overall survival function for the dialysis patient data Kaplan–Meier estimate of the overall survival function for the dialysis patient data Kaplan–Meier estimate of the overall survival function for the dialysis patient data Kaplan–Meier estimate of the overall survival function for the dialysis patient data

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A Novel Transwell Blood Brain Barrier Model Using Primary Human Cells

Frontiers in cellular neuroscience

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Edited by: Stefania Ceruti, University of Milan, Italy Reviewed by: Förster Carola, Universitätsklinikum Würzburg, Germany Babette Weksler, Cornell University, United States *Correspondence: Nicole L Stone Edited by: Stefania Ceruti, University of Milan, Italy Reviewed by: Förster Carola, Universitätsklinikum Würzburg, Germany Babette Weksler, Cornell University, United States Babette Weksler, Cornell University, United States *Correspondence: Nicole L. Stone [email protected]; [email protected] Specialty section: This article was submitted to Non-Neuronal Cells, a section of the journal Frontiers in Cellular Neuroscience Specialty section: This article was submitted to Non-Neuronal Cells, a section of the journal Frontiers in Cellular Neuroscience Received: 21 February 2019 Accepted: 08 May 2019 Published: 06 June 2019 A Novel Transwell Blood Brain Barrier Model Using Primary Human Cells Nicole L. Stone*, Timothy J. England and Saoirse E. O’Sullivan Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, University of Nottingham, Nottingham, United Kingdom Structural alterations and breakdown of the blood brain barrier (BBB) is often a primary or secondary consequence of disease, resulting in brain oedema and the transport of unwanted substances into the brain. It is critical that effective in vitro models are developed to model the in vivo environment to aid in clinically relevant research, especially regarding drug screening and permeability studies. Our novel model uses only primary human cells and includes four of the key cells of the BBB: astrocytes, pericytes, brain microvascular endothelial cells (HBMEC) and neurons. We show that using a larger membrane pore size (3.0 µM) there is an improved connection between the endothelial cells, astrocytes and pericytes. Compared to a two and three cell model, we show that when neurons are added to HBMECs, astrocytes and pericytes, BBB integrity was more sensitive to oxygen-glucose deprivation evidenced by increased permeability and markers of cell damage. Our data also show that a four cell model responds faster to the barrier tightening effects of glucocorticoid dexamethasone, when compared to a two cell and three cell model. These data highlight the important role that neurons play in response to ischaemia, particularly how they contribute to BBB maintenance and breakdown. We consider that this model is more representative of the interactions at the neurovascular unit than other transwell models and is a useful method to study BBB physiology. ORIGINAL RESEARCH published: 06 June 2019 doi: 10.3389/fncel.2019.00230 Keywords: blood-brain barrier, transwell, in vitro, BBB model, BBB permeability, primary human cells, stroke INTRODUCTION The blood brain barrier (BBB) is a unique interface that separates the peripheral blood supply and neuronal tissue. Structurally, the BBB is comprised of specialized brain microvascular endothelial cells (HBMECs), perivascular cells (pericytes) and astrocytes (Abbott et al., 2006, 2010). Neurons and microglia also contribute to the maintenance of the BBB and form what is known as the neurovascular unit (NVU) (Abbott et al., 2006). Pericytes contribute 22–32% of the cerebral vasculature and together with vascular smooth muscle cells and endothelial cells they maintain vascular function (Martini and Bartholomew, 2017). In the CNS, pericytes are present at a higher ratio to HBMECs in the brain compared to the periphery and recent studies have shown the extensive role of pericytes in BBB development and maintenance (Kacem et al., 1998; Armulik et al., 2011; Sweeney et al., 2016). As well as offering mechanical support, they also regulate vessel contractility, endothelial proliferation, blood flow and angiogenesis (Bergers and Song, 2005; The blood brain barrier (BBB) is a unique interface that separates the peripheral blood supply and neuronal tissue. Structurally, the BBB is comprised of specialized brain microvascular endothelial cells (HBMECs), perivascular cells (pericytes) and astrocytes (Abbott et al., 2006, 2010). Neurons and microglia also contribute to the maintenance of the BBB and form what is known as the neurovascular unit (NVU) (Abbott et al., 2006). Pericytes contribute 22–32% of the cerebral vasculature and together with vascular smooth muscle cells and endothelial cells they maintain vascular function (Martini and Bartholomew, 2017). In the CNS, pericytes are present at a higher ratio to HBMECs in the brain compared to the periphery and recent studies have shown the extensive role of pericytes in BBB development and maintenance (Kacem et al., 1998; Armulik et al., 2011; Sweeney et al., 2016). As well as offering mechanical support, they also regulate vessel contractility, endothelial proliferation, blood flow and angiogenesis (Bergers and Song, 2005; Received: 21 February 2019 Accepted: 08 May 2019 Published: 06 June 2019 Citation: Citation: Stone NL, England TJ and O’Sullivan SE (2019) A Novel Transwell Blood Brain Barrier Model Using Primary Human Cells. Front. Cell. Neurosci. 13:230. doi: 10.3389/fncel.2019.00230 June 2019 | Volume 13 | Article 230 Frontiers in Cellular Neuroscience | www.frontiersin.org 1 Transwell Blood Brain Barrier Model Stone et al. Dore-Duffy, 2008). Pericytes have also been shown to secrete angiogenic factors such as vascular endothelial growth factor (VEGF), that support endothelial cell survival and proliferation (Darland et al., 2003). In pathologies such as ischaemic stroke, large gaps can develop between adjacent pericytes, increasing barrier permeability and vessel leakage. These alterations in pericyte morphology, coupled with an upregulation in the expression of adhesion molecules and leukocyte integrin ligands, contribute to the extravasation of peripheral leukocytes into the brain following ischaemic insult (Pieper et al., 2013). Thus, pericytes play a large role in cerebral vascular function under normal physiological conditions as well as vascular dysfunction in hypoxia. Dore-Duffy, 2008). Pericytes have also been shown to secrete angiogenic factors such as vascular endothelial growth factor (VEGF), that support endothelial cell survival and proliferation (Darland et al., 2003). In pathologies such as ischaemic stroke, large gaps can develop between adjacent pericytes, increasing barrier permeability and vessel leakage. These alterations in pericyte morphology, coupled with an upregulation in the expression of adhesion molecules and leukocyte integrin ligands, contribute to the extravasation of peripheral leukocytes into the brain following ischaemic insult (Pieper et al., 2013). Thus, pericytes play a large role in cerebral vascular function under normal physiological conditions as well as vascular dysfunction in hypoxia. development of co-culture transwell systems which exhibited greater barrier strength, exhibited by higher transepithelial resistance (TEER) and lower permeability than single HBMEC models, see Figure 1. More recent transwell systems typically use three cell types originating from either bovine, porcine or rodent origin, see Table 1 (Gaillard et al., 2000; Nakagawa et al., 2009; Thomsen et al., 2015). Whilst modeling using non-human cells is cheaper and easier to obtain, they are not comparable to human cells, with many studies showing key differences in morphology and function, particularly their sensitivity to glutamate and expression of efflux transporter proteins (Oberheim et al., 2009; Warren et al., 2009; Zhang et al., 2016). More complex BBB models are also available, such as spheroid or microfluidic models and offer a closer representation of the in vivo environment. Citation: However, these set ups are difficult and expensive to assemble (Ruck et al., 2015). Therefore, there is a need to develop a multicellular transwell model that incorporates multiple NVU cell types to study their interactions, particularly the role of neurons and their influence on barrier strength in both physiological and disease states. Transwell systems still offer a distinct advantage in that they are relatively easy to setup and control, as well as offering a range of endpoints to study. Measuring TEER in these types of models is commonplace as it provides a reliable, non-invasive quantitative measure of barrier integrity, enabling repeated measurements to be taken over the desired time period with minimal damage to cells (Srinivasan et al., 2015). Further to this, transwell models enable access to both the apical and basolateral (basal) compartments for drug application and medium sampling as well as being able to visualize cells over the course of the experiment. Several studies have also highlighted the roles of neurons and glia in BBB development and maintenance. Neural progenitor cells present in the ventricular neuroepithelium have been shown to aid endothelial cell recruitment during early BBB development, which is largely governed by the Wnt signaling pathway (Risau et al., 1986). Specifically, Wnt signaling in early CNS development is responsible for vascular stabilization and angiogenesis (Liebner et al., 2008). Further to this, early neuronal signaling has been shown to be essential for the maturation of the BBB, specifically, tight junction (TJ) organization. A study carried out using rat microvascular endothelial cells and neuronal progenitor cells, showed that in the presence of neural progenitor cells, endothelial cells established regular TJ formation including: claudin 5, zonula occludens (ZO-1) and occludin (Weidenfeller et al., 2007). After maturation, maintenance of the BBB and preservation of brain homeostasis is largely dependent on adequate perfusion to neuronal tissue and neuronal signaling to cerebral vessels, a process known as hyperaemia (Attwell et al., 2010). Studies have shown that neuronal-astrocyte crosstalk is important for appropriate vessel contractility and blood flow, depending on metabolic demand (Zonta et al., 2003; Attwell et al., 2010; Macvicar and Newman, 2015). In cerebral ischaemia, astrocytes sense elevations in Ca2+ ions and increases in extracellular glutamate released by neurons and respond accordingly, secreting a range of vasoactive substances to help mitigate the effects of the blood vessel occlusion (Macvicar and Newman, 2015). Citation: Altogether, interactions between both neural and vascular cells within the NVU is considered to be paramount in BBB functionality because together they induce and strengthen barrier properties; helping to maintain its key features including low paracellular permeability and functional tightness (Abbott et al., 2010). Breakdown of the BBB in conditions such as ischaemic stroke can lead to severe consequences to brain homeostasis, therefore, modeling these interactions is necessary to understand the complex signaling networks between these cell types and how they are influenced in disease states. g p Our aim was therefore to create a novel four cell human BBB model to study changes in permeability post oxygen-glucose deprivation (OGD) and for use in in vitro pharmacology. We initially focused on model development, refining a protocol first outlined by Hind (2014) by optimizing the inserts themselves, insert coating, cell seeding densities and cell culture timelines. Finally, we incorporated a method of seeding neurons on plastic coverslips which were placed on the bottom of 12 well cell culture plates. Thus, our model maintains the ease of the transwell setup but utilizes four primary human cells, making it a closer representation of the human in vivo environment. Frontiers in Cellular Neuroscience | www.frontiersin.org MATERIALS AND METHODS Primary cells (astrocytes, pericytes, HBMECs, and neurons) and specialized cell culture medium (astrocyte medium, pericyte medium, endothelial cell medium, and neuronal medium) were obtained from ScienCell, United States supplied by Caltag Medsystems, United Kingdom. Poly-L- lysine and porcine fibronectin were also obtained from ScienCell, United States supplied by Caltag Medsystems, United Kingdom. Collagen coated inserts, 3.0 µm, 12 mm were obtained from Corning, United Kingdom. Plastic coverslips (Thermanox R⃝ 13 mm diameter), Accutase dissociation reagent and glucose free RPMI medium were obtained from Thermo Fisher Scientific, United Kingdom. To date, a number of BBB models have been developed ranging from HBMEC monolayers to more sophisticated spheroid and chip style models, see Table 1. After the successful isolation of brain endothelial cells, the first, most simplistic BBB models were developed utilizing HBMECs as a single monolayer in the abluminal side of transwell inserts, see Table 1 (Borges et al., 1994; Hartz et al., 2010). Later addition of other BBB cell types (namely astrocytes and pericytes), led to the June 2019 | Volume 13 | Article 230 2 Transwell Blood Brain Barrier Model Stone et al. TABLE 1 | Different models of the blood brain barrier; their features, advantages, and disadvantages. Model type Typical components Advantages Limitations Representative of BBB phenotype References Single-cell transwell systems (non-co-culture) A monolayer of HBMECs cultured in the apical compartment of the transwell insert. Very easy to set up. Minimal cost. Low labor intensity. Useful if wanting to study endothelial cells alone. TEER is typically low. Cobblestone appearance of HBMECs, barrier formation. Little information on the impact of additional cell types. Borges et al., 1994; Hartz et al., 2010 Co-culture /multicellular transwell systems HBMECs cultured on the apical side of the transwell insert and astrocytes and/or pericytes cultured on the underside of the transwell insert. Time and cost effective. Higher TEER. Greater barrier stability. Some models are not fully in contact. Closer representation of the BBB with the addition of important cell types. Able to study interactions between cell types and how they influence BBB phenotype. Hind, 2014; Wang et al., 2015; Appelt-Menzel et al., 2017 Spheroid 3D organization of cells typically using matrigel. Typically consists of HBMECs and astrocytes and/or pericytes with some models containing neuronal cell types. 3D Cell model. No scaffold. Reduced de-differentiation. Cannot measure permeability with this model. Expensive and greater skill required. Microvessels wrap around endothelial cells and provide structural support. MATERIALS AND METHODS Helps to induce tight junction proteins. Closely represents the in vivo set up with cells in direct contact with each other. Applications include: cancer drug and neurotoxicity screening. Cho et al., 2017; Nzou et al., 2018 Microfluidic systems/3D chip-style models 3D organization of cells with the added benefit of a “flow” system to mimic cerebral blood flow. Typically consists of HBMECs and astrocytes and/or pericytes with some models containing neuronal cell types. Advantage of mimicking sheer stress which is essential for HBMECs optimum phenotype. Difficult to set up and maintain adequate flow unless linked to a computer system. Useful to assess the impact of blood flow on cell development and optimum phenotype. Also useful in studying cell migration and metastatic progression. Yeon et al., 2012; Wang et al., 2017 HBMECs = human brain microvascular endothelial cells, TGFβ = transforming growth factor beta, TEER = transepithelial resistance, BBB = blood brain barrier. TABLE 1 | Different models of the blood brain barrier; their features, advantages, and disadvantages. HBMECs = human brain microvascular endothelial cells, TGFβ = transforming growth factor beta, TEER = transepithelial resistance, BBB = blood brain barrier. FIGURE 1 | Schematic representation of the BBB model development. (A) A co-culture cell model containing HBMECs and astrocytes. (B) HBMECs seeded on the apical side, astrocytes seeded on the underside of the insert and pericytes seeded on the plate bottom. (C) HBMECs seeded on the apical side of the insert, pericytes seeded on the underside of the insert and astrocytes seeded on the plate bottom. (D) HBMECs seeded on the apical side of the insert with mixed culture of astrocytes and pericytes on the underside of the insert. (E) HBMECs seeded on the apical side of the insert with mixed culture of astrocytes and pericytes on the underside of the insert and neurons seeded on a poly-L-lysine coated coverslip on the plate bottom. FIGURE 1 | Schematic representation of the BBB model development. (A) A co-culture cell model containing HBMECs and astrocytes. (B) HBMECs seeded on the apical side, astrocytes seeded on the underside of the insert and pericytes seeded on the plate bottom. (C) HBMECs seeded on the apical side of the insert, pericytes seeded on the underside of the insert and astrocytes seeded on the plate bottom. Cannot measure permeability with this model. Expensive and greater skill required. Pore Size, Insert Size and Coating FIGURE 2 | Model protocol development (A) measured transepithelial resistance (TEER) as a marker of barrier tightness comparing a 12 well plate transwell set up vs. a 24 well plate transwell set up and insert pore size 3.0 µm vs. 0.4 µm. HBMECs seeded on the apical side of the insert, astrocytes underneath and pericytes on the plate bottom. (B) The organization of cells was optimized by comparing the TEER generated by a mixed culture of astrocytes and pericytes, pericytes or astrocytes alone on the underside of transwell inserts and astrocytes or pericytes on the cell culture plate bottom. HBMECs were seeded on the apical side of the insert. Data given as mean ± SEM, n = 4–6 from two experimental repeats. Statistical analysis conducted using 2-way ANOVA with Turkey’s multiple comparisons test, ∗∗P < 0.01 and ∗∗∗P < 0.001 mixed culture astrocytes and pericytes vs. pericytes underside the insert and astrocytes on plate bottom. #P < 0.05 mixed culture astrocytes and pericytes vs. astrocytes underside the insert and pericytes on plate bottom. , g Initially, pore sizes of Corning, United Kingdom inserts were compared (0.4 µm vs. 3.0 µm) as well as cell culture plates (12 well vs. 24 well). This was to determine the best initial setup that provided the highest and most stable barrier resistance, as well as giving the best cell contact. During protocol development, we found addition of pericytes in the smaller 24 well plates yielded poor results and insufficient TEER, suggesting inadequate barrier formation. Possibly as a result of inadequate cellular growth in such a small surface area and environment. Therefore, 24 well plates were switched back to 12 well plates, which resulted in substantially higher TEER readings. Following work carried out by Niego and Medcalf (2013), we also found that inserts with a 3.0 µm pore size had higher TEER values than 0.4 µm inserts, suggesting that increased contact between the cells in the apical and basolateral sides of the insert resulted in greater barrier strength, see Figure 2A. The final set up offered a closer replication of the organization held at the in vivo BBB, as cells would be in direct contact allowing them to exchange vital growth factors required for cellular growth and development. Pore Size, Insert Size and Coating We found that mixed culture of pericytes and astrocytes exhibited significantly higher TEER values when compared to the set-up with pericytes seeded on the plate bottom and astrocytes underneath the insert or astrocytes on the plate bottom and pericytes underneath the insert on days 3 and 4, P < 0.05 and P < 0.01, respectively (Figure 2B). Furthermore, this set up was also considered the most stable, as shown by steadier TEER readings and was altogether more physiologically relevant. This set up was therefore taken forward in subsequent four cell protocol development. MATERIALS AND METHODS (D) HBMECs seeded on the apical side of the insert with mixed culture of astrocytes and pericytes on the underside of the insert. (E) HBMECs seeded on the apical side of the insert with mixed culture of astrocytes and pericytes on the underside of the insert and neurons seeded on a poly-L-lysine coated coverslip on the plate bottom. June 2019 | Volume 13 | Article 230 3 Frontiers in Cellular Neuroscience | www.frontiersin.org Transwell Blood Brain Barrier Model Stone et al. Cells were maintained in a humidified incubator (37◦C, 5% CO2). Astrocytes and pericytes were cultured and used between passages 4 and 6. Human brain microvascular endothelial cells (HBMECs) were used between passages 3 and 5 and neurons were used at passage 1. During subculture, flasks containing HBMECs were coated with 2 µg·cm2 of fibronectin before reviving or splitting cells as per manufacturers recommendations. Cells were passaged at 80–90% confluency. Inserts contained 1.2 mL of medium in the basolateral compartment and 800 µL in the apical compartment. STX-3 probes and Ohms meter were obtained from World Precision Instruments, United Kingdom. Dexamethasone was obtained from Sigma, United Kingdom and dissolved in DMSO at a stock concentration of 10 mM and subsequently diluted in cell culture medium. GasPakTM EZ anaerobe container systems were obtained from BD, United Kingdom. Model Validation Our model was based on an initial co-culture set up established by Hind (2014) and previous models by Allen and Bayraktutan (2009). Our model was modified and developed in a number of preliminary experiments including comparison of insert pore sizes, insert coating, cell organization and addition of multiple cell types. Frontiers in Cellular Neuroscience | www.frontiersin.org Pericyte Seeding On day 2, plates were removed from the incubator and the astrocyte medium was removed with care so as to not disturb the layer of cells on the basolateral side of the insert. Inserts were then inverted again and 100 µL of 6.25 × 104 pericyte cell suspension was added to the astrocyte cell layer on the basolateral side of the transwell inserts, giving an approximate ratio of 5:1 astrocytes to pericytes (Pardridge, 1999). Plate lids were quickly replaced and returned to the incubator for 2–3 h. After this time, transwell inserts were reverted and any excess medium was removed by aspiration and a mixture of astrocyte and pericyte medium (1:1) was added to the apical and basolateral compartments. Insert Coating and Astrocyte Seeding g y g On day one, the basolateral side of transwell inserts were coated with poly-L-Lysine and astrocytes were seeded on the basolateral side of the inserts, see Figure 3. Briefly, 3.0 µm, 12 mm collagen coated inserts (Corning, United Kingdom) were carefully removed from outer packaging and placed into 12 well cell culture plates using sterile forceps. A solution of poly-L- Lysine (2 µg/cm2) was prepared in sterile water, homogenously mixed and carefully pipetted using a Pasteur pipette to just cover the basolateral of the insert, see Figures 4A,i. Plates containing inserts were then returned to the incubator, 37◦C, 5% CO2 for 1 h as per supplier recommendations. After 1 h, plates were removed from the incubator and washed twice with sterile water to remove any residual poly-L-lysine. All remaining liquid was removed by careful aspiration. Transwell inserts were then flipped inside the plate and the lid removed (Figure 4B). On the newly coated inserts, 100 µL of astrocyte cell suspension in astrocyte medium (3.13 × 105 cells) was pipetted quickly Four Cell Method Overview After optimization, our four cell BBB model consisted of four major NVU cell types arranged in a transwell permeability set-up (see Figure 1E). The assembly of this involves seeding different cell types at different times on the apical and basolateral sides of the transwell insert. During this time, neurons are seeded on plastic coverslips placed on the bottom of a separate 12 well plate to develop neurite before putting both parts of the model together on the final day of model establishment. Cell culture medium in both compartments was replaced every other day and the final set up was left to equilibrate for 2 days before commencing experiments. Greater than 85% of inserts are feasible for use in experiments and the model remained viable for up to 5 days. Addition of Multiple Cell Types and Cell Positioning Despite these improvements on the co-culture model, the need for additional cell types was critical to create a closer representation of the in vivo BBB. We established three different set ups as shown in Figure 1. In one, astrocytes were seeded on the basolateral side of the inserts and pericytes on the bottom of the culture dish (Figure 1B), in another pericytes were seeded on the basolateral of the inserts whilst astrocytes were seeded on the bottom of the culture dish (Figure 1C) and finally the last set up involved a mixed culture of astrocytes and pericytes seeded on the basolateral side of the insert (Figure 1D). In all models tested, HBMECs were seeded in the apical side of the transwell insert. To test the viability of adding neurons to the model, we originally seeded neurons on the bottom of the 12 well plate in which the inserts were hung. This, however, was Frontiers in Cellular Neuroscience | www.frontiersin.org June 2019 | Volume 13 | Article 230 Frontiers in Cellular Neuroscience | www.frontiersin.org 4 Transwell Blood Brain Barrier Model Stone et al. not feasible as the TEER probes touched the bottom of the plate causing unwanted damage to the cells. Therefore, we decided to utilize coverslips that could be positioned on the plate bottom, but not take up the entirety of the well, allowing the probe to sit where the cells were not present. After testing both poly-L-lysine coated glass and plastic coverslips, we found that plastic coverslips coated were the most effective in neuronal adhesion and this method was used in the final model. onto the basolateral side of the transwell and the lid carefully replaced (see Figures 4C,ii). Plates were returned to the cell culture incubator for 2–3 h for the cells to adhere. After this time, transwell inserts were reverted and any excess medium was removed by aspiration. Medium was topped up in the apical and basolateral compartments, see Figure 4iii. Again, plates were returned to the incubator. HBMEC Seeding Once astrocytes and pericytes reached 90% confluency (approxi- mately day 4 from model initiation, see Figure 3), the astrocyte:pericyte (1:1) medium in the apical compartment was removed and 100 µL of HBMEC cell suspension (7.5 × 104) in HBMEC medium was added to the apical compartment of transwell inserts and cells were left to adhere for a minimum of 5 h, then medium was topped up to 700 µL with endothelial cell medium and plates returned to the incubator. Neuronal Seeding On the same day as HBMEC seeding, plastic coverslips (13 mm diameter) were coated with poly-L-lysine and placed in the cell culture incubator for a minimum of 1 h, as per supplier recommendations. Plates containing coverslips were carefully removed from the incubator and coverslips were washed twice with sterile water and left to air dry in the cell culture hood. Following this, cryopreserved neurons were revived into 3 mL of neuronal medium (to give a total cell suspension of 4 mL) and 100 µL of cell suspension was added to each coverslip (thus seeded at a density of approximately 2.5 × 104 cells per cm2 within the optimum range according to the manufacturer’s FIGURE 3 | Timeline showing stages of model establishment. On day 1, inserts were coated and astrocyte seeded on the basolateral side of transwell inserts and on day 3 pericytes were seeded on the basolateral side of inserts to form a mixed culture. On day 6 HBMECs were seeded on the apical side of inserts and neurons were seeded on coated plastic coverslips in a separate 12 well plate. On day 10/11, inserts are carefully lifted out of their current plate and placed into the second 12 well plate containing the neurons seeded on coverslips. After 2 days, TEER measurements are taken to ensure adequate barrier formation. ∗In our lab OGD experiments were commenced at this point and were viable for 4–5 days. FIGURE 3 | Timeline showing stages of model establishment. On day 1, inserts were coated and astrocyte seeded on the basolateral side of transwell inserts and on day 3 pericytes were seeded on the basolateral side of inserts to form a mixed culture. On day 6 HBMECs were seeded on the apical side of inserts and neurons were seeded on coated plastic coverslips in a separate 12 well plate. On day 10/11, inserts are carefully lifted out of their current plate and placed into the second 12 well plate containing the neurons seeded on coverslips. After 2 days, TEER measurements are taken to ensure adequate barrier formation. ∗In our lab OGD experiments were commenced at this point and were viable for 4–5 days. June 2019 | Volume 13 | Article 230 Frontiers in Cellular Neuroscience | www.frontiersin.org 5 Transwell Blood Brain Barrier Model Stone et al. FIGURE 4 | Model setup (A–D) and (i–v). Neuronal Seeding (A/i) Inserts are placed into 12 well plate, coated with poly-L-lysine and washed, ensuring all of the liquid is removed. (B/ii) Inserts are carefully flipped inside the plate and the plate removed. 100 µL of relevant cell suspension is carefully placed on the underside of the insert. (C/ii) The bottom of the cell culture plate acts as a “lid” and is replaced as quickly as possible, plates are then returned to the incubator for the cells to adhere for 3–4 h. (iv) In a separate 12 well plate, coverslips are placed in the bottom of the culture dish, coated with poly-L-lysine and seeded with neuronal cell suspension. (v) Once all cells have been seeded on transwells, inserts are carefully transferred to plates containing neurons on coverslips. FIGURE 4 | Model setup (A–D) and (i–v). (A/i) Inserts are placed into 12 well plate, coated with poly-L-lysine and washed, ensuring all of the liquid is removed. (B/ii) Inserts are carefully flipped inside the plate and the plate removed. 100 µL of relevant cell suspension is carefully placed on the underside of the insert. (C/ii) The bottom of the cell culture plate acts as a “lid” and is replaced as quickly as possible, plates are then returned to the incubator for the cells to adhere for 3–4 h. (iv) In a separate 12 well plate, coverslips are placed in the bottom of the culture dish, coated with poly-L-lysine and seeded with neuronal cell suspension. (v) Once all cells have been seeded on transwells, inserts are carefully transferred to plates containing neurons on coverslips. Frontiers in Cellular Neuroscience | www.frontiersin.org June 2019 | Volume 13 | Article 230 6 Transwell Blood Brain Barrier Model Stone et al. recommendations) (Figures 4D,iv). Medium was topped up after 2 h and half of the medium replaced every 2–3 days. After light microscope observation, neurons began showing extensive neurite growth at approximately day 5. At this point HBMECs will have almost formed a confluent monolayer above the astrocytes and pericytes. Transwells were then carefully lifted out of their current 12 well plate using sterile forceps and placed into the 12 well plate containing the neuronal coverslips. Fresh HBMEC medium was applied to the apical compartment and a mix of pericyte, astrocyte and neuronal medium (1:1:2, respectively) was added to the basolateral compartment. Neuronal Seeding This was to maintain a low concentration of fetal bovine serum optimum for neuronal maintenance, whilst also preserving growth of astrocytes and pericytes. As all cells were confluent and the barrier was adequately formed, conditions were able to be maintained in the different compartments. reading for an insert with just cell culture medium was taken and subtracted from each reading (readings were repeated twice to ensure reproducibility), this was then multiplied by 1.12 to address the cell culture insert area (cm2) (Hind, 2014). Evaluation of Barrier Integrity g y Transepithelial resistance (TEER) was measured prior to commencing OGD experiments to ensure model barrier integrity; inserts should exhibit a TEER value of ≥45 /cm2 (Figure 4v). Light microscope observation was also carried out to ensure cell confluency and successful neurite formation. To ensure consistency, TEER measures should always be read at least 24 h after a medium change. Briefly, STX3 electrodes were sterilized by placing the tips of the probe in 70% ethanol, and then equilibrated for 15 min in endothelial cell culture medium at room temperature. The STX probe was then connected to an EVOM2 meter (Both World Precision Instruments, United Kingdom) and inserted into the transwell insert. The electrode has two parts that are uneven in length, the longer part of the electrode was placed so it gently touched the bottom of the cell culture plate, whilst the shorter electrode rested slightly above the insert dish, not quite making contact the HBMEC cell layer. Care should be taken to avoid disrupting the neurons on the bottom of the cell plate, see technical comments and limitations. As TEER values are very susceptible to change, it is important to keep the electrode upright and avoid tilting as this can cause fluctuation in the TEER values. A background When taking TEER values, ensure that the larger part of the STX probe does not touch the neurons cultured on the coverslip. This is especially important if multiple readings are being made (recommended). Utilization of neurons after primary experiments have been completed is also possible. Staining can be done on the coverslips using a variety of techniques including propidium iodide (PI) and DAPI staining, neurons can be lysed and intracellular assays can be performed. Oxygen-Glucose Deprivation (OGD) Protocol Data analysis was carried out using GraphPad prism software (La Jolla, CA, United States). Data are presented as mean ± SEM and analyzed using two-way ANOVA, followed by Sidak’s or Turkey’s multiple comparisons test. ∗P < 0.05 was considered significant. ( ) An oxygen-glucose deprivation (OGD) protocol was used to increase barrier permeability, simulating the effects of ischaemic stroke in vitro (Hind et al., 2015, 2016). Normal cell culture medium was removed from transwell inserts and replaced with glucose free RPMI medium (Thermo Fisher Scientific, United Kingdom) and placed in a 0% O2 environment (GasPakTM anaerobe pouch Beckton Dickinson, Oxford, United Kingdom) for 20 min to ensure anaerobic conditions for a further 4 h. There was no initial pre- conditioning period. Reperfusion was initiated by removing plates from the anaerobe pouch and returning cells to their normal medium (HBMEC medium in the apical compartment and in the basolateral compartment a mix of pericyte, astrocyte and neuronal medium, 1:1:2, respectively). TEER was measured at baseline (0 h), immediately post OGD (4 h), 24, 48, and 72 h. Dexamethasone Protocol Dexamethasone is a synthetic glucocorticoid and several groups have shown that is able to artificially improve barrier strength (Shi and Zheng, 2005; Pyrgos et al., 2010; Hind, 2014). Therefore, we used dexamethasone as a positive control to investigate any potential difference in the response of the three versus four cell model to a drug application. Baseline TEER readings were recorded and medium replaced, then dexamethasone was added to the apical compartment of the transwell insert, giving a final concentration of 1 µM. TEER was measured at 2, 4, and 24 h. Technical Comments and Limitations A critical step for setting up the four cellular model is timing and the revival and seeding of human neurons. Addition of the cells at incorrect timings will result in the model not working as effectively and TEER values will be lower than anticipated. We have therefore outlined a timeline for setting the model up (Figure 2), steps 4 and 5 can vary depending on the time taken for barrier formation to take place and for neurons to form neurite. Improper technique when seeding neurons on the coverslips will result in a lack of uniformity and inadequate neurite formation. Ensure coverslips are adequately air dried and neuronal cell suspension is carefully but adequately mixed during the revival and seeding process. Avoid removing neurons from the incubator for long periods. Frontiers in Cellular Neuroscience | www.frontiersin.org Protocol Development During BBB model development various set ups were compared including; insert pore size, plate size, and cell organization. Figure 2 highlights stages in protocol development and their respective TEER values, prior to the addition of neurons into the model. Figure 2A shows that a larger pore size (3.0 µm) exhibited greater barrier integrity (as shown by greater TEER readings) than the smaller pore size (0.4 µm). Furthermore, the June 2019 | Volume 13 | Article 230 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 7 Transwell Blood Brain Barrier Model Stone et al. 12 well inserts displayed considerably greater TEER readings than the 24 well inserts. Continuing model development using 12 3.0 µm inserts, Figure 2B compares three different cell culture set-ups days after model establishment. On days three and four, the inserts containing a mixed culture of astrocytes and pericytes displayed significantly higher TEER readings than set- ups containing astrocytes or pericytes seeded on the underside of the inserts or the cell culture plate bottom, P < 0.05 and P < 0.01, respectively. light microscope images of neurons in the four-cell model before and immediately after the OGD protocol, respectively. In Figure 5C neuronal clumping is clearly visible along with apparent neurite fragmentation compared to Figure 5B showing healthy neurons prior to OGD. Dexamethasone Application Dexamethasone increased barrier tightness in all three models, as shown by increases in TEER and exhibiting overall significance as a result of drug interaction in the three cell and four cell model, P < 0.05. The two-cell model was the most unstable out of the three models, as shown by greater fluctuations and variability in TEER measurements (Figure 6A). The three-cell model was considerably more stable but differences in TEER between dexamethasone treated and control were only observed after 2 h (Figure 6B). The four-cell model was the fastest to exhibit an increase in barrier tightness (i.e., increased TEER) as a result of dexamethasone application (Figure 6C) and this reached significance compared to the vehicle control at 2 and 24 h (P < 0.05). OGD Model Simulation To assess the effect of having different cells present, changes in TEER from models D and E shown in Figure 1, were compared following an OGD protocol. Figure 5A highlights the different responses of a three cell and four cell model in response to a 4 h OGD protocol, followed by a reperfusion period. The three-cell mode exhibited approximately a 30% drop in TEER from baseline after 4 h OGD. This contrasts to the four-cell model which exhibited a 50% drop in TEER post OGD and was significantly different to the three-cell model P < 0.05. After OGD, when reperfusion was initiated, TEER was able to return to baseline in the three-cell model, however, BBB permeability only marginally recovered by 20% in the four-cell model. This was significantly different at 24 h (P < 0.01) but not 48 or 72 h. Images 5B and C show DISCUSSION The BBB can be compromised in a range of different conditions, including but not limited to ischaemic stroke, Alzheimer’s disease, cancer, and multiple sclerosis (MS). Research into these disorders that affect the BBB is plagued by translational difficulty, resulting in many potential compounds and/or therapies failing to surpass phase I/II clinical trials. This is at least partly due to a lack of suitable in vitro models that can predict drug effectiveness pre-clinically. Most, if not all, current BBB models exhibit “pitfalls” whether that be cost, time or resources. Models that offer the closest representation of the BBB are often complex and expensive to replicate, adding to the cost of the drug screening process. To help improve the translatability of in vitro data, we developed a transwell style model that incorporates four primary human cell types, representing the NVU more than other BBB models currently available. We found that our novel four-cell model was superior in modeling ischaemic stroke and drug application in vitro compared to a three-cell and a two-cell model as shown through changes in TEER as a measure of barrier integrity and dexamethasone application. FIGURE 5 | (A) Effect of a 4 h oxygen-glucose deprivation (OGD) protocol on transepithelial resistance (TEER) as a marker of barrier tightness in a three cell model (HBMECs, astrocytes, and pericytes) and a four cell model (HBMECs, astrocytes, pericytes, and neurons). Neuronal images taken from the four cell model (B) before OGD 40× and (C) neuronal images immediately post OGD 40×. Data given as mean ± SEM, n = 3–6 from two experimental repeats, calculated as a % change from baseline TEER readings. Statistical analysis was conducted using 2-way ANOVA with Sidak’s multiple comparisons test, ∗P < 0.05 and ∗∗P < 0.01 was considered significant. Implications for Drug Testing The effect of dexamethasone was assessed in three transwell models; a two cell, three cell and four cell model. Greater instability in barrier strength and a slower response was exhibited by the two-cell model after dexamethasone application. This could also suggest that models containing just two cell types, in this case astrocytes and HBMECs, would also react differently to other drug applications and are therefore are not sufficient to truly model drug interactions at the BBB. Whilst the three-cell model shared the same trend in increasing barrier strength, it exhibited more stable TEER values compared to the two-cell model and dexamethasone FIGURE 5 | (A) Effect of a 4 h oxygen-glucose deprivation (OGD) protocol on transepithelial resistance (TEER) as a marker of barrier tightness in a three cell model (HBMECs, astrocytes, and pericytes) and a four cell model (HBMECs, astrocytes, pericytes, and neurons). Neuronal images taken from the four cell model (B) before OGD 40× and (C) neuronal images immediately post OGD 40×. Data given as mean ± SEM, n = 3–6 from two experimental repeats, calculated as a % change from baseline TEER readings. Statistical analysis was conducted using 2-way ANOVA with Sidak’s multiple comparisons test, ∗P < 0.05 and ∗∗P < 0.01 was considered significant. June 2019 | Volume 13 | Article 230 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 8 Transwell Blood Brain Barrier Model Stone et al. FIGURE 6 | The effect of dexamethasone on transepithelial resistance (TEER) as a measure of barrier tightness in (A) a two cell (astrocytes and HBMECs), (B) three cell (astrocytes, HBMECs, and pericytes), and (C) four cell model (astrocytes, HBMECs, pericytes, and neurons). Dexamethasone (1 µM) was added to the luminal side and used as positive control that is known to decrease permeability, thus increase TEER. Data represented as mean ± SEM, n = 4 from two experimental repeats. Statistical analysis was conducted using 2-way ANOVA with Sidak’s multiple comparisons test, ∗P < 0.05 was considered significant. VEGF, which influence barrier forming properties and early angiogenesis (Engelhardt, 2003; Eichmann and Thomas, 2013). These comparison data highlight the variations in data obtained from models containing different cell types and the impact this can have on drug screening. This stresses the importance of having a more representative BBB model containing additional cells present at the NVU. Implications for Protocol Testing p g Currently there are a wide range of in vitro BBB models available, but despite promising developments in modeling the BBB, there are still gaps in model design, primarily the inability to include all cell types present in the NVU. Whilst most transwell systems incorporate astrocytes and HBMECs, only more recent studies have introduced pericytes or neurons into these model designs. To gain a better understanding of how these cells contribute to the breakdown of the BBB in ischaemic conditions, we subjected our three cell and four cell models to an OGD protocol and measured TEER overtime to assess changes in barrier integrity. Interestingly, we found that with the presence of neurons our model exhibited a larger decrease in TEER compared to the three- cell model, which only contained astrocytes, pericytes and HBMECs. Similarly, whilst the three-cell model was able to recover 24 h post OGD the four-cell model only marginally recovered by approximately 20%, highlighting the role of and sensitivity of neurons in the level of damage ensued by the OGD protocol. Altogether, we have shown that with the addition of neurons our model became more vulnerable to damage; exhibiting a greater loss of barrier strength shown by a decrease in TEER, supporting previous work which showed that ischaemic neurons disrupt the endothelial barrier through increasing VEGF secretion (Li et al., 2014). Thus, omitting neurons from a BBB modeling stroke would underestimate the damage caused and contribution of neurons to the breakdown of the BBB post ischaemia. FIGURE 6 | The effect of dexamethasone on transepithelial resistance (TEER) as a measure of barrier tightness in (A) a two cell (astrocytes and HBMECs), (B) three cell (astrocytes, HBMECs, and pericytes), and (C) four cell model (astrocytes, HBMECs, pericytes, and neurons). Dexamethasone (1 µM) was added to the luminal side and used as positive control that is known to decrease permeability, thus increase TEER. Data represented as mean ± SEM, n = 4 from two experimental repeats. Statistical analysis was conducted using 2-way ANOVA with Sidak’s multiple comparisons test, ∗P < 0.05 was considered significant. REFERENCES differentiation-dependent and is associated with endothelial survival. Dev. Biol. 264, 275–288. doi: 10.1016/j.ydbio.2003.08.015 Abbott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R., and Begley, D. J. (2010). Structure and function of the blood-brain barrier. Neurobiol. Dis. 37, 13–25. doi: 10.1016/j.nbd.2009.07.030 Desai, S. Y., Marroni, M., Cucullo, L., Krizanac-Bengez, L., Mayberg, M. R., Hossain, M. T., et al. (2002). Mechanisms of endothelial survival under shear stress. Endothel. J. Endothel. Cell Res. 9, 89–102. doi: 10.1080/10623320212004 Abbott, N. J., Rönnbäck, L., and Hansson, E. (2006). Astrocyte–endothelial interactions at the blood–brain barrier. Nat. Rev. Neurosci. 7, 41–53. doi: 10. 1038/nrn1824 Dohgu, S., Takata, F., Yamauchi, A., Nakagawa, S., Egawa, T., Naito, M., et al. (2005). 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Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells. Stem Cell Rep. 8, 894–906. doi: 10.1016/j.stemcr.2017.02.021 Engelhardt, B. (2003). Development of the blood-brain barrier. Cell Tissue Res. 314, 119–129. doi: 10.1007/s00441-003-0751-z Ferland-McCollough, D., Slater, S., Richard, J., Reni, C., and Mangialardi, G. (2017). Pericytes, an overlooked player in vascular pathobiology. Pharmacol. Ther. 171, 30–42. doi: 10.1016/j.pharmthera.2016.11.008 Armulik, A., Genové, G., and Betsholtz, C. (2011). Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215. doi: 10.1016/j.devcel.2011.07.001 Attwell, D., Buchan, A. M., Charpak, S., Lauritzen, M., MacVicar, B. A., and Newman, E. A. (2010). Glial and neuronal control of brain blood flow. Nature 468, 232–243. doi: 10.1038/nature09613 Gaillard, P. J., Voorwinden, L. H., Nielsen, J. L., Ivanov, A., Atsumi, R., Engman, H., et al. (2000). Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur. J. Pharm. Sci. 12, 215–222. doi: 10.1016/ S0928-0987(00)00123-8 Bergers, G., and Song, S. (2005). FUNDING This work was supported by the Biotechnology and Biological Sciences Research Council (Grant No. BB/M008770/1). AUTHOR CONTRIBUTIONS NS performed the research. TE and SO’S designed the research study. NS and SO’S analyzed the data. NS, TE, and SO’S wrote the manuscript. DATA AVAILABILITY The overall function of the NVU is the perfusion of brain tissue to supply neurons with essential nutrients and the ability of neurons to regulate this blood flow. Glia, namely astrocytes, act as mediators between the vascular and neural compartments (Lo et al., 2015). Pericytes provide an extra level of communication between the endothelia and astrocytes as well as serving a prominent immune function (Darland et al., 2003; Armulik et al., 2011; Kovac et al., 2011). During cerebral ischaemia, these complex interactions are disrupted, and homeostasis is lost as a consequence of functional, morphological and metabolic changes within the NVU (Lo et al., 2015). It is important to model how these cells interact in both normoxic and ischaemic conditions to study the pathophysiology of ischaemic stroke. Finally, transwell systems offer noticeable advantages over the more complex models as they maintain the ease simpler cell culture set up The datasets generated for this study are available on request to the corresponding author. Limitations and Future Development Limitations and Future Development Although our model now includes four cell present in the NVU, our model does not incorporate flow which is an important feature to maintain the BBB phenotype in vitro. Studies have shown that sheer stress is critical to increase cell longevity and influence cell phenotype, regulate BBB transport, preventing de-differentiation (Desai et al., 2002; Chiu et al., 2005; Partyka et al., 2017). Culturing HBMECs under physiological shear stress, is particularly important in a ischaemic stroke setting because there is an interruption in blood flow. Microfluidic systems that mimic physiological flow have the advantage in that they can simulate continuous flow improving translation to the environment (Partyka et al., 2017; Wang et al., 2017). treated wells were overall significantly different to the vehicle control. Also, by introducing pericytes (generating a three cell model) there was a large increase in baseline TEER from 30 to 40 , again highlighting the role of pericytes in strengthening vascular stability at the BBB and the need for their presence in BBB models (Bergers and Song, 2005; Dohgu et al., 2005; Nakagawa et al., 2007; Ferland-McCollough et al., 2017) Interestingly, the four-cell model exhibited a significant increase in barrier tightness (as shown by an increase in TEER) compared to the vehicle control at just 2 h after dexamethasone application. Although neurons in this model do not directly interact with the BBB, neurons have been shown to secrete a number of vasoactive substances, including Equally, there is increasing evidence of the role of microglial cells in BBB breakdown. 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Detection of MspI polymorphism and the single nucleotide polymorphism (SNP) of GH gene in camel breeds reared in Egypt

African journal of biotechnology

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Full Length Research Paper Growth hormone (GH) is an anabolic hormone synthesized and secreted by the somatotroph cells of the anterior lobe of the pituitary gland in a circadian and pulsatile manner, the pattern of which plays an important role in postnatal longitudinal growth and development, tissue growth, lactation, reproduction as well as protein, lipid and carbohydrate metabolism. The aim of this study was to detect the genetic polymorphism of GH gene in five camel breeds reared in Egypt which are Sudany, Somali, Mowaled, Maghrabi and Falahy, using polymerase chain reaction- restriction fragment length polymorphism (PCR- RFLP) technique. Also, this work aimed to identify the single nucleotide polymorphism (SNP) between different genotypes detected in these camel breeds. The amplified fragment of camel GH at 613-bp was digested with the restriction enzyme MspI and the result reveals the presence of three different genotypes; CC, CT and TT in tested breeds. Significant differences were recorded in the genotype frequencies between these camel breeds. The result shows that the Maghrabi breed that is classified as a dual purpose camels had higher frequency for allele C (0.75) than those in the other tested four breeds. The sequence analysis declared the presence of a SNP (C264T) in the amplified fragment which is responsible for the elimination of the restriction site C^CGG and consequently the appearance of two different alleles C and T. The nucleotide sequences of camel GH alleles T and C were submitted to nucleotide sequences database NCBI/Bankit/GenBank and have accession numbers: KP143517 and KP143518, respectively. It is concluded that only one SNP C→T was detected in GH gene among the five tested camel breeds reared in Egypt and this nucleotide substitution can be used as a marker for the genetic biodiversity between these camel breeds. Also, due to the possible association between allele C and higher growth rate, we can used it in marker assisted selection (MAS) for camels in breeding program as a way for enhancement of growth trait in camel breeds reared in Egypt. Key words: Camel breeds in Egypt, GH, PCR, RFLP, SNPs. Vol. 14(9), pp. 752-757, 4 March, 2015 DOI: 10.5897/AJB2014.14374 Article Number: BC6F11D50954 ISSN 1684-5315 Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB African Journal of Biotechnology Vol. 14(9), pp. 752-757, 4 March, 2015 DOI: 10.5897/AJB2014.14374 Article Number: BC6F11D50954 ISSN 1684-5315 Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB African Journal of Biotechnology African Journal of Biotechnology DNA sequencing The PCR products representing each detected genotype of GH gene in different camel breeds, were purified and sequenced by Macrogen Incorporation (Seoul, Korea). Sequence analysis and alignment were carried out using NCBI/BLAST/blastn suite to detect each single nucleotide substitution between different detected genotypes. Results of endonuclease restriction were carried out using FastPCR. The nucleotide sequences of different alleles for camel GH gene were submitted to GenBank (NCBI, BankIt). RFLP Genotyping In order to detect variants of GH gene in different camel breeds, the PCR products were digested using restriction enzyme; MspI (Fermentas). 10 µl of PCR product was digested with 1 µl of FastDigest restriction enzymes for 5 min at 37°C. The restriction fragments were subjected to electrophoresis in 2% agarose/ ethidium bromide gel (GIBCO, BRL, England) in 1x TBE buffer (0.09 M Tris-boric acid and 0.002 M EDTA). Gels were visualized under UV light and documented in FX Molecular Imager apparatus (BIO-RAD). Genetic polymorphism can be identified by several techniques; one of the most commonly used methods is PCR-RFLP. It is a powerful method for identifying nucleo- tide sequence variation in amplified DNA and can detect single base substitutions in enzymatic restriction sites (Amie Marini et al., 2012). The present study aimed to detect the genetic polymorphism of GH gene in some camel breeds reared in Egypt using PCR-RFLP technique and identify SNPs between different genotypes detected in these breeds. Polymerase chain reaction (PCR) A PCR cocktail containing 1.0 mM forward and reverse primers specific for growth hormone gene, 0.2 mM dNTPs, 10 mM Tris (pH 9), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin (w/v), 0.1% Triton X- 100 and 1.25 units of Taq polymerase was used. The cocktail was aliquot into PCR tubes with 100 ng of camel DNA. The reaction was run at 94°C for 5 min, 35 cycles of 94°C for 1 min, 55°C for 30 s, 72°C for 45 s and a final extension at 72°C for 5 min. The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide to check amplification product. The primers used in this study were designed on the basis of DNA sequence of the camel GH gene (Ishag et al., 2010). The primer sequences are: G CC G GG C GC C C g ( g , ) p q F: 5′-GTCCTGTGGACAGCTCAC-3′ and R: 5′- TGTCCTCCTCACTGCTTTA-3′ g ( g , ) p q F: 5′-GTCCTGTGGACAGCTCAC-3′ and R: 5′- TGTCCTCCTCACTGCTTTA-3′ g ( g , ) p q F: 5′-GTCCTGTGGACAGCTCAC-3′ and R: 5′- TGTCCTCCTCACTGCTTTA-3′ INTRODUCTION and the Sahel region have about 70% (12.6 million) of the world's Dromedary camels. Somalia and Sudan together own about half of this number (Kesseba et al., 1991). In Egypt, the camel population was about 120 thousand head, (SADS, 2009). It was reported that many camel The population of old world camelidae in the world is estimated to be 18.5 million heads. Dromedary camels comprise 95% of them while the remaining 5% are Bactrian camel. Bactrian camels are found mainly in the cold high altitudes of Asia. The Near East, North Africa The population of old world camelidae in the world is estimated to be 18.5 million heads. Dromedary camels comprise 95% of them while the remaining 5% are Bactrian camel. Bactrian camels are found mainly in the cold high altitudes of Asia. The Near East, North Africa 653 El-Aziem et al. El-Aziem et al. Thereafter, it was diluted to the working concentration of 50 ng/μl to be used for polymerase chain reaction. breeds are reared in Egypt but the main camel breeds are Maghrabi (a dual purpose animal; (meat and milk) and Falahy, Sudany, Somali and Mowaled (meat type animal) (Mahrous et al., 2005). Camels are economically important animals in Egypt where they are dual purpose animals. In the Nile Valley and Delta, they are mainly raised for meat production and for some agricultural labors. In the desert, they are raised equally for meat and milk production, while some for labors and transport, and some especially for camel racing. The main biological role of growth hormone is to control postnatal growth, although it also has effects on metabolic regulation, lactation and reproduction (Louveau and Gondret, 2004; Daverio et al., 2012). Growth hormone gene, with its functional and positional potential role in the regulation of growth and metabolism in animals, has been widely used for marker in several livestock species, including the cattle (Dybus, 2002; Ge et al., 2003; Beauchemin et al., 2006; Katoh et al., 2008), sheep (Marques et al., 2006) and goat (Malveiro et al., 2001; Boutinaud et al., 2003). The camel growth hormone gene extends over about 1900 bp, and as other mammalian GH genes, it is organized in five exons separated by four introns (Maniou et al., 2004). It is a 22 KDa single chain polypeptide primarily produced and secreted by the somatotrophs of the anterior pituitary gland in circadian and pulsatile manner (Dybus, 2002). Blood samples and DNA extraction covering a total transcribed area of about 1.7 kb in sheep and humans (Byrne el al., 1987; Vize and Wells,1987), 2.7 kb in cattle (Yao et al.,1996) and 1.9 kb in camel (Maniou, 2003) and horse (Zhang et al., 2004). Camels provide mankind with a range of products and services; like wool, meat, milk and draught power. They have been domesticated about 3000 years ago and are present in high numbers in the arid parts of Africa, particularly in the arid lowlands of Eastern Africa (Somalia, Sudan, Ethiopia, Kenya and Djibouti) (Schwartz and Dioli, 1992). The dromedary camel contributes significantly to family food security in semi dry and dry climates, and is a major component of the agro- pastoral systems in vast pastoral areas in Asia and Africa (Ishag et al., 2010). Camels are economically important animals in Egypt where they are dual purpose animals (meat and milk). Improvement of camel productivity and detection of biodiversity between different camel breeds has necessitated the genotyping of the productivity trait genes in these breeds. The development in molecular genetics techniques has made it possible to identify differences between individuals at the DNA level. Recently, genetic polymorphisms at candidate genes affecting economic traits have stimulated substantial research interest because of their impending utilization as an aid to genetic selection and to demarcate evolutionary relationships in different livestock breeds (Sodhi et al., 2007). We aimed in this work to detect the genetic polymorphism of GH gene in some camel breeds reared in Egypt using PCR-RFLP technique and identify SNPs between different genotypes detected in these breeds. The primers used in this study flanked 613-bp fragments consist of 36 base pairs from exon 1, 243 base pairs from intron 1, 161 base pair from exon 2 and 173 base pairs Figure 2. Different genotypes obtained after digestion of PCR products of camel GH gene with MspI; TT: homozygous genotype with one undigested fragment at 613-bp, TC: heterozygous genotype with three fragments at 613-, 349- and 264-bp and CC: homozygous genotype with two digested fragments at 349- and 264-bp. M: 100-bp ladder marker. from intron 2 of camel GH gene. The amplified fragments obtained from all tested camels were of 613-bp (Figure 1). These PCR amplified fragments (613-bp) were digested with MspI endonuclease. Blood samples and DNA extraction Growth hormone has proven to be the major regulator of postnatal growth and metabolism in mammals and thus affects growth rate, body composition, health, milk production and aging by modulating the expression of many genes (Carnicella et al., 2003; Ge et al., 2003). Growth hormone has also been suggested as responsible for more important and prolonged cell proliferation in hypertrophied animals (Schoenfeld, 2010). The growth hormone gene in mammals was comprised of five exons and four introns (Maniou et al., 2004). The coding region of GH consisted of five exons, Blood samples were collected from jugular vein of 15 individual camels from each tested breed; Fallahi, Maghrabi, Mowalled, Sudany and Somali. Genomic DNA was extracted from the whole blood according to the method described by Miller et al. (1988) with minor modifications. Briefly, blood samples were mixed with cold 2x sucrose-triton x-100 and centrifuged at 5000 rpm for 15 min at 4°C. The nuclear pellet was suspended in lysis buffer, sodium dodecyl sulfate and proteinase K and incubated overnight in a shaking water bath at 37°C. Nucleic acids were extracted with saturated NaCl solution and then washed with 70% ethanol. The DNA pellet was dissolved in 1x TE buffer and its concentration was determined by using NanoDrop1000 (Thermo Scientific) Spectrophotometer. *Corresponding author. E-mail: [email protected]. Fax: 202 33370931. *Corresponding author. E-mail: [email protected]. Fax: 202 33370931. emains permanently open access under the terms of the Creative Commons Attribution License 4.0 Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 754 Afr. J. Biotechnol. Figure 1. Electrophoretic pattern of PCR-amplified fragment from GH gene in camels. M: 100-bp ladder marker. Lanes 1-6: 613-bp PCR product amplified from camel DNA. Figure 1. Electrophoretic pattern of PCR-amplified fragment from GH gene in camels. M: 100-bp ladder marker. Lanes 1-6: 613-bp PCR product amplified from camel DNA. Figure 2. Different genotypes obtained after digestion of PCR products of camel GH gene with MspI; TT: homozygous genotype with one undigested fragment at 613-bp, TC: heterozygous genotype with three fragments at 613-, 349- and 264-bp and CC: homozygous genotype with two digested fragments at 349- and 264-bp. M: 100-bp ladder marker. Blood samples and DNA extraction Depending on the presence or absence of the restriction site (C^CGG) at position 264^265, we can easily differentiate between three different genotypes: CC with two digested frag- ments at 349-and 264-bp, TT with undigested one fragment at 613-bp and CT with three fragments at 613-, 349- and 264-bp (Figure 2). 755 El-Aziem et al. Table 1. Genotype and allele frequencies of the GH gene in different camel breeds. Table 1. Genotype and allele frequencies of the GH gene in different camel breeds. Camel breed Genotype frequencies Allele frequencies CC CT TT C T Falahy 0.20 0.40 0.40 0.40 0.60 Maghrabi 0.50 0.50 0.00 0.75 0.25 Mowaled 0.10 0.40 0.50 0.30 0.70 Somali 0.09 0.18 0.73 0.18 0.82 Sudany 0.23 0.62 0.15 0.54 0.46 Total 0.22 0.42 0.36 0.43 0.57 Figure 3. Nucleotide sequence of allele C with nucleotide C at position 264 in the amplified fragment. GTCCTGTGGACAGCTCACCAGCTGTGATGGCTGCAGGTAAGTGCCCTAAAATCCCCTTAGGCTTGATGTGTACG GAAGGGTGATGTGGGGGCCCTGCAGATGGATGGGGCACTAACCTTGGTCTTTGGGGCTTCTGAATGTGAGCGT GGATATCTATGCCCACACATTTGGCTACATTTTAGAAAGGAAGGGCCCCTGGAGCACAGAGAGGGCTGGCAGG AGACGAGGCCTCTGGCTCTCCAGGCCCCTTCCTCGCTGGCCCTCCGGTTCTCTCTCTAGGCCCTCGGACCTCC GTGCTCCTGGCTTTCACCCTGCTCTGCCTGCCCTGGCCTCAGGAGGCGGGTGCCTTCCCAGCCATGCCTCTGT CCAGCCTGTTTGCCAACGCTGTGCTCCGCGCCCAGCACCTGCACCAGCTGGCTGCTGACACCTACAAAGAGTTT GTAAGCTCCTCAGGGATGGGTGCTAGTGGGGGGTGGCAGTAAGGGGTGAACCCACCCCCCTCTGCATAATGGG AGGAAACTAACAAGTTCAGGGGTATCTTATCCAAGTGAAGATGCTGTCAGGTGAGCATAAACTGAGGGGGGCTG TTCTGCATAAAGCAGTGAGGAGGACA Camel breed Genotype frequencies Allele frequencies CC CT TT C T Falahy 0.20 0.40 0.40 0.40 0.60 Maghrabi 0.50 0.50 0.00 0.75 0.25 Mowaled 0.10 0.40 0.50 0.30 0.70 Somali 0.09 0.18 0.73 0.18 0.82 Sudany 0.23 0.62 0.15 0.54 0.46 Total 0.22 0.42 0.36 0.43 0.57 GTCCTGTGGACAGCTCACCAGCTGTGATGGCTGCAGGTAAGTGCCCTAAAATCCCCTTAGGCTTGATGTGTACG GAAGGGTGATGTGGGGGCCCTGCAGATGGATGGGGCACTAACCTTGGTCTTTGGGGCTTCTGAATGTGAGCGT GGATATCTATGCCCACACATTTGGCTACATTTTAGAAAGGAAGGGCCCCTGGAGCACAGAGAGGGCTGGCAGG AGACGAGGCCTCTGGCTCTCCAGGCCCCTTCCTCGCTGGCCCTCCGGTTCTCTCTCTAGGCCCTCGGACCTCC GTGCTCCTGGCTTTCACCCTGCTCTGCCTGCCCTGGCCTCAGGAGGCGGGTGCCTTCCCAGCCATGCCTCTGT CCAGCCTGTTTGCCAACGCTGTGCTCCGCGCCCAGCACCTGCACCAGCTGGCTGCTGACACCTACAAAGAGTTT GTAAGCTCCTCAGGGATGGGTGCTAGTGGGGGGTGGCAGTAAGGGGTGAACCCACCCCCCTCTGCATAATGGG AGGAAACTAACAAGTTCAGGGGTATCTTATCCAAGTGAAGATGCTGTCAGGTGAGCATAAACTGAGGGGGGCTG TTCTGCATAAAGCAGTGAGGAGGACA GTCCTGTGGACAGCTCACCAGCTGTGATGGCTGCAGGTAAGTGCCCTAAAATCCCCTTAGGCTTGATGTGTACG GAAGGGTGATGTGGGGGCCCTGCAGATGGATGGGGCACTAACCTTGGTCTTTGGGGCTTCTGAATGTGAGCGT GGATATCTATGCCCACACATTTGGCTACATTTTAGAAAGGAAGGGCCCCTGGAGCACAGAGAGGGCTGGCAGG AGACGAGGCCTCTGGCTCTCCAGGCCCCTTCCTCGCTGGCCCTCCGGTTCTCTCTCTAGGCCCTCGGACCTCC GTGCTCCTGGCTTTCACCCTGCTCTGCCTGCCCTGGCCTCAGGAGGCGGGTGCCTTCCCAGCCATGCCTCTGT CCAGCCTGTTTGCCAACGCTGTGCTCCGCGCCCAGCACCTGCACCAGCTGGCTGCTGACACCTACAAAGAGTTT GTAAGCTCCTCAGGGATGGGTGCTAGTGGGGGGTGGCAGTAAGGGGTGAACCCACCCCCCTCTGCATAATGGG AGGAAACTAACAAGTTCAGGGGTATCTTATCCAAGTGAAGATGCTGTCAGGTGAGCATAAACTGAGGGGGGCTG TTCTGCATAAAGCAGTGAGGAGGACA GTCCTGTGGACAGCTCACCAGCTGTGATGGCTGCAGGTAAGTGCCCTAAAATCCCCTTAGGCTTGATGTGTACG GAAGGGTGATGTGGGGGCCCTGCAGATGGATGGGGCACTAACCTTGGTCTTTGGGGCTTCTGAATGTGAGCGT GGATATCTATGCCCACACATTTGGCTACATTTTAGAAAGGAAGGGCCCCTGGAGCACAGAGAGGGCTGGCAGG AGACGAGGCCTCTGGCTCTCCAGGCCCCTTCCTCGCTGGCCCTCCGGTTCTCTCTCTAGGCCCTCGGACCTCC GTGCTCCTGGCTTTCACCCTGCTCTGCCTGCCCTGGCCTCAGGAGGCGGGTGCCTTCCCAGCCATGCCTCTGT CCAGCCTGTTTGCCAACGCTGTGCTCCGCGCCCAGCACCTGCACCAGCTGGCTGCTGACACCTACAAAGAGTTT GTAAGCTCCTCAGGGATGGGTGCTAGTGGGGGGTGGCAGTAAGGGGTGAACCCACCCCCCTCTGCATAATGGG AGGAAACTAACAAGTTCAGGGGTATCTTATCCAAGTGAAGATGCTGTCAGGTGAGCATAAACTGAGGGGGGCTG TTCTGCATAAAGCAGTGAGGAGGACA Figure 3. Nucleotide sequence of allele C with nucleotide C at position 264 in the amplified fragment. GTCCTGTGGACAGCTCACCAGCTGTGATGGCTGCAGGTAAGTGCCCTAAAATCCCCTTAGGCTTGATGTGTACG GAAGGGTGATGTGGGGGCCCTGCAGATGGATGGGGCACTAACCTTGGTCTTTGGGGCTTCTGAATGTGAGCGT GGATATCTATGCCCACACATTTGGCTACATTTTAGAAAGGAAGGGCCCCTGGAGCACAGAGAGGGCTGGCAGG AGACGAGGCCTCTGGCTCTCCAGGCCCCTTCCTCGCTGGCCCTTCGGTTCTCTCTCTAGGCCCTCGGACCTCC GTGCTCCTGGCTTTCACCCTGCTCTGCCTGCCCTGGCCTCAGGAGGCGGGTGCCTTCCCAGCCATGCCTCTGT CCAGCCTGTTTGCCAACGCTGTGCTCCGCGCCCAGCACCTGCACCAGCTGGCTGCTGACACCTACAAAGAGTTT GTAAGCTCCTCAGGGATGGGTGCTAGTGGGGGGTGGCAGTAAGGGGTGAACCCACCCCCCTCTGCATAATGGG AGGAAACTAACAAGTTCAGGGGTATCTTATCCAAGTGAAGATGCTGTCAGGTGAGCATAAACTGAGGGGGGCTG TTCTGCATAAAGCAGTGAGGAGGACA Figure 4. Nucleotide sequence of allele T with nucleotide T at position 264 in the amplified fragment. tiation between these three different genotypes C/C (Figure 5), C/T (Figure 6) and T/T (Figure 7). The nucleotide sequences of camel GH alleles T and C were submitted to nucleotide sequences database NCBI/ Bankit/GenBank and have accession numbers: KP143517 and KP143518, respectively. In the present study, sequence analysis revealed a single nucleotide polymorphism C→T, where nucleotide "C" furnished cutting site for MspI restriction enzyme. Restriction reaction resulted three different genotypes; homozy- gous without restriction (TT), homozygous with restriction (CC) and heterozygous animals (T/C). The results show significant differences in the allele frequencies between the breeds. This finding agrees with the results of Shah (2006) which explained the significant differences in allele frequencies among Pakistani camel breeds and also the similar results were reported by Ishag et al. Blood samples and DNA extraction Two SNPs (C419T and T450C) were detected in the Saheli breed and T450C SNP was associated with increased estimated body weight. Both male and female Saheli camels with the CC genotype had higher body weights than the CT and TT genotypes (P≤0.05). The SNP T450C, which was detected only in camels of the Saheli breed, was correlated with greater body weight. growth hormone gene:Sequence, organization and SNP identification. Small Rumin. Res. 103: 108- 111. Dybus A (2002). Associations of growth hormone (GH) and prolactin (PRL) genes polymorphism with milk production traits in Polish Black and White cattle. J. Anim. Sci. 20:203-212 and White cattle. J. Anim. Sci. 20:203-212 Ge W, Davis M, Hines H, Irvin K, Simmen R (2003). Association of single nucleotide polymorphisms in the growth hormone and growth hormone receptor genes with blood serum insulin-likegrowth factor I concentration and growth traits in Angus cattle. J. Anim. Sci. 81:641- 648. Ishag IA, Reissmann M, Peters KJ, Musa LM, Ahmed MK (2010). Phenotypic and molecular characterization of six Sudanese camel breeds. South Afr. J. Anim. Sci. 40:319-326. Katoh K, Kouno S, Okazaki A, Suzuki K, Obara Y (2008). Interaction of GH polymorphism with body weight and endocrine functions in Japanese black calves. Domest. Anim. Endocrinol. 34(1):25-30. p ( ) Kesseba AM, Wardeh FM, Wilson RT, Zaied AA (1991). Camel applied research and development network. Proceedings of International conference on camel production and improvement, 10-13 December, Tobruk, Libya, 21-36. In our study, the frequency of allele C in the Maghrabi breed that is classified as a dual purpose camels was higher (0.75) than those in the other tested four breeds. This allele may be associated with body weight as reported by Shah (2006), Ishag et al. (2010) and Afifi et al. (2014). Consequently, this allele may be considered as a useful marker in the selection of camels for higher growth rate and meat production. It is concluded that only one SNP C→T was detected in GH gene among the five tested camel breeds reared in Egypt and this nucleotide substitution can be used as a marker in different genetic studies including the detection of genetic biodiversity and establishment of phylogenic tree for different camel breeds reared in Egypt. Blood samples and DNA extraction (2010) in Sudanese camel breeds where they The result shows that all tested camel breeds have the three genotypes with different frequencies with the exception of Maghrabi breed which carries only CC and CT genotypes with absence of TT genotype (Table 1). The highest frequencies of three genotypes were present in Maghrabi breed for CC homozygous genotypes (0.50), Sudany breed for CT heterozygous genotype (0.62) and Somali breed for TT homozygous genotype (0.73). The frequencies for alleles C and T ranged from 0.18 to 0.75 and from 0.25 to 0.82, respectively, in tested camel breeds. These three detected genotypes are resulted from the presence of two different alleles; C (Figure 3) and T (Figure 4) in tested camel animals. The sequence analysis of the purified PCR products representing these three detecting genotypes CC, CT and TT declared the presence of a single nucleotide polymorphism (C→T) at position 264 which is responsible for the elimination of restriction site C^CGG and consequently the differen- Afr. J. Biotechnol. 756 Figure 5. Genotype C/C. Figure 6. Genotype C/T. Figure 7. Genotype T/T. Figure 5. Genotype C/C. Figure 5. Genotype C/C. Figure 5. Genotype C/C. Figure 6. Genotype C/T. Figure 6. Genotype C/T. Figure 7. Genotype T/T. Figure 7. Genotype T/T. Figure 7. Genotype T/T. relatively light weight had slightly higher T allele frequencies (0.57 and 0.48, respectively) than those of the other four breeds which are classified as pack camels. On the other hand, other breeds (Kenani, Lahwee, Rashaidi and Kabbashi) have higher body weights with low T allele frequencies (0.30 to 0.33) and studied the RFLP and SNP of GH gene in six Sudanese camel breeds; Kenani, Lahwee, Rashaidi, Anafi, Bishari and Kabbashi. The sequence comparison of Sudanese camel GH sequences with the GenBank sequence identified one SNP 419C>T in intron 1. The Bishari and Anafi breeds that are classified as riding camels with El-Aziem et al. 757 growth hormone gene:Sequence, organization and SNP identification. Small Rumin. Res. 103: 108- 111. high C allele frequencies (0.67 to 0.70). The relationship between GH polymorphism and body weight was evaluated in four Arabian camel breeds (Majaheem, Saheli, Waddah and Homor) by Afifi et al. (2014). Thirteen (13) SNPs (two insertion and 11 substitution) were detected in the Majahem breed and one was detected in the Waddah and Homor breeds each at position 419 (C419T). Blood samples and DNA extraction Also, due to the possible association between allele C and higher growth rate, this can be used in marker assisted selection (MAS) for camels and enter the camels possess this allele in breeding program as a way for enhancement of growth trait in camel breeds reared in Egypt. Louveau I, Gondret F (2004). Regulation of development and metabolism of adipose tissue by growth hormone and the insulin-like growth factor system. Domest. Anim. Endocrinol. 27:241-255. Mahrous KF, Abd-El Mordy M, Abd El-Aziem SH, Hemdan DM (2005). Genetic variations in one-humped camels present in Egypt. J. Genet. Eng. Biotechnol. (NRC) 3(1):15-29. g Malveiro E, Pereira M, Marques PX, Santos IC, Belo C, Renaville R (2001). Polymorphisms at the five exons of the growth hormone gene in the algarvia goat: Possible association with milk traits. Small Rumin. Res. 41(2):163-170. Maniou Z (2003). Molecular evaluation of pituitary growth hormone (AJ575419). PhD Thesis, Biological Sciences, University of Sussex, Brighton, UK. Maniou Z, Wallis OC, Wallis M (2004). Episodic molecular evolution of pituitary growth hormone in Cetartidactyla. J. Mol. Evol. 58(6):743- 753. Marques MR, Santos IC, Carolino N, Belo CC, Renaville R, Cravador A (2006). Effects of genetic polymorphisms at the growth hormone gene on milk yield in Serra da Estrela sheep. J. Dairy Res. 73(4):394- 405. Miller SM, Dykes DD, Polesky HF (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16(3):1215. Schoenfeld BJ (2010). The mechanism of muscle hypertrophy and their application to resistance training. J. Strength Cond. Res. 24(10):2857-2872 The authors did not declare any conflict of interest. Shah MG (2006). Differentiation of six Pakistani camel breed by phenotype and molecular genetics analysis. University of Agriculture, Faisalabad, Ph.D. Thesis. Conflict of interests ( ) Schwartz H, Dioli M (1992). The one humped camel in eastern Africa: A pictorial guide to diseases, health care and management. Verlag Josef Margraf, Weikersheim, FR Germany. The authors did not declare any conflict of interest. REFERENCES Sodhi M, Mukesh M, Prakash B, Mishra B, Sobti R, Karn S, Singhand S, Ahlawat S (2007). MspI allelic pattern of bovine growth hormone gene in Indian Zebu cattle (Bosindicus) breeds. Biochem. Genet. 45:145-153 Afifi M, Metwali EMR, Brooks PH (2014). Association between growth hormone single nucleotide polymorphism and body weight in four Saudi camel (Camelus dromedarius) breeds. Pak. Vet. J. 34(4):494- 498. Sustainable Agricultural Development Strategy Towards 2030 (SADS) (2009). Agricultural Research and Development Council. Arab Republic of Egypt, Ministry of Agriculture and Land Reclamation. Oct. 2009. Amie Marini AB, Aslinda K, Mohd Hifzan R, Muhd Faisal AB, Musaddin K (2012). HaeIII-RFLP polymorphism of growth hormone gene in Savanna and Kalahari goats. Malays. J. Anim. 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Carnicella D, Dario C, Bufano G (2003). Polimorfismo del gene GH performance productive. Large Anim. Rev. 3:3-7. D i S Di R F Vid l Ri j L (2012) Th ll (L l ) Byrne C, Wilson B, Ward K (1987). The isolation and characterization of the ovine growth hormone gene. J. Biol. Sci. 40:459-468. Zhang Y, Xiraigol U, Dugarjav M (2004). Equuscaballus growth hormone gene, complete CDs (AY837571). Department of Biology, Inner Mongolia Agric Univ, Huhhot, Inner Mongolia, PR, China. Carnicella D, Dario C, Bufano G (2003). Polimorfismo del gene GH performance productive. Large Anim. Rev. 3:3-7. Daverio S, Di Rocco F, Vidal-Rioja L (2012). The llama (Lama glama)

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Expression of GITR Enhances Multiple Myeloma Cell Sensitivity to Bortezomib

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Expression of GITR Enhances Multiple Myeloma Cell Sensitivity to Bortezomib * [email protected] * [email protected] OPEN ACCESS OPEN ACCESS Citation: Zhao Y, Zhang K, Li G, Zhang X, Shi D (2015) Expression of GITR Enhances Multiple Myeloma Cell Sensitivity to Bortezomib. PLoS ONE 10(5): e0127334. doi:10.1371/journal.pone.0127334 Academic Editor: Claire M. Edwards, University of Oxford, UNITED KINGDOM Academic Editor: Claire M. Edwards, University of Oxford, UNITED KINGDOM Copyright: © 2015 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Abstract Recently tumor necrosis factor receptor super family member 18 (TNFRSF18, also called GITR) has been identified as a novel tumor suppressor gene in Multiple Myeloma (MM), undergoing aberrant DNA methylation-mediated gene expression silencing. Furthermore, the expression of GITR blocks canonical NF-κB activation in MM cells in response to TNFα. Bortezomib, a proteasome inhibitor, can induce NF-κB activation, which may signif- icantly influence the drug response in MM patients. In this study, we aim to elucidate if GITR status is associated with response to Bortezomib in MM cells through regulating GITR mediated NF-κB blockade. We found that GITR was significantly downregulated in MM patients and cell lines. Overexpression of GITR inhibited non-canonical NF-κB activa- tion induced by TNFα. Moreover, NF-κB inhibitor induced apoptosis in GITR-deficient MM cells in response to TNFα. In addition, overexpression of GITR could inhibit Bortezomib- induced NF-κB activation and enhance the cytotoxicity of Bortezomib in GITR-deficient MM cell line (MM1.S). In contrast, knockdown of GITR attenuated the cytotoxic effect of Bortezomib on GITR proficient MM (RPMI) cell line and increased NF-κB activation. Final- ly, overexpression of GITR enhanced the sensitivity to Bortezomib in co-culture with bone marrow stromal cells and significantly reduced the tumor growth in MM1.S xenograft mice. In conclusion, we demonstrated that GITR expression can enhance the sensitivity to Bor- tezomib by inhibiting Bortezomib-induced NF-κB activation. OPEN ACCESS Citation: Zhao Y, Zhang K, Li G, Zhang X, Shi D (2015) Expression of GITR Enhances Multiple Myeloma Cell Sensitivity to Bortezomib. PLoS ONE 10(5): e0127334. doi:10.1371/journal.pone.0127334 Data Availability Statement: All relevant data are within the paper and its Supporting Information files. In addition, expression of GITR correlates with Bortezomib sensi- tivity in MM cells, supported by overexpression and knockdown of GITR affecting the cytotoxic- ity of Bortezomib in MM cell lines. Furthermore, we also demonstrated overexpression of GITR impaired the interaction between MM cells and stromal cells and significantly decreased MM cell growth upon the treatment of Bortezomib. Finally, we showed that GITR expression could also enhance the effect of Bortezomib on inhibition of MM tumor growth in MM1.S xenograft mice model. These findings imply that GITR status is critical to response to Bortezomib in mye- loma cells through regulating NF-κB pathway. Results GITR is downregulated in MM patients First, using Real Time-PCR, we evaluated the expression of GITR in primary CD138+ bone marrow derived plasma cells from 16 MM patients by comparing to pooled normal bone mar- row derived plasma cells (N = 20). We found that GITR levels of MM patients were significant- ly lower than the pooled normal groups (Fig 1), which is consistent with previous report [10]. These observations suggest that deregulation of GITR is very prevalent in MM. Fig 1. Expression of GITR in MM patients. mRNA of GITR from 16 MM patients and 20 pooled normal bone marrow specimens were assessed by real time-PCR. 18S was considered as the internal control. doi:10.1371/journal.pone.0127334.g001 In this present study, we hypothesized that deregulation of GITR may play a pivotal role in modulating drug response in MM. Here, we showed that GITR is significantly downregulated in MM patients. Inhibition of NF-κB activity can significantly result in TNFα-induced apoptosis in GITR-deficient MM cell lines. In addition, expression of GITR correlates with Bortezomib sensi- tivity in MM cells, supported by overexpression and knockdown of GITR affecting the cytotoxic- ity of Bortezomib in MM cell lines. Furthermore, we also demonstrated overexpression of GITR impaired the interaction between MM cells and stromal cells and significantly decreased MM cell growth upon the treatment of Bortezomib. Finally, we showed that GITR expression could also enhance the effect of Bortezomib on inhibition of MM tumor growth in MM1.S xenograft mice model. These findings imply that GITR status is critical to response to Bortezomib in mye- loma cells through regulating NF-κB pathway. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Tumor Necrosis Factor receptor super family members (TNFRSFs) play an important role in the immune responses and inflammatory reactions [1–3]. One of TNFRSFs, TNFRSF18 (GITR), a recently identified novel tumor suppressor on chromosome 1p36, loss of which might be highly related to pathogenesis in differential human cancers [4–9]. It has been reported that GITR defi- ciency could result in increased cell proliferation and reduced apoptosis in human Multiple Mye- loma (MM) [10]. NF-κB transcription factors play a key role in the survival and proliferation of many kinds of B-cell tumors, especially for multiple myeloma [11]. It has also been shown that mutations involved in the NF-κB pathway are present in 15–20% of MM tumors [12]. These Funding: This work was supported by National Natural Science Foundation of China grant NSFC81272472. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 1 / 12 GITR Sensitizes MM Cells to Bortezomib mutations can lead to activation of the canonical and non-canonical NF-κB pathway [13]. There- fore, targeting the NF-κB pathway is an attractive therapy approach for MM [14]. In previous re- port, it has been shown that GITR expression also impacts the NF-κB activation in response to GITR ligand [10]. These findings above indicate that GITR might also be important to drug re- sponse through modulating NF-κB pathway since NF-κB inhibitors were developed to treat MM patients in the past years. fore, targeting the NF-κB pathway is an attractive therapy approach for MM [14]. In previous re- port, it has been shown that GITR expression also impacts the NF-κB activation in response to GITR ligand [10]. These findings above indicate that GITR might also be important to drug re- sponse through modulating NF-κB pathway since NF-κB inhibitors were developed to treat MM patients in the past years. In this present study, we hypothesized that deregulation of GITR may play a pivotal role in modulating drug response in MM. Here, we showed that GITR is significantly downregulated in MM patients. Inhibition of NF-κB activity can significantly result in TNFα-induced apoptosis in GITR-deficient MM cell lines. GITR is downregulated in MM patients First, using Real Time-PCR, we evaluated the expression of GITR in primary CD138+ bone marrow derived plasma cells from 16 MM patients by comparing to pooled normal bone mar- row derived plasma cells (N = 20). We found that GITR levels of MM patients were significant- ly lower than the pooled normal groups (Fig 1), which is consistent with previous report [10]. These observations suggest that deregulation of GITR is very prevalent in MM. Fig 1. Expression of GITR in MM patients. mRNA of GITR from 16 MM patients and 20 pooled normal bone marrow specimens were assessed by real time-PCR. 18S was considered as the internal control. doi:10 1371/journal pone 0127334 g001 Fig 1. Expression of GITR in MM patients. mRNA of GITR from 16 MM patients and 20 pooled normal bone marrow specimens were assessed by real time-PCR. 18S was considered as the internal control. doi:10 1371/journal pone 0127334 g001 doi:10.1371/journal.pone.0127334.g001 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 2 / 12 GITR Sensitizes MM Cells to Bortezomib Inhibition of NF-κB activity enhanced TNF-induced apoptosis in GITR- deficient MM cells Previous study showed that loss of GITR resulted in increased NF-κB activity in MM cells [10]. Since increased NF-κB activity has been shown to play an important role in pathogenesis of MM disease, it is very important to determine if loss of GITR mediated NF-κB activation is crucial for MM pathogenesis. To examine this, we performed the cell viability and apoptosis assay in 5 MM cell lines in response to NF-κB inhibitor in the presence of TNFα. We found that NF-κB in- hibitor, BAY-11-7085, significantly inhibited cell growth of low GITR expressing cell lines, MM1.S, OPM1, U266 and INA6. In contrast, RPMI cell line, which showed higher GITR level, was not sensitive to BAY-11-7085 in the presence of TNFα (Fig 3A and 3B). These results sug- gest that loss of GITR mediated NF-κB activation might be important to MM tumorigenesis. Overexpression of GITR inhibits TNFα-induced non-canonical NF-κB activation Previous study showed that overexpression of GITR could block canonical NF-κB activation [10]. Herein, to further determine the association of NF-κB activity with GITR expression, we examined NF-κB activation in response to TNFα in three MM cell lines with differential levels of GITR by DNA binding ELISA assay. As shown in Fig 2A, MM1.S and OPM1 cells with low GITR expression showed strong p65 activation. However, there was very weak NF- κB activation observed in RPMI8226 cells, which express high level of GITR. In addition, using western blot assay, we also found that GITR expression impacted p52 nuclear translo- cation in stimulation with TNFα (Fig 2B). These results suggest that GITR expression nega- tively correlate with both canonical and non-canonical NF-κB activation in MM cells. To further determine the effect of GITR expression on non-canonical NF-κB pathway, we over- expressed GITR in MM1.S, a GITR-deficient cell line, and found that overexpression of GITR significantly impaired p52/RelB NF-κB translocation into the nuclear in a time-depen- dent manner (Fig 2C). Furthermore, immunofluorescence staining showed that p52 localiza- tion was attenuated upon TNFα stimulation in GITR-overexpressed MM1.S cells (Fig 2D and S1 Fig). Combined with the previous reports, these results suggest that GITR plays an in- hibitory role in both canonical and non-canonical NF-κB activation. PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 GITR Sensitizes MM Cells to Bortezomib Fig 2. Overexpression of GITR impacts TNFα-induced non-canonical NF-κB activation. A. MM1.S, OPM1 (GITR low) and RPMI8226 (G were exposed to TNFα (10ng/ml) for 60 minutes. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor b consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experi **P<0.01 and ***P<0.001 compared with indicated groups. B. MM1.S, OPM1 and RPMI8226 cells were exposed to TNFα (10ng/ml) for 60 m and whole cell lysates were subjected to western blot using anti-p65, -p52 and Actin antibodies. C. Cells were exposed to TNFα (10ng/ml) for minutes. Nuclear protein and cytoplasmic extraction were subjected to western blot using anti-p52, -RelB and -nucleolin antibodies. D. pCMV GITR-MM1.S cells were harvested at 24 hours after treatment with and without TNF-α (10ng/ml) for 60 minutes. Immunocytochemical analys using anti-phospho-NF-κB-p52 antibody, with DAPI used to stain nuclei. doi:10.1371/journal.pone.0127334.g002 Fig 2. Overexpression of GITR impacts TNFα-induced non-canonical NF-κB activation. A. MM1.S, OPM1 (GITR low) and RPMI8226 (GITR high) cells were exposed to TNFα (10ng/ml) for 60 minutes. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups. B. MM1.S, OPM1 and RPMI8226 cells were exposed to TNFα (10ng/ml) for 60 minutes. Nuclear and whole cell lysates were subjected to western blot using anti-p65, -p52 and Actin antibodies. C. Cells were exposed to TNFα (10ng/ml) for 15, 30, or 60 minutes. Nuclear protein and cytoplasmic extraction were subjected to western blot using anti-p52, -RelB and -nucleolin antibodies. D. pCMV-GITR and GITR-MM1.S cells were harvested at 24 hours after treatment with and without TNF-α (10ng/ml) for 60 minutes. Immunocytochemical analysis was assessed using anti-phospho-NF-κB-p52 antibody, with DAPI used to stain nuclei. doi:10.1371/journal.pone.0127334.g002 Fig 2. Overexpression of GITR impacts TNFα-induced non-canonical NF-κB activation. A. MM1.S, OPM1 (GITR low) and RPMI8226 (GITR high) cells were exposed to TNFα (10ng/ml) for 60 minutes. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups. B. Expression of GITR is associated with the sensitivity to Bortezomib in MM cells It has been reported that Bortezomib induces NF-κB activation in MM, we proposed to deter- mine if GITR status can affect MM cells responded to Bortezomib. To explore this, we investi- gated the sensitivity to Bortezomib by knocking down GITR in RPMI cell line in comparison with control cell line. We found that knockdown of GITR significantly impaired the sensitivity of RPMI to Bortezomib-induced apoptosis (Fig 4B, 4C and 4D). The knockdown efficiency was confirmed by real time PCR, showing mRNA of GITR decreased to about 20% of control cell line (Fig 4A). In contrast, overexpression of GITR in MM1.S, a GITR negative cell line, sig- nificantly enhanced Bortezomib-induced apoptosis and reduced MM1.S cell growth in vitro (Fig 5A, 5B and 5C, S2 Fig). Notably, we found that overexpression of GITR inhibited Bortezo- mib-induced NF-κB activation, supported by decreased NF-κB p65/p50 translocation into the nuclear upon Bortezomib treatment (Fig 5D). Since previous study showed that GITR may also activate p53 pathway, we also overepxressed GITR in U266, a p53 mutated MM cell line, to examine the apoptotic effect of GITR expression in the presence of Bortezomib. Indeed, we found that expression of GITR could also enhance Bortezomib-induced apoptosis in PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 3 / 12 MM1.S, OPM1 and RPMI8226 cells were exposed to TNFα (10ng/ml) for 60 minutes. Nuclear and whole cell lysates were subjected to western blot using anti-p65, -p52 and Actin antibodies. C. Cells were exposed to TNFα (10ng/ml) for 15, 30, or 60 minutes. Nuclear protein and cytoplasmic extraction were subjected to western blot using anti-p52, -RelB and -nucleolin antibodies. D. pCMV-GITR and GITR-MM1.S cells were harvested at 24 hours after treatment with and without TNF-α (10ng/ml) for 60 minutes. Immunocytochemical analysis was assessed using anti-phospho-NF-κB-p52 antibody, with DAPI used to stain nuclei. doi:10.1371/journal.pone.0127334.g002 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 4 / 12 GITR Sensitizes MM Cells to Bortezomib Fig 3. GITR expression correlates with NF-κB activation and sensitivity to NF-κB inhibitor. A. Sensitivity of MM cells to NF-κB inhibitor-BAY-11-7085 was assessed in five MM cell lines. Cell viability was determined by CellTiter-Glo assay. Veh indicating DMSO treated cells. Com indicates combination treatment of TNF with BAY-11-7085. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with Veh groups. B. MM cell lines were exposed to 10ng/ml TNF with/without BAY-11-7085. Cells were lysed after 12 hours incubation and subjected to Immunoblotting using anti-PARP1 and Actin antibodies. doi:10.1371/journal.pone.0127334.g003 Fig 3. GITR expression correlates with NF-κB activation and sensitivity to NF-κB inhibitor. A. Sensitivity of MM cells to NF-κB inhibitor-BAY-11-7085 was assessed in five MM cell lines. Cell viability was determined by CellTiter-Glo assay. Veh indicating DMSO treated cells. Com indicates combination treatment of TNF with BAY-11-7085. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with Veh groups. B. MM cell lines were exposed to 10ng/ml TNF with/without BAY-11-7085. Cells were lysed after 12 hours incubation and subjected to Immunoblotting using anti-PARP1 and Actin antibodies. doi:10.1371/journal.pone.0127334.g003 doi:10.1371/journal.pone.0127334.g003 doi:10.1371/journal.pone.0127334.g003 GITR-U266 cells, indicating GITR mediated inhibition of NF-κB activation is crucial for sensi- tivity of MM cells to Bortezomib (S3 Fig). Furthermore, overexpression of GITR also sensitized MM1.S cells to Bortezomib when co-cultured with bone marrow derived stromal cells (BMSC), suggesting GITR may also play an important role in counteracting the BMSC-mediated surviv- al signals in MM microenvironment (Fig 5E). Expression of GITR enhances the cytotoxicity effect of Bortezomib in vivo The number of dead cells were assessed by FACS based PI single staining and quantified by Flowjo software. Data represent mean ± SD, * P<0.05, and **P<0.01 compared with indicated groups. Fig 4. Knockdown of GITR reduced the sensitivity to Bortezomib in RPMI cell line. A. Knockdown efficacy of GITR gene was assessed by real time-PCR. Scr (Scramble) indicates the non-targeting SiRNA control. B. Knockdown of GITR reduced sensitivity to Bortezomib in RPMI cell line. Cell viability was evaluated by CellTiter-Glo assay after 48 hours incubation. Data represent mean ± SD, * P<0.05, and **P<0.01. C. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-cleaved caspase-3 and Actin antibodies. D. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by FACS based PI single staining and quantified by Flowjo software. Data represent mean ± SD, * P<0.05, and **P<0.01 compared with indicated groups. doi:10.1371/journal.pone.0127334.g004 of CD138+ plasma cells in the untreated GITR expressing group was less than the controls, sug- gesting GITR expression had cytotoxicity effect on MM cells. In addition NF-κB activity was also assessed in these isolated bone marrow plasma cells from each group, showing expression of GITR effectively inhibited Bortezomib-induced NF-κB activation in vivo (Fig 6C). Expression of GITR enhances the cytotoxicity effect of Bortezomib in vivo To determine if GITR expression can also have effect on Bortezomib-induced MM tumor growth inhibition in mice model, we assessed the tumor growth upon the treatment of Bortezo- mib in MM1.S xenograft immune deficiency mice by tail vein injection. The expression of GITR in extracted bone marrow CD138+cells was confirmed by quantitative RT-PCR (Fig 6A). Using flow cytometry assay to assess the number of CD138+ cells, we found that overexpression of GITR significantly improved the Bortezomib-induced MM tumor inhibition comparing to the control group after 4 weeks treatment with Bortezomib (Fig 6B and S4 Fig). Notably, the number 5 / 12 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 GITR Sensitizes MM Cells to Bortezomib Fig 4. Knockdown of GITR reduced the sensitivity to Bortezomib in RPMI cell line. A. Knockdown efficacy of GITR gene was assessed by real time-PCR. Scr (Scramble) indicates the non-targeting SiRNA control. B. Knockdown of GITR reduced sensitivity to Bortezomib in RPMI cell line. Cell viability was evaluated by CellTiter-Glo assay after 48 hours incubation. Data represent mean ± SD, * P<0.05, and **P<0.01. C. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-cleaved caspase-3 and Actin antibodies. D. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by FACS based PI single staining and quantified by Flowjo software. Data represent mean ± SD, * P<0.05, and **P<0.01 compared with indicated groups. Fig 4. Knockdown of GITR reduced the sensitivity to Bortezomib in RPMI cell line. A. Knockdown Fig 4. Knockdown of GITR reduced the sensitivity to Bortezomib in RPMI cell line. A. Knockdown efficacy of GITR gene was assessed by real time-PCR. Scr (Scramble) indicates the non-targeting SiRNA control. B. Knockdown of GITR reduced sensitivity to Bortezomib in RPMI cell line. Cell viability was evaluated by CellTiter-Glo assay after 48 hours incubation. Data represent mean ± SD, * P<0.05, and **P<0.01. C. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-cleaved caspase-3 and Actin antibodies. D. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated for 24 hours. PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 GITR Sensitizes MM Cells to Bortezomib Fig 5. Overexpression of GITR enhanced sensitivity to Bortezomib-induced apoptosis in MM1.S cells. A. Empty vector and GITR-transfected MM1.S cells were treated with Bortezomib for 48 hours. Cell viability was assessed by CellTiter-Glo assay. Data represent mean ± SD, **P<0.01 compared with indicated groups. B. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-PARP1 and Actin antibodies. C. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by PI single staining and quantified by Flowjo software. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. D. NF-κB activity was evaluated in control and GITR expressing MM1.S cells. Nuclear protein lysates were subjected to western blot using anti-p65 and nucleolin antibodies. E. Empty vector and GITR-transfected MM1.S cells were treated with 50nM Bortezomib for 48 hours in co-cultured with or without BMSC. After 48 hours incubation, the cell viability was assessed by CellTiter- Glo assay. Data represent mean ± SD, *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups. doi:10.1371/journal.pone.0127334.g005 Fig 5. Overexpression of GITR enhanced sensitivity to Bortezomib-induced apoptosis in MM1.S cells. A. Em Fig 5. Overexpression of GITR enhanced sensitivity to Bortezomib-induced apoptosis in MM1.S cells. A. Empty vector and GITR-transfected MM1.S cells were treated with Bortezomib for 48 hours. Cell viability was assessed by CellTiter-Glo assay. Data represent mean ± SD, **P<0.01 compared with indicated groups. B. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-PARP1 and Actin antibodies. C. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by PI single staining and quantified by Flowjo software. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. D. NF-κB activity was evaluated in control and GITR expressing MM1.S cells. Nuclear protein lysates were subjected to western blot using anti-p65 and nucleolin antibodies. E. Empty vector and GITR-transfected MM1.S cells were treated with 50nM Bortezomib for 48 hours in co-cultured with or without BMSC. After 48 hours incubation, the cell viability was assessed by CellTiter- Glo assay. Discussion Multiple myeloma is a hematologic cancer, and characterized by uncontrolled plasma cell pro- liferation in human bone marrow [15]. Bortezomib, one of the most commonly used protea- some inhibitor, has also been approved for the treatment of myeloma [16, 17]. In pre-clinical studies Bortezomib showed a number of different anti-myeloma effects including disruption of the cell cycle and induction of apoptosis [18, 19]. However, only 30–40% relapsed and refracto- ry MM patients were sensitive to Bortezomib in phase II/III clinical trials. In this study, we found that the expression of a TNFRSFs member, GITR, positively correlated with the response to Bortezomib. These observations were verified by both in vitro and in vivo cytotoxicity assay, showing deregulation of GITR could significantly impair the sensitivity to Bortezomib in MM cell lines. In addition, we also showed that the expression of GITR enhanced the cellular re- sponse to Bortezomib through inhibiting Bortezomib-induced NF-κB activation. Therefore, we provide a novel molecular mechanism of MM cells resistance to Bortezomib. 6 / 12 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 Data represent mean ± SD, *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups. doi:10.1371/journal.pone.0127334.g005 Recently, GITR has been identified as a novel tumor suppressor, affecting NF-κB activation in MM. Notably, the expression of GITR significantly correlates with the stage of MM disease, suggesting the role of GITR in MM disease progression [10]. These findings led us to hypothe- size that GITR status might be associated with the sensitivity of Bortezomib in MM patients. Indeed, our study showed that GITR played an important role in Bortezomib response, sup- ported by GITR overexpression enhanced the apoptosis in Bortezomib treated MM cells. Bortezomib treatment can induce NF-κB activation in parallel with I-κB α degradation [20]. The activated NF-κB pathway might affect the sensitivity to Bortezomib treatment in 7 / 12 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 GITR Sensitizes MM Cells to Bortezomib Fig 6. Overexpression of GITR enhanced Bortezomib induced tumor growth inhibition in MM1.S xenograft mice. A. Empty control and GITR expressing cells were isolated from MM1.S xenograft mice and subjected to mRNA extraction. The expression of GITR was examined by real time-PCR. GAPDH was considered as an internal control. B. CD138+ human plasma cells were isolated from femur of the four groups of investigated mice. The number of CD138+ cell was assessed by flow cytometry. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. C. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. ***P<0.001 compared with indicated groups. doi:10.1371/journal.pone.0127334.g006 Fi 6 O i f GITR h d B ib i d d h i hibi i i MM1 S f i A E l d GITR Fig 6. Overexpression of GITR enhanced Bortezomib induced tumor growth inhibition in MM1.S xenograft mice. A. Empty control and GITR expressing cells were isolated from MM1.S xenograft mice and subjected to mRNA extraction. The expression of GITR was examined by real time-PCR. GAPDH was considered as an internal control. B. CD138+ human plasma cells were isolated from femur of the four groups of investigated mice. The number of CD138+ cell was assessed by flow cytometry. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. C. NF-κB activity was evaluated by DNA binding ELISA assay. PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 PI staining for cell death assessment For cell death analysis by flow cytometry, cells were seeded in a 6 cm2 dish at 60% confluency. The cells were concomitantly treated with Bortezomib overnight and stained with 10ng/ml propidium iodide (PI). The number of dead cells was determined using a FACS caliber flow cy- tometer (Becton Dickinson, Oxford, UK) and quantified by FlowJo7.6.5 software. Ethics statement Bone marrow samples from patients with MM and healthy donors were obtained under The Second Hospital of Jilin University IRB approval with written informed consent. In animal studies, mice were treated, monitored, and sacrificed in accordance with approved protocol of The Second Hospital of Jilin University Animal Care and Use Committee. Real time-PCR and siRNA transfection Real time-PCR for GITR was performed on an Applied Biosystems AB7500 Real Time PCR system. All PCR reactions were run in triplicate, and GITR expression relative to 18s was calcu- lated using the 2–ΔΔCt method. The delivery of siRNA into RPMI cells was performed using Lipofectamine 2000 reagent (Invitrogen). Cells were transfected with siRNAs at a final concen- tration of 50nM. The siRNAs used in this study were: Scrambled ON-TARGETplus nontarget- ing pool (Dharmacon #D001810-10), and SMARTpool ON-TARGETplus GITR siRNA (Dharmacon #L-006449-00-0005). Cultured cell lines and primary tumor samples 5 human myeloma cell lines, MM1.S,RPMI, U266(ATCC, Manassas, VA), INA6 and OPM1 (provided by Dr. Y. Zhang, National institute of Health, Bethesda, MD) and human bone mar- row stromal cell line (Lonza, Walkersville, MD) were used in this study. All human MM cell lines were cultured as described previously [23]. Primary CD138+ MM cells were obtained from BM samples of MM patients (n = 16). CD138+ Plasma cells were obtained by using CD138+ microbeads selection (Miltenyi Biotec, Auburn, CA). Similarly, CD138+ plasma cells were isolated from the BM of 20 healthy donors and pooled together as normal controls. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. ***P<0.001 compared with indicated groups. doi:10.1371/journal.pone.0127334.g006 MM patients [21, 22]. Recently, it has been shown that GITR expression impacts NF-κB activa- tion by inhibiting phosphorylation of IKK-beta in MM [10]. Although it is still not clear how GITR modulates NF-κB activity, it raises up a question if loss of GITR influences MM sensitivi- ty to Bortezomib via NF-κB pathway. In fact, by using MM1.S cell line, we found that overex- pression of GITR could inhibit Bortezomib-induced NF-κB activation. Furthermore, the MM1.S-GITR xenograft mice also showed decreased NF-κB activity in CD138+ plasma cells compared to MM1.S control cells. These findings also suggest that GITR-mediated NF-κB ac- tivity is critical for sensitivity to Bortezomib treatment. However, we can’t completely exclude the possibility that p53 activation may also play a role in modulating Bortezomib response since it has been shown that GITR can also lead to more or less p53 activation. PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 8 / 12 GITR Sensitizes MM Cells to Bortezomib In summary, we report here, that Bortezomib-induced NF-κB activation can be inhibited by overexpression of GITR. Loss of GITR reduced the sensitivity to Bortezomib in MM. More- over, expression of GITR enhanced the Bortezomib-induced apoptosis in MM cell lines. There- fore, we propose there that GITR status might be used to predict the clinical therapeutic response to Bortezomib in relapsed MM patients. Cell viability assay For cell viability assays, 3000 cells were plated in sterile 96-well plates and cultured overnight. Compounds were then added in serial dilutions. The Bay 11–7084 was purchased from Santa Cruz (CAT# sc-202490). Cellular viability was determined after 48 hours incubation by the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Plates were measured on a THERMO max microplate reader. For stromal co-culture cell assay, BMSCs were obtained from bone marrow of MM patients. Briefly, bone marrow aspirates were subjected to Ficoll- Paque gradient centrifugation (GE Healthcare, Little Chalfont, United Kingdom), and mono- nuclear cells (MNCs) were collected and expanded in human complete MesenCult medium (STEMCELL Technologies, Canada) for 2 weeks. Then a confluent monolayer was generated by plating 10×105 BMSCs in a 96-well plate for additional 48 hours. MM.1S cells were treated with Bortezomib and plated in co-culture with BMSCs for 48 hours at 37°C. Cell Viability was assessed by the CellTiter-Glo Luminescent intensity. In vivo tumor progression of MM cells 5 SCID mice for each group were injected with 5X106 empty control MM1.S and GITR-MM1.S cells. The treatments for each group were as follows: (1) No treatment (control), (2) Bortezomib, 1 mg/kg body weight on 7, 10, 13, 17, 20, and 24 days after MM1.S cells injection. Body weight and the number of tumor cells were measured at least twice a week. All the mice were sacrificed by CO2 asphyxiation after 24 days treatment with/without Bortezomib. Bone marrow tissue of femur as well as peripheral blood was isolated from the four groups. Mice bone marrow tissues were crushed and filtered into single cell suspensions. Anti human CD138 monoclonal antibody was used to examine MM cells growth by flow cytometry as previously described [10]. Analysis of NF-κB activity NF-κB activity was assessed by Active Motif TransAM NF-κB Family Kit, (Active Motif North America, Carlsbad, CA). Briefly, MM.1S cells (control MM1.S and GITR-transfected MM1.S) were treated with Bortezomib. NF-kB-p65 binding to the related DNA sequence on the oligo- nucleotide coated-plates was studied from nuclear extracts, following the manufacturer’s pro- cedure. For analysis of NF-κB activity in xenograft mice, the whole bone marrow, including femur, skull and vertebrae were crashed and filtered into single cell suspension. To obtain the pure CD138+ cell fraction, these cells were isolated by CD138 microbeads selection (Miltenyi Biotec, Auburn, CA). Immunoblotting Immunoblotting was carried out using standard techniques. Briefly, cells were lysed in ice-cold 1x RIPA lysis buffer and protein concentrations determined. Aliquots (50μg) of protein were dena- tured in Laemmli loading buffer and separated on precast 4–10% NuPAGE Novex 4–12% Bis- Tris Protein Gels, (Novex-Invitrogen). The antibodies used for immunoblotting included anti— caspase3, p65, p50, Parp-1 RelB (Cell Signaling Technology, Danvers, MA), and Actin (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclear extracts of the cells were prepared using the Nucle- ar Extraction Kit (Panomics, Redwood City, CA) and subjected to immunoblotting with anti-p65, -p50, (Cell Signaling Technology), and anti-nucleolin (Santa Cruz Biotechnology) antibodies. Immunofluorescence staining Immunocytochemical analysis was performed according to the protocol from Cell Signaling Technology Company. In brief, cells were fixed with 16% formaldehyde in phosphate-buffered saline and permeabilized with 0.3% Triton X-100. Cells were blocked in 1X PBS/5% normal goat serum PBS and incubated with primary antibody in 1X PBS/1% BSA/0.3% Triton X-100 at 4°C overnight. In the next day, cells were washed by PBS for three times and incubated with Alexa Fluor 647 goat anti-rabbit secondary antibody (CAT# A-21245, Life Technology) for 2hours. Finally the cell staining was observed under fluorescence microscopy. 9 / 12 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 GITR Sensitizes MM Cells to Bortezomib Supporting Information S1 Fig. Overexpression of GITR inhibits TNFα-induced p52 nuclear translocation. pCMV-GITR and GITR-MM1.S cells were harvested at 24 hours after treatment with 10ng/ml TNF-α for 60 minutes. Immunocytochemical analysis was assessed using anti-phospho-NF- κB-p52 antibody and DAPI for nuclei staining. Representative images are shown with higher magnification. S2 Fig. Overexpression of GITR enhances Bortezomib-induced cell death inMM1.S cell line. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bor- tezomib and incubated for 24 hours. The dead cells were assessed by PI single staining. Data are shown as representative dot plot of flow cytometry analysis in Fig 5C. (TIF) S3 Fig. Overexpression of GITR enhances Bortezomib-induced apoptosis in U266 cell line. Empty control and GITR expressing U266 cells were exposed to 5nM Bortezomib and incubat- ed overnight. Cells were lysed and subjected to Immunoblotting using anti-PARP1, IκB, Myc and Actin antibodies. The expression of GITR enhanced Bortezomib-induced apoptosis in GITR-U266 cells, indicating GITR mediated inhibition of NF-κB activation is crucial for sensi- tivity of MM cells to Bortezomib. (TIF) S4 Fig. Expression of GITR enhances the cytotoxicity effect of Bortezomib in xenograft mice. CD138+ human plasma cells were isolated from femur of the four groups of investigated mice. Data represent the dot plot of flow cytometry analysis in Fig 6B. (TIF) S4 Fig. Expression of GITR enhances the cytotoxicity effect of Bortezomib in xenograft mice. CD138+ human plasma cells were isolated from femur of the four groups of investigated mice. Data represent the dot plot of flow cytometry analysis in Fig 6B. (TIF) Author Contributions Conceived and designed the experiments: DLS. Performed the experiments: YHZ. Analyzed the data: YHZ KZ XYZ. Contributed reagents/materials/analysis tools: YHZ KZ GQL. Wrote the paper: DLS. Statistical analysis The statistical significance between groups was assessed with Student’s t-test. Experiments were repeated in triplicates. A value of P<0.05 was considered statistically significant and 10 / 12 PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 GITR Sensitizes MM Cells to Bortezomib indicated by one asterisks. P <0.01or <0.001 was represented by two asterisks or three asterisks respectively. Error bars shown in the figures represent standard deviations. Statistical analyses were carried out using Microsoft Excel software. PLOS ONE | DOI:10.1371/journal.pone.0127334 May 14, 2015 References Frequent allelic imbalance suggests involvement of a tumor suppressor gene at 1p36 in the pathogenesis of human lung cancers. Genes Chromosomes Cancer 2000; 28: 342–346. PMID: 10862041 8. Poetsch M, Dittberner T, Woenckhaus C. Frameshift mutations of RIZ, but no point mutations in RIZ1 exons in malignant melanomas with deletions in 1p36. Oncogene 2002; 21: 3038–3042. PMID: 12082534 9. Katzenberger T, Kalla J, Leich E, Stocklein H, Hartmann E, Barnickel S, et al. 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Supplemental Figures from PINK1 Is a Negative Regulator of Growth and the Warburg Effect in Glioblastoma

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A 0 1000 2000 3000 4000 5000 6000 Neg Control Pos Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Luciferase expression (RLU/ug) * * * * * * * * * * * * * * B p53 -/- astrocytes Activated Ras expressing astrocytes Supplemental Fi Gene Trap Retrovirus Genomic Locus Random Integration Fusion mRNA Reporter Proteins LTR SA Luciferase IRES Puro PA LTR Non Transformed Cell Transformed Cell P P Luciferase IRES Puromycin Exon AAAAAAAAAA Luciferase IRES Puromycin Exon SD SA Splicing Event After Transcription E E E E 30 40 50 60 vasion * (3) (T) A B C D E 0 50 100 150 Agar Colonies C NMA Gene-trapped Clones E 100 200 300 400 500 olony formation ve to non-treated control NMA * * * 0 Gy 5 Gy * * * F A 0 1000 2000 3000 4000 5000 6000 Neg Control Pos Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Luciferase expression (RLU/ug) * * * * * * * * * * * * * * B p53 -/- astrocytes Activated Ras expressing astrocytes Supplemental Fi Gene Trap Retrovirus Genomic Locus Random Integration Fusion mRNA Reporter Proteins LTR SA Luciferase IRES Puro PA LTR Non Transformed Cell Transformed Cell P P Luciferase IRES Puromycin Exon AAAAAAAAAA Luciferase IRES Puromycin Exon SD SA Splicing Event After Transcription E E E E 30 40 50 60 vasion * (3) (T) A B C D E 0 50 100 150 Agar Colonies C NMA Gene-trapped Clones E 100 200 300 400 500 olony formation ve to non-treated control NMA * * * 0 Gy 5 Gy * * * F Supplemental Figure 1 A 0 1000 2000 3000 4000 5000 6000 Neg Control Pos Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Luciferase expression (RLU/ug) * * * * * * * * * * * * * * B p53 -/- astrocytes Activated Ras expressing astrocytes Retrovirus Genomic Locus Random Integration Fusion mRNA Reporter Proteins P P Luciferase IRES Puromycin Exon AAAAAAAAAA Luciferase IRES Puromycin Exon SD SA Splicing Event After Transcription E E E E G 0 10 20 30 40 50 60 NMA p53 -/- GT-6 GT-8 RasB8 P(0) GT-21 GT % Invasion * B8 P(3) p53 -/- (T) A B C D E 0 50 100 150 Soft Agar Colonies C NMA Gene-trapped Clones NMA RasB8 P(0) GT-21 D Control p53 -/- GT-6 GT-8 Control p53 -/- GT-6 GT-8 0 100 200 300 400 % Colony formation relative to non-treated control NMA * * 0 Gy 5 Gy E * * * * * * Control RasB8 P(0) GT-21 GT-25 Control RasB8 P(0) GT-21 GT-25 0 100 200 300 400 500 % Colony formation relative to non-treated control NMA * * * 0 Gy 5 Gy * * * F B B 0 1000 2000 3000 4000 5000 6000 ntrol ntrol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Luciferase expression (RLU/ug) * * * * * * * * * * * * * * B Reporter Proteins C E D G 0 NMA p53 -/- GT-6 GT-8 RasB8 P(0) GT-21 GT-25 NMA RasB8 P(0) GT-21 G NMA RasB8 P(0) GT-21 G Supplemental Figure 2 Supplemental Figure 2 Supplemental Figure 2 A D shRNA Con PINK1 shRNA PINK1 Rescue U87 T98G A172 U251 G179 GSC 144 0 500 1000 1500 2000 RFU E B 0 200 400 600 % ROS Relative to non treated control 1.3 Fold reduction **p<0.01 3.2 Fold reduction ****p<0.0001 0 2 4 6 0 100 200 300 Days Cell Count (x1000) F *** G 5 ake trol shRNA con + EV PINK1 shRNA + EV shRNA con + SOD2 PINK1 shRNA + SOD2 H I *** Vehicle (DMSO) 10uM Mitotemp 10uM D 8 s) shRNA con PINK1 shRNA 0.0 0.5 1.0 1.5 Relative mitochondrial DNA content C shRNA con PINK1 shRNA 0 5 10 15 20 Average Cell Diameter (um) L EV + EV SOD2 + SOD2 shRNA control + - + - + - PINK1 shRNA - + - + - + M MTCO1 LDHA PINK1 Cyto Mito 0 2000 4000 6000 8000 RFU *** *** *** *** *** DCFH2 Control Probe B shRNA con PINK1 shRNA 0.0 0.5 1.0 1.5 Relative mitochondrial DNA content A MTCO1 LDHA PINK1 Cyto Mito C shRNA con PINK1 shRNA 0 5 10 15 20 Average Cell Diameter (um) B C A D shRNA Con PINK1 shRNA PINK1 Rescue U87 T98G A172 U251 G179 GSC 144 0 500 1000 1500 2000 RFU E 0 200 400 600 % ROS Relative to non treated control 1.3 Fold reduction **p<0.01 3.2 Fold reduction ****p<0.0001 0 2 4 6 0 100 200 300 Days Cell Count (x1000) F *** G 0 1 2 3 4 5 Fold glucose uptake compared to control shRNA con + EV PINK1 shRNA + EV shRNA con + SOD2 PINK1 shRNA + SOD2 H I *** Vehicle (DMSO) 10uM Mitotemp 10uM DPI 0 2 4 6 8 Lactate (uM / million cells) J K 0 1 2 3 4 5 HK Activity (Fold Change) 0 50 100 150 PKM Activity (%) shRNA con PINK1 shRNA 0.0 0.5 1.0 Relative mitocho DNA conten shRNA con PINK1 shRNA 0 5 10 15 Average Cell Diamete L shRNA con + EV PINK1 shRNA + EV shRNA con + SOD2 PINK1 shRNA + SOD2 HA-SOD2 Beta-Actin shRNA control + - + - + - PINK1 shRNA - + - + - + M MTCO1 LDHA PINK1 0 2000 4000 6000 8000 RFU *** *** *** *** *** *** *** *** *** DCFH2 Control Probe D shRNA Con PINK1 shRNA PINK1 Rescue U87 T98G A172 U251 G179 GSC 144 0 500 1000 1500 2000 RFU D E 0 200 400 600 % ROS Relative to non treated control 1.3 Fold reduction **p<0.01 3.2 Fold reduction ****p<0.0001 shRNA control + - + - + - PINK1 shRNA - + - + - + shRNA Con PINK1 shRNA PINK1 Rescue U87 T98G A172 U251 G179 GSC 144 0 500 1000 1500 RFU E 0 200 400 600 % ROS Relative to non treated control 1.3 Fold reduction **p<0.01 3.2 Fold reduction ****p<0.0001 V hi l (DMSO) 10uM Mitotemp 10uM DPI shRNA control + - + - + - PINK1 shRNA - + - + - + E 0 2 4 6 0 100 200 300 Days Cell Count (x1000) F *** G 0 1 2 3 4 5 Fold glucose uptake compared to control shRNA con + EV PINK1 shRNA + EV shRNA con + SOD2 PINK1 shRNA + SOD2 H I *** Vehicle (DMSO) 10uM Mitotemp 10uM 0 2 4 6 8 Lactate (uM / million cells) J K 0 1 2 3 4 5 HK Activity (Fold Change) 0 50 100 150 PKM Activity (%) L shRNA con + EV PINK1 shRNA + EV shRNA con + SOD2 PINK1 shRNA + SOD2 HA-SOD2 Beta-Actin PINK1 shRNA + + + M 0 2000 4000 6000 8000 RFU *** *** *** *** *** *** *** *** *** DCFH2 Control Probe 0 2 4 6 0 100 200 300 Cell Count (x1000) *** G F 0 2000 4000 6000 8000 RFU *** *** DCFH2 Control Probe shRNA con + EV PINK1 shRNA + EV shRNA con + SOD2 PINK1 shRNA + SOD2 L G F M I I 0 2 4 6 Days I 0 2 4 6 8 Lactate (uM / million cells) K *** *** 0 1 2 3 4 5 Fold glucose uptake compared to control H *** *** H shRNA con + EV K J F J 0 1 2 3 4 5 HK Activity (Fold Change) *** *** ( K 0 50 100 150 PKM Activity (%) *** *** Supplemental Figure 3 C A B Control PINK1 shRNA 0 1 2 3 4 5 Densitometry: HIF1A/B-ACTIN ** 1% Hypoxia D F Fold mRNA Change PINK1 KD/Control LDHA HK2 GLUT1 PDK1 -4 -2 0 2 4 6 * * * * PKM2 PKM1 B-ACTIN NHA PKM1 pos con PKM2 pos con % Invasion (relative to normoxia control) shRNA control + siRNA contr PINK1 shRNA shRNA control + HIF1a siRNA Normoxia 1% Hypoxia 0 100 200 300 400 500 * * * * * * E PINK1 HIF1A siRNA Control siRNA - + + - + - - - HIF1A VINCULIN NHA Control PINK1 shRNA 1% Hypoxia Control PINK1 KD Normoxia Hypoxia 0 5 10 15 Fold mRNA Increase * * * * * * PDK1 LDHA PKM2 PDK1 LDHA PKM2 Supplemental Figur C A B Control PINK1 shRNA 0 1 2 3 4 5 Densitometry: HIF1A/B-ACTIN ** Fold mRNA Change PINK1 KD/Control LDHA HK2 GLUT1 PDK1 -4 -2 0 2 4 6 * * * * PKM2 PKM1 B-ACTIN NHA PKM1 pos con PKM2 pos con C B A 1% Hypoxia D F % Invasion (relative to normoxia control) shRNA control + siRNA contro PINK1 shRNA Normoxia 1% Hypoxia 0 100 200 300 400 500 * * * * * * E PINK1 HIF1A siRNA Control siRNA - + + - + - - - HIF1A VINCULIN NHA Control PINK1 shRNA 1% Hypoxia Control PINK1 KD Normoxia Hypoxia 0 5 10 15 Fold mRNA Increase * * * * * * PDK1 LDHA PKM2 PDK1 LDHA PKM2 F D E E H 0 24 48 72 96 120 0 20 40 60 80 100 Hours Cell Count (x1000 ) ** ** shRNA control + control siRNA PINK1 shRNA shRNA control + HIF1 siRNA PINK1 shRNA + HIF1 siRNA Normoxia G 0 24 48 72 96 120 0 20 40 60 80 100 120 140 160 Hours Cell Count (x1000 ) ** ** shRNA control + control siRNA PINK1 shRNA shRNA control + HIF1 siRNA PINK1 shRNA + HIF1 siRNA Hypoxia H 0 24 48 72 96 120 0 20 40 60 80 100 120 140 160 Hours Cell Count (x1000 ) ** ** shRNA control + control siRNA PINK1 shRNA shRNA control + HIF1 siRNA PINK1 shRNA + HIF1 siRNA Hypoxia 0 24 48 72 96 120 0 20 40 60 80 100 Hours Cell Count (x1000 ) ** ** shRNA control + control siRNA PINK1 shRNA shRNA control + HIF1 siRNA PINK1 shRNA + HIF1 siRNA Normoxia G H G ** Supplemental Figure 4 Supplemental Figure 4 A * * * Normoxia Hypoxia Minus NAC Plus NAC C KD C KD C KD C KD 0 2 4 6 8 10 Fold ROS increase C U87 T98G SF188 B 0 40 80 120 Doubling Times (H) ** ** Normoxia Hypoxia Minus NAC Plus NAC C KD C KD C KD C KD HIF1A - - + + - - + + Hypoxia - + - + - + - + NAC control PINK1 shRNA B-ACTIN A HIF1A - - + + - - + + Hypoxia - + - + - + - + NAC control PINK1 shRNA B-ACTIN PINK1 shRNA * * * Normoxia Hypoxia Minus NAC Plus NAC C KD C KD C KD C KD 0 2 4 6 8 10 Fold ROS increase C U87 T98G SF188 B 0 40 80 120 Doubling Times (H) ** ** Normoxia Hypoxia Minus NAC Plus NAC C KD C KD C KD C KD C B A B Supplemental Figure 5 0 20 40 60 80 100 0 50 100 150 200 SF188 Cells OCR (pmol/min) Time (min) O F R Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * Control PINK1 0 20 40 60 80 100 0 50 100 150 200 Time (min) OCR (pmol/min) Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * T98G Cells O F R Control PINK1 0 500 1000 1500 2000 2500 RFU U87 SF188 T98G Control PINK1 C D E F 0 50 100 150 % Invasion (relative to control) U87 SF188 T98G * * * 150 ) Control PINK1 200 250 ) * * * B Supplemental Figure 5 0 500 1000 1500 2000 2500 RFU U87 SF188 T98G Control PINK1 A B Supplemental Figure 5 0 500 1000 1500 2000 2500 RFU U87 SF188 T98G Control PINK1 0 50 100 150 % Invasion (relative to control) U87 SF188 T98G * * * Control PINK1 A 0 50 100 150 % Invasion (relative to control) U87 SF188 T98G * * * Control PINK1 B B A 0 20 40 60 80 100 0 50 100 150 200 SF188 Cells OCR (pmol/min) Time (min) O F R Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * Control PINK1 0 20 40 60 80 100 0 50 100 150 200 Time (min) OCR (pmol/min) Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * T98G Cells O F R Control PINK1 0 U87 SF188 T98G C D E F 0 U87 SF188 T98G 0 50 100 150 HK Activity (relative to control) Control PINK1 U87 SF188 T98G * * * Control PINK1 Control PINK1 Control PINK1 0 50 100 150 200 250 PKM Activity (relative to control) * * * 0 20 40 60 80 100 0 50 100 150 200 SF188 Cells OCR (pmol/min) O F R Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * C 0 20 40 60 80 100 0 50 100 150 200 OCR (pmol/min) Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * T98G Cells O F R C D 0 20 40 60 80 100 0 50 100 150 200 SF188 Cells OCR (pmol/min) Time (min) O F R Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * Control PINK1 D 0 20 40 60 80 100 0 50 100 150 200 OCR (pmol/min) Basal ATP Turnover (Coupled) Max Respiratory Non Mitochondrial * * * * * * T98G Cells O F R C D C C OCR (pmol/min) OCR (pmol/min) E F 0 50 100 150 HK Activity (relative to control) Control PINK1 U87 SF188 T98G * * * Control PINK1 Control PINK1 Control PINK1 0 50 100 150 200 250 PKM Activity (relative to control) * * * F E A B 0 2 4 6 0 5 10 15 20 T98G Cells Days Lactate (uM / million cells) Control PINK1 C D * * * E F 10 15 20 (uM / million cells) SF188 Cells * 0 2 4 6 0 10 20 30 40 Days Glucose (mM) Control PINK1 * * * 20 30 40 lucose (mM) T98G Cells SF188 Cells * * * Supplemental Figure 6 1 2 3 4 5 6 0 5 10 15 20 25 Days U87 Cells Control PINK1 ** ** 1 2 3 4 5 6 15 20 25 30 Days U87 Cells Control PINK1 * * Lactate (uM / million cells) Glucose (mM) A B Supplemental Figure 6 1 2 3 4 5 6 0 5 10 15 20 25 D U87 Cells Control PINK1 ** ** 1 2 3 4 5 6 15 20 25 30 U87 Cells Control PINK1 * * Lactate (uM / million cells) Glucose (mM) Supplemental Figure 6 B A A Control PINK1 0 2 4 0 5 10 15 20 T98G Cells Days Lactate (uM / million cells) Control PINK1 C D * * * E F 0 2 4 6 0 5 10 15 20 Days Lactate (uM / million cells) Control PINK1 SF188 Cells * * 0 2 4 6 0 10 20 30 40 Days Glucose (mM) Control PINK1 * * * 0 2 4 6 0 10 20 30 40 Days Glucose (mM) PINK1 Control T98G Cells SF188 Cells * * * Days Days 0 2 4 0 5 10 15 20 T98G Cells Days Lactate (uM / million cells) Control PINK1 C D * * * E F 10 15 20 M / million cells) SF188 Cells * 0 2 4 6 0 10 20 30 40 Days Glucose (mM) Control PINK1 * * * 20 30 40 cose (mM) T98G Cells SF188 Cells * * * Days Days 0 2 4 0 5 10 15 20 T98G Cells Days Lactate (uM / million cells) Control PINK1 C D * * * 0 2 4 6 0 10 20 30 40 Days Glucose (mM) Control PINK1 * * * T98G Cells Days Days 0 2 4 6 0 5 10 15 20 T98G Cells Lactate (uM / million cells) D * * * D C 0 2 4 6 0 10 20 30 40 Days Glucose (mM) Control PINK1 * * * T98G Cells C 6 6 E F 0 2 4 6 0 5 10 15 20 Days Lactate (uM / million cells) Control PINK1 SF188 Cells * * 0 2 4 6 0 10 20 30 40 Days Glucose (mM) PINK1 Control SF188 Cells * * * E 0 2 4 6 0 10 20 30 40 Days Glucose (mM) PINK1 Control SF188 Cells * * * F 0 2 4 6 0 5 10 15 20 Days Lactate (uM / million cells) Control SF188 Cells * * F E 2 4 6 Days 2 6 Supplemental Figure 7 Supplemental Figure B 1 3 5 0 500 1000 1500 2000 Days Cell Count (x1000) GBM 12 Control GBM 12 PINK1 GBM 8 Control GBM 8 PINK1 *** *** B A A Control PINK1 Control PINK1 V5-PINK1 B-ACTIN C D 1 3 5 0 500 1000 1500 Days Cell Count (x1000) GBM 12 PINK1 GBM 8 Control GBM 8 PINK1 *** 0.0 0.5 1.0 1.5 Fold Increase Hexokinase Activity ** ** 0 5 10 15 20 Lactate (uM / million cells) ** ** E F GBM 8 GBM 12 GBM 8 GBM 12 Control PINK1 0 50 100 150 % Invasion (relative to control) GBM 8 GBM 12 ** ** Control PINK1 Control PINK1 0.0 0.5 1.0 1.5 Fold glucose uptake compared to control ** ** G 0 25 50 75 100 125 % Superoxide Anion Reduction H * * U87 T98G C D 0 50 100 150 % Invasion (relative to control) GBM 8 GBM 12 ** ** D C 0.0 0.5 1.0 1.5 Fold Increase Hexokinase Activity ** ** 0 5 10 15 20 Lactate (uM / million cells) ** ** E F GBM 8 GBM 12 GBM 8 GBM 12 Control PINK1 C C G 0 25 50 75 100 125 % Superoxide Anion Reduction H * * U87 T98G F E G H Supplemental Figure 8 Supplemental Figure A GSC-179 Day C 1 3 5 7 GSC-144 Day C 1 3 5 7 PINK1 B-ACTIN U118 Day C 1 3 5 7 U251 Day C 1 3 5 7 PINK1 B-ACTIN Control PINK1 siRNA 0 2 4 6 8 0 200 400 600 800 G179 Days Cell Count (x1000) 8 0 2 4 6 0 200 400 600 800 G144 Days Cell Count (x1000) * * * * 0 2 4 6 8 0 200 400 600 800 1000 U118 Days Cell Count (x1000) * * 0 2 4 6 8 0 200 400 600 800 1000 U251 Days Cell Count (x1000) * * B C D E H F 0 1 2 3 4 NADP+/NADPH Ratio U118 U251 G179 G144 * * * * Control PINK1 KD I 0.0 0.2 0.4 0.6 2.0 4.0 6.0 8.0 GSH/GSSG (uM) Control + + - - + + - - + + - - + + - - PINK1 - - + + - - + + - - + + - - + + U118 U251 G179 G144 GS GS J 20 40 60 80 H:GSSG Ratio Control G * * * * 0 5000 10000 15000 ROS (RFU) NHA U118 U251 G179 G144NHA U118 U251 G179 G144 Control PINK1 KD * * * * * + PEG-SOD 0 500 1000 1500 2000 2500 RFU NHA U118 U251 G179 G144 Supplemental Figure A GSC-179 Day C 1 3 5 7 GSC-144 Day C 1 3 5 7 PINK1 B-ACTIN U118 Day C 1 3 5 7 U251 Day C 1 3 5 7 PINK1 B-ACTIN Control PINK1 siRNA 0 2 4 6 8 0 200 400 600 800 G179 Days Cell Count (x1000) 8 0 2 4 6 0 200 400 600 800 G144 Days Cell Count (x1000) * * * * 0 2 4 6 8 0 200 400 600 800 1000 U118 Days Cell Count (x1000) * * 0 2 4 6 8 0 200 400 600 800 1000 U251 Days Cell Count (x1000) * * B C D E H F o * * * * Control G 0 5000 10000 15000 ROS (RFU) NHA U118 U251 G179 G144NHA U118 U251 G179 G144 Control PINK1 KD * * * * * + PEG-SOD 0 500 1000 1500 2000 2500 RFU pp g 0 2 4 6 8 0 200 400 600 800 G179 Cell Count (x1000) 8 0 2 4 6 0 200 400 600 800 G144 Cell Count (x1000) * * * * B C A GSC-179 Day C 1 3 5 7 GSC-144 Day C 1 3 5 7 PINK1 B-ACTIN U118 Day C 1 3 5 7 U251 Day C 1 3 5 7 PINK1 B-ACTIN B A 0 2 4 6 8 Days 8 0 2 4 6 Days 0 2 4 6 8 0 200 400 600 800 1000 U118 Cell Count (x1000) * * 0 2 4 6 8 0 200 400 600 800 1000 U251 Cell Count (x1000) * * D E rol D Control PINK1 siRNA 0 2 4 6 8 0 200 400 600 Days Cell Count * * 0 2 4 6 8 0 200 400 600 Days Cell Count ( * * F G 0 5000 10000 15000 ROS (RFU) NHA U118 U251 G179 G144NHA U118 U251 G179 G144 Control PINK1 KD * * * * * + PEG-SOD 1000 1500 2000 2500 RFU C P 0 2 4 6 8 0 200 400 Days Cell Coun * * F G 0 5000 10000 15000 ROS (RFU) NHA U118 U251 G179 G144NHA U118 U251 G179 G144 Control PINK1 KD * * * * * + PEG-SOD 1000 1500 2000 2500 RFU F G H 0 1 2 3 4 NADP+/NADPH Ratio U118 U251 G179 G144 * * * * Control PINK1 KD I 0.0 0.2 0.4 0.6 2.0 4.0 6.0 8.0 GSH/GSSG (uM) Control + + - - + + - - + + - - + + - - PINK1 - - + + - - + + - - + + - - + + U118 U251 G179 G144 GS GSS J 0 20 40 60 80 GSH:GSSG Ratio Control PINK1 KD U118 U251 G179 G144 * * * * NHA U118 U251 G179 G144NHA U118 U251 G179 G144 0 500 1000 RF NHA U118 U251 G179 G144 H 0 1 2 3 4 NADP+/NADPH Ratio U118 U251 G179 G144 * * * * Control PINK1 KD I 2.0 4.0 6.0 8.0 GSSG (uM) U118 U251 G179 G144 GSH GSSG 0 500 1000 R NHA U118 U251 G179 G144 H U118 U251 G179 G144 0.0 0.2 0.4 0.6 GSH/GS Control + + - - + + - - + + - - + + - - PINK1 - - + + - - + + - - + + - - + + J 0 20 40 60 80 GSH:GSSG Ratio Control PINK1 KD U118 U251 G179 G144 * * * * Supplemental Figure 9 U118 * 0 Gy 2 Gy B 0 200 400 600 800 U251 0 Gy 2 Gy C D 0 500 1000 1500 G179 0 Gy 2 Gy * * 0 200 400 600 800 1000 G144 * 0 Gy 2 Gy Activated Caspase (RFU) Activated Caspase (RFU) Activated Caspase (RFU) 0 100 200 300 400 500 Colony Forming Units (CFU) 0 Gy 2 Gy E F U118 * * G H 0 100 200 300 Colony Forming Units (CFU) 0 Gy 2 Gy G179 * * Control siRNA PINK1 siRNA 0 50 100 150 200 250 Colony Forming Units (CFU) 0 Gy 2 Gy G144 * * 0 100 200 300 400 Colony Forming Units (CFU) 0 Gy 2 Gy U251 * * Supplemental Figure 9 Supplemental Figure 9 A 0 200 400 600 800 1000 U118 Activated Caspase (RFU) * 0 Gy 2 Gy B 0 200 400 600 800 U251 0 Gy 2 Gy C D 0 500 1000 1500 G179 0 Gy 2 Gy * * 0 200 400 600 800 1000 G144 * 0 Gy 2 Gy Activated Caspase (RFU) Activated Caspase (RFU) Activated Caspase (RFU) 0 100 200 300 400 500 Colony Forming Units (CFU) 0 Gy 2 Gy E F U118 * * G H 300 nits (CFU) G179 * * 200 250 nits (CFU) G144 * * 0 100 200 300 400 Colony Forming Units (CFU) 0 Gy 2 Gy U251 * * Supplemental Figure 9 A 0 200 400 600 800 1000 U118 Activated Caspase (RFU) * 0 Gy 2 Gy B 0 200 400 600 800 U251 0 Gy 2 Gy C D 0 500 1000 1500 G179 0 Gy 2 Gy * * 0 200 400 600 800 1000 G144 * 0 Gy 2 Gy Activated Caspase (RFU) Activated Caspase (RFU) Activated Caspase (RFU) D B A * y 2 Gy 0 200 400 600 800 0 Gy 2 Gy 0 500 1000 1500 0 Gy 2 Gy 0 200 400 600 800 1000 Activated Caspase (RFU) Activated Caspase (RFU) Activated Caspase (RFU) 0 100 200 300 400 500 Colony Forming Units (CFU) 0 Gy 2 Gy E F U118 * * G H 0 100 200 300 Colony Forming Units (CFU) 0 Gy 2 Gy G179 * * Control siRNA PINK1 siRNA 0 50 100 150 200 250 Colony Forming Units (CFU) 0 Gy 2 Gy G144 * * 0 100 200 300 400 Colony Forming Units (CFU) 0 Gy 2 Gy U251 * * 0 100 200 300 400 500 Colony Forming Units (CFU) 0 Gy 2 Gy E F U118 * * 0 100 200 300 400 Colony Forming Units (CFU) 0 Gy 2 Gy U251 * * F G H 0 100 200 300 Colony Forming Units (CFU) 0 Gy 2 Gy G179 * * 0 50 100 150 200 250 Colony Forming Units (CFU) 0 Gy 2 Gy G144 * * H G Control siRNA PINK1 siRNA A B Supplementa Medulloblastoma Subgroup RNA Expression (Log2 Expression) 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 Normal Cerebellum WNT SHH Group 3 Group 4 * * * * n=11 n=14 n=51 n=52 n=71 C Het loss (n=103) Diploid (n=173) Gain (n=9) -4 -2 0 2 4 Z-Score RNA Seq Expression PINK1 Copy Number Vs. A 0 1000 2000 3000 4000 5000 6000 Neg Control Pos Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Luciferase expression (RLU/ug) * * * * * * * * * * * * * * B p53 -/- astrocytes Activated Ras expressing astrocytes Supplemental Fi Gene Trap Retrovirus Genomic Locus Random Integration Fusion mRNA Reporter Proteins LTR SA Luciferase IRES Puro PA LTR Non Transformed Cell Transformed Cell P P Luciferase IRES Puromycin Exon AAAAAAAAAA Luciferase IRES Puromycin Exon SD SA Splicing Event After Transcription E E E E 30 40 50 60 vasion * (3) (T) A B C D E 0 50 100 150 Agar Colonies C NMA Gene-trapped Clones E 100 200 300 400 500 olony formation ve to non-treated control NMA * * * 0 Gy 5 Gy * * * F mRNA *** U87 Control U87 PINK1 H&E PINK1 KI67 Supplemental Figure 10 A B Supplemental Het loss (n=103) Diploid (n=173) Gain (n=9) -4 -2 0 2 4 Z-Score RNA Seq Expression PINK1 Copy Number Vs. mRNA *** U87 Control U87 PINK1 H&E PINK1 KI67 B Supplemental F Het loss (n=103) Diploid (n=173) Gain (n=9) -4 -2 0 2 4 Z-Score RNA Seq Expression PINK1 Copy Number Vs. mRNA *** A B Z-Score RNA Seq Expression PINK1 Copy Number Vs. mRNA H&E PINK1 KI67 Medulloblastoma Subgroup RNA Expression (Log2 Expression) 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 Normal Cerebellum WNT SHH Group 3 Group 4 * * * * n=11 n=14 n=51 n=52 n=71 C C Supplemental Table 1: Identification of Gene-trapped clones with Inverse PCR Background Clone Gene Gene Symbol Function NM_Accession Number Site of Integration Link with Glioma p53 -/- GT-6 Mitochondrial ribosomal protein S6 Mrps6 Protein synthesis within the mitochondrion. NM_080456.1 Intron 1 N p53 -/- GT-8 Storkhead box 1 Stox1 DNA binding protein NM_001033260.1 Intron 1 N Ras GT-15 RAP1 GTPase activating protein Rap1gap GTPase activating protein NM_001081155.1 Intron 1 Y Ras GT-16 Inhibitor of kappa light polypeptide gene enhancer in B- cells, kinase beta Ikbkb Acts as part of the IKK complex in the conventional pathway of NF-kappa-B activation and phosphorylates inhibitors of NF-kappa-B NM_001159774.1 Intron 2 Y Ras GT-19 Electron-transfer- flavoprotein, beta polypeptide Etfb Shuttles electrons between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism NM_026695.2 Intron 1 Y Ras GT-21 PTEN induced putative kinase 1 Pink1/ Park6 This gene encodes a serine/threonine protein kinase that localizes to mitochondria. NM_026880.2 Intron 1 N Ras GT-25 Suppressor of cytokine signaling 2 Socs2 STAT-induced STAT inhibitor (SSI), also known as suppressor of cytokine signaling NM_001168655.1 Intron 1 N Supplemental Table 1: Identification of Gene-trapped clones with Inverse PCR

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https://dro.deakin.edu.au/articles/journal_contribution/Regulation_of_carbon_dioxide_and_methane_in_small_agricultural_reservoirs_Optimizing_potential_for_greenhouse_gas_uptake/20686099/1/files/36900724.pdf

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Regulation of carbon dioxide and methane in small agricultural reservoirs: optimizing potential for greenhouse gas uptake

Biogeosciences

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Correspondence: Jackie R. Webb ([email protected]) Correspondence: Jackie R. Webb ([email protected]) Received: 2 July 2019 – Discussion started: 29 July 2019 Revised: 2 October 2019 – Accepted: 7 October 2019 – Published: 8 November 2019 Received: 2 July 2019 – Discussion started: 29 July 2019 Received: 2 July 2019 – Discussion started: 29 July 2019 Revised: 2 October 2019 – Accepted: 7 October 2019 – Published: 8 November 2019 farm reservoirs can be greatly minimized with overall im- provements in water quality and consideration to position and hydrology within the landscape. Abstract. Small farm reservoirs are abundant in many agri- cultural regions across the globe and have the potential to be large contributing sources of carbon dioxide (CO2) and methane (CH4) to agricultural landscapes. Compared to nat- ural ponds, these artificial waterbodies remain overlooked in both agricultural greenhouse gas (GHG) inventories and in- land water global carbon (C) budgets. Improved understand- ing of the environmental controls of C emissions from farm reservoirs is required to address and manage their poten- tial importance in agricultural GHG budgets. Here, we con- ducted a regional-scale survey (∼235 000 km2) to measure CO2 and CH4 surface concentrations and diffusive fluxes across 101 small farm reservoirs in Canada’s largest agricul- tural area. A combination of abiotic, biotic, hydromorpho- logic, and landscape variables were modelled using general- ized additive models (GAMs) to identify regulatory mech- anisms. We found that CO2 concentration was estimated by a combination of internal metabolism and groundwater- derived alkalinity (66.5 % deviance explained), while mul- tiple lines of evidence support a positive association be- tween eutrophication and CH4 production (74.1 % deviance explained). Fluxes ranged from −21 to 466 and 0.14 to 92 mmol m−2 d−1 for CO2 and CH4, respectively, with CH4 contributing an average of 74 % of CO2-equivalent (CO2-e) emissions based on a 100-year radiative forcing. Approxi- mately 8 % of farm reservoirs were found to be net CO2-e sinks. From our models, we show that the GHG impact of Biogeosciences, 16, 4211–4227, 2019 https://doi.org/10.5194/bg-16-4211-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Biogeosciences, 16, 4211–4227, 2019 https://doi.org/10.5194/bg-16-4211-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction The expansion of agriculture and urban land use has intro- duced a new type of lentic system that remains relatively un- explored – small artificial waterbodies (Clifford and Heffer- nan, 2018). These artificial aquatic systems have been cre- ated through human modification of the hydrological land- scape and include small farm reservoirs and urban ponds. Farm reservoirs are earthen excavations designed to store water for later use (BC Ministry of Agriculture, 2013). The global abundance of these systems remains uncertain (Ver- poorter et al., 2014), but statistical extrapolation suggest there may be around 16 million artificial reservoirs world- wide (Lehner et al., 2011). Regional-scale inventories indi- cate that collectively upwards of 8 million farm reservoirs exist in the USA (Brunson, 1999; Smith et al., 2002), China (Chen et al., 2019), India (Anbumozhi et al., 2001), South Africa (Mantel et al., 2017), and Australia alone (Lowe et al., 2005; MDBA, 2008; Grinham et al., 2018a). The den- sity of farm reservoirs can exceed 30 % of agricultural area in some regions such as China where food demand is high (Chen et al., 2019). Small agricultural reservoirs are esti- Jackie R. Webb1, Peter R. Leavitt1,2,3, Gavin L. Simpson1,2, Helen M. Baulch4, Heather A. Haig1, Kyle R. Hodder5, and Kerri Finlay1 1Department of Biology, University of Regina, Regina, SK, S4S0A2, Canada 2Institute of Environmental Change and Society, University of Regina, Regina, Saskatchewan, S4S 0A2, Canada 3Institute for Global Food Security, Queen’s University Belfast, Belfast, Northern Ireland, BT9 5DL, UK 4School of Environment and Sustainability, Global Institute for Water Security, University of Saskatchewan, 11 Innovation Boulevard, Saskatoon, SK S7N3H5, Canada 5Department of Geography and Environmental Studies, University of Regina, Regina, SK, S4S0A2, Canada Published by Copernicus Publications on behalf of the European Geosciences Union. ublished by Copernicus Publications on behalf of the European Geosciences Union. J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4212 mated to cover 77 000 km2 globally and are being created at rates up to 60 % of existing reservoirs per annum in some regions (Downing et al., 2008). Given their abundance, these artificial systems may contribute substantially to landscape biogeochemical cycles, including fluxes of GHG. In particu- lar, very little is known of the capability of these systems to act as GHG sinks to partially offset the otherwise strong car- bon efflux associated with intensive agriculture (Robertson et al., 2000). 2014; Grinham et al., 2018a; Ollivier et al., 2019). All studies found large fractions of CH4 being released and large mean CO2 emissions on the order of 24 and 99 mmol m−2 d−1, comparable to the global average flux rate of very small natural ponds (35 mmol m−2 d−1, Holgerson and Raymond, 2016). However, carbon fluxes from farm reservoirs remain unaccounted for in agricultural GHG inventories and global inland water carbon budgets. To facilitate their inclusion in agricultural and global budgets, we need to further constrain flux rates and mechanisms across a broad geographic area. Small waterbodies have recently been recognized as sub- stantial contributors to global carbon emissions from inland waters. Current assessments estimate that diffusive CO2 and CH4 emissions from small ponds (< 0.001 km2) account for 15 % and 40 % of global emissions from lakes, respectively (Holgerson and Raymond, 2016). Other estimates suggest emissions from small lakes and impoundments (0.001 to 0.01 km2) could constitute 40 % of global CO2 emissions and 20 % of global CH4 emissions from lentic ecosystems (Del- Sontro et al., 2018). Extreme CO2 and CH4 supersaturation is characteristic of small waterbodies due to greater contact with the sediment and littoral zone (Downing et al., 2008; Holgerson, 2015), often making them disproportionately im- portant in landscape carbon (C) budgets (Hamilton et al., 1994; Premke et al., 2016; Kuhn et al., 2018). Conversely, ponds may have the capacity to store landscape-significant amounts of carbon, with burial rates 20–30 times higher than wetlands and large lakes (Gilbert et al., 2014; Taylor et al., 2019). Published by Copernicus Publications on behalf of the European Geosciences Union. While these assessments have stimulated a growing area of research on small waterbodies, much work is still needed to revise estimates of their carbon emissions due to limited knowledge on their regional distribution and variabil- ity, as well as their overall global extent (Verpoorter et al., 2014). This is particularly true for greenhouse gas (GHG) emissions from human-created small waterbodies. Here, we present a large-scale assessment of CO2 and CH4 concentrations from small farm reservoirs in the Northern Great Plains, the largest agricultural region in Canada. This study builds on from our previous farm reservoir GHG re- search which found an unexpected nitrous oxide (N2O) sink in 67 % of reservoirs (Webb et al., 2019). The hydroclimate, lithology and edaphic features are vastly different compared to previous studies of agricultural areas (Australia, India, USA), with factors that favour CO2 uptake by alkaline sur- face waters (Finlay et al., 2009, 2015) and lead to high vari- ability in CH4 fluxes from regional wetlands (Pennock et al., 2010; Badiou et al., 2019). Our aim was to identify the key environmental conditions regulating CO2 and CH4 fluxes, as well as to evaluate these baseline data in the context of emis- sion mitigation strategies. To achieve this goal, we carried out an extensive survey of CO2 and CH4 concentrations across 101 farm reservoirs and used generalized additive models (GAMs) to assess the effects of abiotic, biotic, hydromor- phological, and land use properties. Our findings show that farm dams were not always strong sources of carbon emis- sions and in certain cases can be carbon neutral or sinks in terms of CO2-equivalent (CO2-e) emissions. By identifying the driving characteristics of farm dams that support reduced C emissions, our findings provide the first step to developing management strategies to help minimize farm carbon emis- sions. Understanding the controls and rates of carbon fluxes from small artificial waterbodies is the first step required to un- derstand their landscape and eventually global importance. Further, estimates of CO2 and CH4 flux are complicated by high variation among reservoirs and regions in the impor- tance of groundwater, littoral macrophytes, and local land use practises (Pennock et al., 2010; Badiou et al., 2019). Ar- tificial reservoirs have the potential to be potent sources of CO2 and CH4 (Downing et al., 2008; Holgerson and Ray- mond, 2016). Published by Copernicus Publications on behalf of the European Geosciences Union. This role can be demonstrated by a carbon budget estimate from an urban pond where carbon emis- sions (both diffusive and ebullitive for CH4) offset carbon burial by > 1000 % (van Bergen et al., 2019). The recent 2019 IPCC Refinement has assigned a CH4 emission fac- tor of 183 kg ha−1 yr−1 to constructed waterbodies; however, data are greatly limited, both geographically and in number (n = 68), such that climatic-zone emission factors cannot be estimated (IPCC, 2019). Currently, only three studies have assessed C fluxes from small agricultural reservoirs at re- gional scales, and these support the notion that they are im- portant landscape sources of GHGs (Panneer Selvam et al., J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4213 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs tle was then shaken for 2 min to ensure gas equilibration in the headspace. Two analytical replicates were extracted and stored in 12 mL evacuated Exetainer vials with double- wadded caps. Headspace concentrations of CO2 and CH4 were measured using gas chromatography with a Scion 456 gas chromatograph (Bruker Ltd.) and calculated using stan- dard curves. Dry molar fractions were corrected for dilution and converted to concentrations according to solubility coef- ficients (Weiss, 1974; Yamamoto et al., 1976). Figure 1. (a) Map of southern Saskatchewan in Canada showing the distribution of studied farm reservoirs, (b) aerial image showing 10 farm reservoirs delineated by white rectangles within a 1 km2 area, and (c) general size and shape of farm reservoirs with two characteristic side mounds of excavated materials. To compare with the literature and assess the source/sink behaviour of the reservoirs, diffusive fluxes of carbon diox- ide and methane fluxes were estimated for each waterbody. Given that the focus of the study was to investigate drivers of CO2 and CH4 concentrations across farm reservoirs, ebulli- tion events were not measured during this survey and as such total CH4 fluxes are likely underestimated. Diffusive fluxes were estimated using water-column concentrations (Cwater) and average farm reservoir gas transfer velocity (kc) using the following equation: Figure 1. (a) Map of southern Saskatchewan in Canada showing the distribution of studied farm reservoirs, (b) aerial image showing 10 farm reservoirs delineated by white rectangles within a 1 km2 area, and (c) general size and shape of farm reservoirs with two characteristic side mounds of excavated materials. (1) fC = kc(Cwater −Cair), (1) fC = kc(Cwater −Cair), although subsequent densities are unknown. We sampled 101 farm reservoirs between July and August 2017, rang- ing in surface area from 158 to 13 900 m2 (Table 1), in- cluding basins in pasture (n = 18), pastures with livestock (n = 62), and cropland (n = 21) sites. Each site was sam- pled once during this period, between the daylight hours of 10:00 and 15:00 Central Standard Time (CST; or GMT−6). Saskatchewan farm reservoirs are typically uniform in shape and morphometry, dug to a depth of 4 to 6 m with steep sides (1.5 : 1 slopes). J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs This metric offers a more attainable measure of ecosystem cli- matic forcing, assuming gas flux persists over time instead of occurring as a single pulse as quantified using traditional global warming potentials (GWP, Myhre et al., 2013). Here, a SGWP multiplier of 45 was applied to all CH4 fluxes in the literature comparison, which is slightly higher than the tradi- tional GWP of 32 over a 100-year time frame (Myhre et al., 2013). For comparing CO2-equivalent fluxes, CH4 fluxes were converted using the 100-year sustained-flux global warm- ing potential (SGWP, Neubauer and Megonigal, 2015). This metric offers a more attainable measure of ecosystem cli- matic forcing, assuming gas flux persists over time instead of occurring as a single pulse as quantified using traditional global warming potentials (GWP, Myhre et al., 2013). Here, a SGWP multiplier of 45 was applied to all CH4 fluxes in the literature comparison, which is slightly higher than the tradi- tional GWP of 32 over a 100-year time frame (Myhre et al., 2013). 2.2 CO2 and CH4 measurements Dissolved gas samples were collected using the in-field headspace extraction method (Webb et al., 2019). Briefly, water was collected from ∼30 cm below the surface using a submersible pump which filled a 1.2 L glass-serum bot- tle, ensuring the bottle overflowed and no air bubbles were present. The bottle was sealed with a rubber stopper fit- ted with two three-way stopcock valves. Using two 60 mL airtight syringes, atmospheric air was added to the bottle whilst simultaneously extracting 60 mL of water. The bot- Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs Most shallow wetlands and lakes in the region exhibit water balances dominated by evaporation and limited inflow from winter precipitation or groundwa- ter (Conly and van der Kamp, 2001; Pham et al., 2009). Farm reservoirs differ from small natural waterbodies in that they have a higher ratio of water volume to surface area, designed to minimize evaporation losses. Despite this fea- ture, arid conditions persisted during the sampling year, with reduced (34 %–65 %) annual rainfall such that many reser- voirs were only half their designed depth. Natural waterbod- ies also tend to be high-pH hard-water systems, owing to the soils which consist of glacial till high in carbonates (Last and Ginn, 2005). The same was observed for the majority of farm reservoirs, with an average pH of 8.75 (Table 1). where fC is the flux of CO2 or CH4 (mmol m−2 d−1) and Cair is the ambient air concentration. The average global mixing ratios for the sampling period of 406 and 1.85 µatm were used for ambient concentrations for CO2 and CH4, respectively (Mauna Loa NOAA station, June to Au- gust 2017). Site-specific gas transfer velocity (kc) was deter- mined from 30 individual floating-chamber (area = 0.23 m2, volume = 0.046 m3) measurements carried out on a subset of 10 reservoirs. During each 10 min deployment, changes in gas concentrations were measured at 2.5 min intervals by taking samples using syringes and dispensing gases into pre- evacuated 12 mL vials. The flux (mmol m−2 d−1) was cal- culated from the observed rate of change in the dry mole fraction of the respective gas (Lorke et al., 2015). The gas transfer velocity normalized to a Schmidt number of 600 (k600) for each respective gas was then determined using measured flux, in situ gas concentrations, atmospheric con- centration, Henry’s constant, and Schmidt numbers, assum- ing a Schmidt exponent of 0.67. The average k600 calculated from the floating-chamber deployments was 1.50 ± 1.34 and 1.64 ± 1.14 m d−1 for CO2 and CH4, respectively (Table 1). where fC is the flux of CO2 or CH4 (mmol m−2 d−1) and Cair is the ambient air concentration. The average global mixing ratios for the sampling period of 406 and 1.85 µatm were used for ambient concentrations for CO2 and CH4, respectively (Mauna Loa NOAA station, June to Au- gust 2017). 2.1 Study site Farm sites were surveyed across the agricultural region of Saskatchewan, Canada (Fig. 1). This region covers an area of 235 000 km2 in the southern half of the province, where agriculture accounts for ∼80 % of land use. The region has a sub-humid to semi-arid climate (Köppen Dfb classifica- tion), with short warm summers (∼18 ◦C) and long winters (∼−17 ◦C) resulting in 4.5 to 5.5 months of ice cover on surface waters (Finlay et al., 2015). Average annual precipi- tation in the area ranges from 354 to 432 mm. Small farm reservoirs (known locally as “dugouts”) are a prominent feature of the landscape, with densities of up to 10 km−2 (Fig. 1b). Up until 1985, over 110 000 farm reser- voirs had been constructed in Saskatchewan (Gan, 2000), www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs Site-specific gas transfer velocity (kc) was deter- mined from 30 individual floating-chamber (area = 0.23 m2, volume = 0.046 m3) measurements carried out on a subset of 10 reservoirs. During each 10 min deployment, changes in gas concentrations were measured at 2.5 min intervals by taking samples using syringes and dispensing gases into pre- evacuated 12 mL vials. The flux (mmol m−2 d−1) was cal- culated from the observed rate of change in the dry mole fraction of the respective gas (Lorke et al., 2015). The gas transfer velocity normalized to a Schmidt number of 600 (k600) for each respective gas was then determined using measured flux, in situ gas concentrations, atmospheric con- centration, Henry’s constant, and Schmidt numbers, assum- ing a Schmidt exponent of 0.67. The average k600 calculated from the floating-chamber deployments was 1.50 ± 1.34 and 1.64 ± 1.14 m d−1 for CO2 and CH4, respectively (Table 1). i CO i l fl C fl where fC is the flux of CO2 or CH4 (mmol m−2 d−1) and Cair is the ambient air concentration. The average global mixing ratios for the sampling period of 406 and 1.85 µatm were used for ambient concentrations for CO2 and CH4, respectively (Mauna Loa NOAA station, June to Au- gust 2017). Site-specific gas transfer velocity (kc) was deter- mined from 30 individual floating-chamber (area = 0.23 m2, volume = 0.046 m3) measurements carried out on a subset of 10 reservoirs. During each 10 min deployment, changes in gas concentrations were measured at 2.5 min intervals by taking samples using syringes and dispensing gases into pre- evacuated 12 mL vials. The flux (mmol m−2 d−1) was cal- culated from the observed rate of change in the dry mole fraction of the respective gas (Lorke et al., 2015). The gas transfer velocity normalized to a Schmidt number of 600 (k600) for each respective gas was then determined using measured flux, in situ gas concentrations, atmospheric con- centration, Henry’s constant, and Schmidt numbers, assum- ing a Schmidt exponent of 0.67. The average k600 calculated from the floating-chamber deployments was 1.50 ± 1.34 and 1.64 ± 1.14 m d−1 for CO2 and CH4, respectively (Table 1). For comparing CO2-equivalent fluxes, CH4 fluxes were converted using the 100-year sustained-flux global warm- ing potential (SGWP, Neubauer and Megonigal, 2015). J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4214 4214 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultu Table 1. Farm reservoir and landscape physical, hydrological, and chemical characteristics of the study sites (n = 101). Units N Mean Median Min Max Area m2 101 1312 1040 158 13 900 Depth m 101 2.08 2.10 0.18 5.10 Buoyancy frequency s−2 99 0.01 0.005 0.00 0.03 δ18O inflow ‰ 101 −13.37 −13.33 −19.39 −8.40 E/I 101 0.46 0.43 0.04 1.58 Water residence time Years 100 0.76 0.66 0.08 2.51 CO2 µM 101 42.2 14.6 1.3 326.1 CH4 µM 101 4.3 1.9 0.1 54.5 Flux CO2 Positive mmol m−2 d−1 47 100.1 58.1 0.1 466.2 Negative mmol m−2 d−1 54 −11.9 −13.3 −21.3 −0.1 Flux CH4 mmol m−2 d−1 101 7.1 3.2 0.4 91.5 k600-CO2 m d−1 15 1.50 0.98 0.20 4.12 k600-CH4 m d−1 23 1.64 1.25 0.38 4.14 Temperature ◦C 101 20.1 19.9 15.7 29.5 Dissolved O2 % 101 92.6 88.9 2.3 344.0 Salinity ppt 101 0.9 0.5 0.1 8.6 pH 101 8.75 8.75 6.95 10.19 Chlorophyll a µg L−1 101 99.1 36.9 2.2 2483 NH3 µg N L−1 100 354.7 100.0 10.0 5930 NOx µg N L−1 98 196.6 34.1 1.2 3188 TP µg P L−1 98 285.2 80.0 8.7 6480 TN µg N L−1 98 3082 2360 417.5 14 280 DOC mg C L−1 99 31.8 29.3 4.6 90.4 Sediment organic carbon % 101 5.2 3.9 0.6 31.4 Sediment organic nitrogen % 101 0.6 0.4 0.1 2.8 Alkalinity mg L−1 96 245.4 219.2 71.0 755.5 Soil CEC meq 100 g−1 98 24 24 10 180 Ksat cm h−1 101 9.9 5.0 0.0 39.7 Elevation m 101 627.6 598.0 484.0 997.0 www.biogeosciences.net/16/4211/2019/ 2.3 Abiotic and biotic variables lected at the centre of each reservoir, with the uppermost 10 cm using an Ekman grab sampler, and were frozen at −10 ◦C until analysis. A range of abiotic and biotic parameters were measured at each site. Water quality variables including temperature (◦C), pH, dissolved O2 (DO; % saturation), conductivity (µS cm−2), and salinity were measured at 0.5 m intervals from the surface to the bottom using a YSI (Yellow Springs Instruments, OH, USA) multi-probe meter. Surface (0.5 m) samples for water chemistry were collected using a sub- mersible pump. Upon collection, samples for dissolved ni- trogen (NO3 + NO2, NH4, total dissolved N; µg N L−1), sol- uble reactive phosphorus (SRP; µg P L−1) and total dissolved P (TDP; µg P L−1), dissolved organic and inorganic car- bon (DOC, DIC; mg C L−1), alkalinity (OH+HCO3 +CO3; mg L−1 as CaCO3), and water isotopes (δ2H, δ18O; ‰) were filtered through a 0.45 µm pore membrane filter. Nutrient and dissolved carbon samples were stored in a dark bottle at 4 ◦C until analysis. Chlorophyll a (Chl a) samples were collected on GF/C glass-fibre filters (nominal pore size 1.2 µm) and frozen (−10 ◦C) until analysis. Sediment samples were col- Most analyses were carried out at the University of Regina Institute of Environmental Change and Society (IECS). Wa- ter nutrient and dissolved carbon concentrations were mea- sured on a Lachat QuikChem 8500 and Shimadzu model 5000A total carbon analyzer, following standard analyti- cal procedures, respectively (Patoine et al., 2006; Finlay et al., 2009). Alkalinity was measured using standard meth- ods of the US Environmental Protection Agency (EPA) on a SmartChem 200 Discrete Analyzer (WestCo) and esti- mated as the concentration of CaCO3 (EPA, 1974). Chl a was analysed using standard trichromatic methods (Finlay et al., 2009). The total carbon and nitrogen content (% dry weight) of freeze-dried sediment samples were determined on a NC2500 Elemental Analyzer (ThermoQuest, CE Instru- ments). www.biogeosciences.net/16/4211/2019/ J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 2.6 Statistical analyses Environmental variables were selected based on known or presumed influence on CO2 and CH4 concentrations in lakes and small waterbodies. Both biotic and abiotic predictors that influence production or consumption of CO2 and CH4 were selected, including DO, alkalinity, NOx (NO2 + NO3), NH4, dissolved inorganic nitrogen (DIN), total dissolved nitrogen (TDN), TDP, Chl a, DOC, conductivity, pH, and sediment organic C : N ratio. The influence of reservoir hydrology and morphology was also examined, including measures of sur- face area, basin permanence, hydrologic regime (E/I), water source (δI), and degree of mixing (or stratification). Finally, potential effects of the surrounding terrestrial landscape were estimated in models using soil properties, elevation, and land use practises to account for any localized landscape drivers. Before testing relationships, all predictors were transformed as needed using either log10 or square root to remove skew- ness. The hydrology of farm reservoirs was estimated through analysis of δ18O and δ2H isotope values of water. Sam- ples were collected from 0.5 m below the surface, filtered (0.45 µm pore) and stored in amber borosilicate jars at 4 ◦C until analysis using a Picarro L2120-I cavity ring-down spectrometer (CRDS). Hydrological parameters, including evaporation-to-inflow ratio (E/I), residence time (years), and inflow volume (m3), deuterium (2H) excess (d-excess), and δ18O inflow (δI) values, were calculated using the cou- pled isotope tracer method (Yi et al., 2008) and conventional isotopic water-balance methods (Gibson et al., 2001). All methods assumed that reservoirs were headwater systems in a hydrological steady state (Yi et al., 2008). Model in- puts included information about the local meteoric water line (LMWL), the trajectory of evaporation along a local evap- orative line (LEL), and regional meteorological conditions. From here, the water mass balance of a given waterbody can be quantified based on its relative position along the LEL (Gibson et al., 2001). The relationships between covariates and CO2 and CH4 were estimated using generalized additive models (GAMs). GAMs provide an ideal approach to model non-linear as- sociations between predictor variables and responses, using the sum of unspecified smooth functions to estimate trends. GAMs are not constrained by prescribed assumptions asso- ciated with parametric models such as linearity of link-scale effects in generalized linear models. Instead, the functional form of the partial relationships between covariates and the response is determined from the data. J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs ca/cansis/nsdb/dss/v3/index.html, last access: 31 Octo- ber 2019), using ArcGIS to extract the soil attributes at each site. Extracted variables included soil salinity; soil pH; soil organic carbon content; saturated hydraulic conductivity (Ksat); cation exchange capacity (CEC); and the total com- position of soil from sand, silt, and clay fractions (%). Reser- voir elevation (m a.s.l.) was determined using ArcGIS and the Canadian Digital Elevation Model (CDEM, v1.1). Lo- cal land use in the immediate area surrounding each reser- voir was categorized into three types based on local obser- vations at the time of sampling. Categories included pasture land used for either livestock grazing or hay harvesting, pas- ture where livestock have direct access to the waterbody, and crop fields. 2.6 Statistical analyses The more flexible mod- elling approach is useful where the effects of covariates on the response are non-linear and has been applied to com- plex aquatic datasets assessing GHGs (Wiik et al., 2018; Webb et al., 2019). GAMs were developed with a gamma distribution for the response and the log link function. Each model included covariates that represented hydromorpholog- ical, abiotic and biotic, and landscape controls. To avoid mul- ticollinearity, correlation coefficients and statistical signifi- cance (p < 0.05) between pairs from Pearson linear correla- tion tests were used to guide covariate choice before model fitting (Tables S1–S3 in the Supplement). Candidate vari- Briefly, the isotopic inflow values were estimated by the intercept between the LMWL and site-specific LEL as deter- mined by the δ18O evaporation value (δE) and δ18O reservoir water value at each site (Yi et al., 2008). The E/I ratio was calculated by using headwater isotopic models of the water mass balance ((δI −δL)·(δE −δL)−1). Hydrologic residence time was estimated from the reservoir volume and the wa- ter isotopic values of waterbodies, inflow, and evaporation. Deuterium excess (d-excess ‰ = δ2H −8 · δ18O) was calcu- lated as an additional indicator of evaporation losses, where lower values (< −10 ‰) indicate isotopic enrichment from precipitation (Brooks et al., 2014). 2.4 Hydromorphology Morphometric parameters of reservoirs were estimated for each site. The depth of each farm reservoir was measured during using a portable ultrasonic depth sounder, taken at the deepest section in the centre of the reservoir. Surface area was determined using Google Earth satellite imagery. Reser- voir volume was calculated using the formula for a prismoid by assuming that all sites maintained their original shape, in- cluding slopes of 1.5 : 1 ratio (Andresen et al., 2015). From these measurements, an Index of Basin Permanence (IBP) was calculated (Kerekes, 1977). The degree of water-column mixing or vertical stratifi- cation was determined by calculating the squared Brunt– Väisälä buoyancy frequency (N2, s−2). The strongest density gradient was calculated based on vertical temperature mea- surements at 0.5 m depth intervals using the package rLake- Analyzer (Read et al., 2012) in R (version 3.5.2; R Core Team, 2018). www.biogeosciences.net/16/4211/2019/ www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 4215 2.5 Landscape properties Landscape soil data were obtained from the National Soil DataBase, Government of Canada (http://sis.agr.gc. 3 Results The region experienced a drier-than-average year during sampling, with recorded average annual precipitation ∼60 % less than the long-term climate average of 390 mm in Regina, Saskatchewan (Government of Canada, https://weather.gc. ca, last access: 31 October 2019). Consequently, while most farm reservoirs were constructed to ∼5 m depth the mean water-column depth was 2.1 m (0.2–5.1, Table 1). Despite this, isotopic analysis of water revealed that 93 % of wa- terbodies exhibited an E/I < 1.0, suggesting that reser- voirs were gaining more water than was lost via evap- oration. In general, water residence time (WRT) was ∼ 8 months, although the range in this value was large (29 d to 2.5 years). Estimates of inflow δ18O (δI) indicated variable water sources, with 79 % derived from rain (> −15.66 ‰), 6 % from snowmelt or groundwater (< −17.9 ‰), and 15 % intermediate between sources (−17.9 ‰ to −15.6 ‰). Figure 2. Kernel density estimates of CO2 and CH4 concentrations measured in 101 farm reservoirs grouped by land use. NOx, buoyancy frequency, and soil CEC, with a generally negative response to increasing DO and elevation. The ef- fect of DO on CO2 was particularly distinct between 25 %– 100 % O2 saturation (Fig. 3a). The interactive effect of hy- drology parameters suggests that sites with elevated rain in- flows (δ18O > −12.5 ‰) and longer WRT will exhibit un- dersaturated CO2 concentrations. Carbon dioxide and methane concentrations spanned 3 or- ders of magnitude across surveyed reservoirs, with concen- trations ranging between 1.3 and 326.1 µM and between 0.1 and 54.5 µM for CO2 and CH4, respectively (Fig. 2). Most waterbodies were alkaline, with a mean pH of 8.8 (7.0 to 10.2) and carbonate alkalinity between 71 and 755 mg L−1 (Table 1). Many waters were highly eutrophic, with means for Chl a of 99 µg L−1 (range 2 to 344 µg L−1), total nitrogen of > 3000 µg N L−1 (418 to 14 280), and total phosphorus of 285 µg P L−1 (9 to 648). Dissolved O2 in the surface layer varied by 3 orders of magnitude among basins with 32 % ex- hibiting oversaturation (> 100 %). Variation in CH4 concentrations among waterbodies was explained by a combination of DO saturation, sediment C/N ratio, DIN, conductivity, the interaction between δI and WRT, and local land use (Fig. 4), with buoyancy fre- quency, soil Ksat, and elevation not significant. J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoir 4216 arbon dioxide and methane in small agricultural reservoirs Figure 2. Kernel density estimates of CO2 and CH4 concentrations measured in 101 farm reservoirs grouped by land use. J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs ables were then selected for each model to test which vari- ables best estimate variability in CO2 and CH4 concentra- tions. All model coefficients were estimated using restricted marginal likelihood with the mgcv package (Wood, 2011; Wood et al., 2016) for R (version 3.5.2; R Core Team, 2018). 3.1 Models Regional variation in CO2 concentrations were best esti- mated in a GAM including pH alone, with 86.3 % of de- viance explained and a strongly declining CO2 at pH above 8 (Fig. S1 in the Supplement). Exclusive of the model with pH, the detailed mechanistic GAM for estimating CO2 con- centrations across farm reservoirs included a combination of DO saturation, alkalinity, NOx, thermal stratification (buoy- ancy frequency), basin hydrology (the interaction between δI and WRT), and landscape features (soil CEC, elevation, soil salinity) (Fig. 3). Overall, the model explained 66.5 % of deviance in CO2 concentrations (Table S4, Fig. S2). All covariates had a significant effect except soil salinity, with DO, alkalinity, and the interaction between δI and WRT being the strongest predictors (p < 0.001). CO2 concentra- tions displayed a positive response with increasing alkalinity, 3 Results Overall, the GAM explained 74.1 % of the deviance in CH4 (Table S5, Fig. S3). Concentrations of CH4 increased with sediment C/N and DIN and decreased with conductivity. The signif- icant unimodal relationship with DO indicates that the high- est observed CH4 concentrations occurred under both anoxic and supersaturated O2 environments (Fig. 4a), while low CH4 levels were seen when inflow was more composed of snowmelt or groundwater (depleted isotope values) and WRT was long (Fig. 4f). In contrast to the CO2 model, soil prop- erties and elevation were not significant drivers, yet local land use was significant, with crop sites having significantly higher CH4 compared to pastures. www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4217 Figure 3. Response patterns of farm reservoir CO2 concentrations with abiotic, biotic, hydromorphological, and landscape variables based on GAMs. CO2 was best estimated by a combination of (a) DO saturation, (b) alkalinity, (c) NOx, (d) buoyancy frequency, (e) interaction between δI and WRT, (f) soil CEC, (g) and elevation, with soil salinity (non-saline, N; weakly saline, W; moderately saline, M; strongly saline, S) (h) and land use (i) not significant. Model deviance explained was 66.5 %. The response patterns shown are the partial effect splines from the GAM (solid line), and the shaded area indicates 95 % credible intervals. See Table S4 and Fig. S2 for summary of model statistics and model fit with observed data. Figure 3. Response patterns of farm reservoir CO2 concentrations with abiotic, biotic, hydromorphological, and landscape variables based on GAMs. CO2 was best estimated by a combination of (a) DO saturation, (b) alkalinity, (c) NOx, (d) buoyancy frequency, (e) interaction between δI and WRT, (f) soil CEC, (g) and elevation, with soil salinity (non-saline, N; weakly saline, W; moderately saline, M; strongly saline, S) (h) and land use (i) not significant. Model deviance explained was 66.5 %. The response patterns shown are the partial effect splines from the GAM (solid line), and the shaded area indicates 95 % credible intervals. See Table S4 and Fig. S2 for summary of model statistics and model fit with observed data. The detailed GAM showed that variance in CO2 concen- trations among farm reservoirs was estimated (66.5 % of deviance) by a combination of predictors related to water- column productivity and microbial metabolism (DO satura- tion, alkalinity, NOx), thermal stratification (buoyancy fre- quency), basin hydrology (the interaction between δI and WRT), and landscape features (soil CEC, elevation) (Fig. 3) but not local soil salinity. This pattern was shown by the DO, alkalinity, δI, and WRT covariates having the most signifi- cant effect at p < 0.001, while CO2 concentrations did not vary significantly between different soil salinity levels (Ta- ble S4, Fig. 3). drivers in detail and from our evidence suggest management strategies that may help reduce the net GHG effect of these farm reservoirs. 4 Discussion Our comprehensive spatial analysis revealed wide variations among CO2 and CH4 concentrations between farm reser- voirs (Fig. 2). Significant modelled environmental drivers suggested CO2 was primarily controlled by pH, with strong independent models indicating mechanisms associated with primary productivity, the hydrological regime, and landscape elevation. In contrast, CH4 was most correlated with internal abiotic and biotic mechanisms. We discuss these potential www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4.1 Environmental drivers of CO2 concentrations Model deviance explained was 74.1 %. The response patterns shown are the partial effect splines from the GAM (solid line), and the shaded area indicates 95 % credible intervals. See Table S5 and Fig. S3 for summary of model statistics and model fit with observed data. Figure 4. Response patterns of farm reservoir CH4 concentrations with abiotic, biotic, hydromorphological, and landscape variables based on generalized additive models (GAMs). CH4 was explained by a combination of (a) DO saturation, (b) sediment C/N, (c) DIN, (d) conductivity, (e) buoyancy frequency (not significant), (f) interaction between δI and WRT, (g) soil Ksat (not significant), (h) elevation (not significant), and (i) local land use. Model deviance explained was 74.1 %. The response patterns shown are the partial effect splines from the GAM (solid line), and the shaded area indicates 95 % credible intervals. See Table S5 and Fig. S3 for summary of model statistics and model fit with observed data. ies (Ollivier et al., 2019; Peacock et al., 2019) and regional prairie lakes (Wiik et al., 2018). In some lakes, high N loading favoured elevated heterotrophy, despite simultane- ous boosts in primary production, which draw down free CO2 (Huttunen et al., 2003; Cole et al., 2000). The effect of a high N influx on CO2 may be heightened in smaller or shallow lentic waters which are more influenced by sedimentary pro- cesses (Torgersen and Branco, 2008). Further, high N avail- ability can increase algal biomass and the deposition of fresh organic matter (OM) made increasingly available for bacte- rial respiration (Cole et al., 2000). As a result, the effect of increased benthic respiration offsets CO2 uptake by primary producers, while extremely high influx of dissolved N can also favour microbial processes such as denitrification which increase CO2 evolution (Bogard et al., 2017). natively, alkalinity buffering can mediate the effect of NEP on CO2 concentrations at both extreme ranges of the DO spectrum (Marcé et al., 2015). Alkalinity buffering is most likely to affect CO2–DO relationships in waters where alka- linity is > 2000 µeq L−1 (Stets et al., 2017), which was the case for ∼90 % of our sites (Table 1; Fig. 3). g Stratification can also weaken the impact of DO as a driver for CO2 by regulating the effect of sediment respiration on epilimnetic chemistry (Huotari et al., 2009; Holgerson, 2015). 4.1 Environmental drivers of CO2 concentrations As seen in other hard-water ecosystems, variations in CO2 were strongly coupled to differences among sites in water- column pH (Finlay et al., 2015; Müller et al., 2016). We demonstrate this relation with the strong correlation observed between CO2 and pH in a separate GAM of only water pH as a covariate, explaining 86.3 % of deviance (Fig. S1). As ex- pected, the role of pH in regulating CO2 content is most pro- nounced at values between 8.6 and 9.0, the transition point where the predominant species of DIC shifts from free CO2 to HCO− 3 (Duarte et al., 2008; Finlay et al., 2015). Above this value, carbonate buffering increasingly regulates pH and restricts CO2 to only trace fractions of total DIC (Stumm and Morgan, 1970). However, direct changes in CO2 con- centrations can also alter water-column pH, such as biolog- ical metabolism (Talling, 2010). Therefore, given the direct chemical relationship between pH and CO2 concentrations (Stumm and Morgan, 1970), we opted to leave pH out of our model to further investigate the underlying biological, chem- ical, hydrological, and land use mechanisms. Carbon dioxide and dissolved oxygen are closely linked by biological metabolism in aquatic systems and diverge when other chemical or physical processes occur. Here, we see evidence for both linked and divergent processes (Fig. 3a). The tight linear relationship between CO2 and O2 at 25 % to 100 % saturation indicates close coupling between the gases. This likely represents control via metabolic processes such as net ecosystem production (NEP) or chemical oxidation of reduced species (Stets et al., 2017). In contrast, relation- ships between CO2 and O2 were less well defined a both high and low oxygen saturations, conditions which may in- dicate a greater contribution from anaerobic production of CO2 (Torgersen and Branco, 2008; Holgerson, 2015). Alter- www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4218 Figure 4. Response patterns of farm reservoir CH4 concentrations with abiotic, biotic, hydromorphological, and landscape variables based on generalized additive models (GAMs). CH4 was explained by a combination of (a) DO saturation, (b) sediment C/N, (c) DIN, (d) conductivity, (e) buoyancy frequency (not significant), (f) interaction between δI and WRT, (g) soil Ksat (not significant), (h) elevation (not significant), and (i) local land use. J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs ductivity) had the most significant effect (p < 0.01), while some of the broader landscape features such as soil Ksat and elevation had no significant effect on CH4 levels. The nutri- ent status of waterbodies is often a primary driver of high CH4 emissions in lakes, impoundments, and ponds (Deemer et al., 2016; Beaulieu et al., 2019; Peacock et al., 2019). Con- sequently, high nutrient availability is likely fuelling elevated values in both O2 saturation and CH4 (Fig. 4a). High CH4 concentrations at low O2 saturation reflect the development of anoxic habitats which favour methanogenesis (Huttunen et al., 2003; Bastviken et al., 2004). This is likely the result of rapid biomass production which both enriches epilimnion with O2 and depletes O2 in the hypolimnion by providing fresh labile organic matter for decomposition. lutes (Junger et al., 2019), and have higher biotic assimilation of nutrients (Devito and Dillon, 1993; Fairchild and Velinsky, 2006). Larger waterbodies may also be able to better medi- ate stream or groundwater C inputs through longer chemi- cal processing times and transformations. For example, agri- cultural reservoirs with the highest WRTs tended to be hy- drologically closed systems (E/I > 1) and any watershed- derived DIC delivered from previous water sources is likely to be consumed by primary production, which encourages atmospheric CO2 uptake (Macrae et al., 2004). Addition- ally, smaller waterbodies with shorter WRT can support higher rates of internal CO2 production due to higher rates of allochthonous DOC mineralization (Weyhenmeyer et al., 2015; Vachon et al., 2017). Groundwater delivery of DIC-rich porewater is the most likely hydrological source resulting in CO2 enrichment of small farm reservoirs. This mechanism is also suggested by the observation that higher reservoir CO2 concentrations are predicted in high-CEC soils. Alkaline high-CEC soils re- tain more calcium ions within clay particles, which releases carbonates and bicarbonates into soil porewater (Kelley and Brown, 1934). Although regional snowmelt and groundwater have similar isotopic signatures (Pham et al., 2009; Jasechko et al., 2017), the positive correlation of CO2 with alkalinity suggests groundwater as the main source. Edaphic sources of inorganic carbon can result in farm waterbodies accumulat- ing dissolved CO2, bicarbonates, and carbonates, and there- fore alkalinity, from the surrounding soils via groundwater discharge (Miller et al., 1985). J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs Other studies have found strong evidence for groundwater inputs driving CO2 super- saturation in small lentic systems (Perkins et al., 2015; Pea- cock et al., 2019) and watershed-derived alkalinity driving CO2 supersaturation in lakes (Marcé et al., 2015). In support of eutrophication-driven CH4 production, our model indicated that high proportions of autochthonous or- ganic matter in sediments were associated with elevated con- centrations of CH4 (Fig. 4b). Overall, sedimentary C/N ra- tios were in the range (8.5 to 13.4) expected for both phy- toplankton and submerged macrophytes (Liu et al., 2018). This suggests that in situ rather than terrestrial organic mat- ter was likely the main source of C fuelling methanogenesis in these reservoirs, although increasing CH4 concentrations with C/N may also represent a larger contribution of terres- trial OM. Strong associations of labile autochthonous C and CH4 production in sediments (Due et al., 2010; Crowe et al., 2011) also suggests a direct link between eutrophication and CH4 production in small farm waterbodies. Thermal stratification of the water column did not signif- icantly influence surface CH4 concentrations in small farm reservoirs (Fig. 4e). This finding contrasts with observations from other small waterbodies where limited mixing favours CH4 accumulation (Kankaala et al., 2013). Although some small systems exhibit diurnal mixing patterns with turnover at night (Glaz et al., 2016), the wide range of buoyancy fre- quency values (0.00 to 0.16) suggests that at least some farm reservoirs are continuously stratified, particularly in deeper ponds (Kankaala et al., 2013), as noted for CO2 distributions (see above and Fig. 3d). Taken together, our findings suggest that variability in the biological production of CH4 likely ex- erts a stronger influence over CH4 concentrations across farm reservoirs than does physical mixing, and this further sup- ports the hypothesis that the prevailing sediment and water chemistry are the primary controls of CH4 concentrations. Finally, landscape elevation had a significant external ef- fect on reservoir CO2 and may represent diverse weak con- trols related to landscape setting. Lower CO2 concentrations at higher elevations are common in perched ecosystems with smaller contributing catchment areas (Diem et al., 2012) and low rates of allochthonous carbon influx (Rose et al., 2015). Conversely, waterbodies low in the landscape may receive more watershed C via groundwater influx due to topograph- ical gradient (Winter and LaBaugh, 2003; van der Kamp and Hayashi, 2009). J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs The effect of elevation could also be related to changes in vegetation composition within the lo- cal landscape, with the lowest lying catchments exhibiting higher abundance of marginal wetland vegetation (Zhang et al., 2010), which favours higher inputs of terrestrial C (Mag- nuson et al., 2006; Abril et al., 2014). Although the hydrological regime of small water bod- ies is rarely measured, we find that water source (rain, snow/groundwater) and reservoir retention time interact to influence CH4 concentrations (Fig. 4f). In particular, CH4 concentrations were lowest when WRT was long (> 1 year) and water was derived mainly from snow or groundwater sources (δ18O depleted). This may be due to a combina- tion of reasons, including the prevalence of sulfate delivered from groundwater (Pennock et al., 2010), dilution of water- body from snowmelt inflow, and sediments depleted in la- bile carbon due to longer biogeochemical processing times 4.1 Environmental drivers of CO2 concentrations Our model shows that those sites that were most strat- ified (elevated buoyancy frequency) exhibited higher CO2 concentrations (Fig. 3d). This pattern contrasts those ob- served in other small lentic systems where elevated epil- imnetic CO2 concentrations were observed during and af- ter the breakdown of water-column stratification (Huotari et al., 2009; Glaz et al., 2016). Preliminary seasonal studies of some farm reservoirs in 2018 show that stratification is strong and persistent throughout the summer, with no ob- vious diurnal mixing events. Such strong stratification can maintain anoxic conditions throughout most of the water col- umn, which supports intense anaerobic respiration and CO2 production. Hydrological controls were found to be important regu- lators of CO2 concentrations in these farm reservoirs. Sites which received most of their inflow from snowmelt or groundwater, and which had short WRT, supported super- saturated CO2 concentrations (Fig. 3f). Such patterns may reflect increased inputs of groundwater which are typically supersaturated with CO2 (Macpherson, 2009). Long WRT is associated with larger, deeper systems. These sites are usu- ally less influenced by the terrestrial–aquatic interface, take longer to concentrate the effect of any catchment-derived so- The positive association between NOx and CO2 found in our reservoirs is consistent with similar patterns seen with dissolved inorganic N species in other artificial waterbod- www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4219 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs in the dams. The potential effect of sulfate limiting methano- genesis is in agreement with the strong negative relationship found between CH4 and conductivity in our model (Fig. 4d). Sulfate makes up a large portion of the ionic composition of groundwater in the Prairie Pothole Region due to pyrite oxidation (Goldhaber et al., 2014). Evidently, the biological influence on CH4 concentrations appears less pronounced in these larger, low-flow dams. CO2 sink behaviour, with most showing net heterotrophy (Fig. 5). Although other studies have noted CO2 sink be- haviour in artificial ponds and reservoirs (Peacock et al., 2019; Ollivier et al., 2019), this is the first study to cap- ture such a high proportion (> 52 %) of CO2 uptake in such systems, with negative fluxes estimated to range between −21 and −0.1 mmol m−2 d−1 (mean −12 mmol m−2 d−1) for CO2 (Table 1). These flux ranges compare to CO2 uptake of −1 to −11 mmol m−2 d−1 in agricultural eutrophic lakes of North America (Finlay et al., 2010; Pacheco et al., 2014). Studies have shown the importance of eutrophication, lead- ing to net autotrophy, in enhancing CO2 uptake and revers- ing carbon fluxes in lakes (Pacheco et al., 2014). However, a global analysis of GHG fluxes from lakes and reservoirs revealed that the consequence of increased CH4 emissions with increasing trophic status often outweighs the impact of negative CO2 fluxes (Deemer et al., 2016). Here, our model shows the potential importance of reservoir placement within the landscape as a way of reducing CO2 emissions via hy- drological and geochemical controls without the added con- sequence of increased CH4 emissions. In contrast to the external drivers found for CO2, local land use had a significant effect on CH4 concentrations in farm reservoirs (Fig. 4i), with significantly higher CH4 lev- els in cropland waterbodies than those in pasture. Catchment land use regulates the physico-chemical properties of ponds (Novikmec et al., 2016) by influencing the degree of local vegetative cover and associated influx of allochthonous C to waterbodies (Whitfield et al., 2011). Similarly, regions with crops undergo more intensive agricultural modification, with fertilization, crop rotations, and mechanical disturbance of soil, which all lead to greater nutrient runoff and soil erosion. J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs Our finding contrasts with those from Australian farm reser- voirs where diffusive CH4 fluxes were 250 % higher in reser- voirs with livestock compared to crops, although the mech- anisms responsible for observed differences were inconclu- sive (Ollivier et al., 2019). This difference could be the re- sult of the intensity of agricultural production, where farm reservoirs supporting high intensity grazing may also expe- rience high CH4 production as demonstrated by a couple of high CH4 concentrations observed in our livestock pasture reservoirs (Fig. 2). In this case it is likely that CH4 levels are more influenced by nutrient loading from the landscape which stimulates eutrophication (Huttunen et al., 2003), as suggested by the biotic variables in our model (Fig. 4). The intensity of agricultural production under different land use types should be an area of further exploration for external controls on farm reservoir GHG production. When CO2 and CH4 fluxes from small artificial waterbod- ies are compared with natural small waterbodies, no appar- ent trend exists in which group produces more or less car- bon emissions (Fig. 5). Natural ponds and constructed wa- terbodies have a similar range in variability of mean fluxes for both gases, while wetlands exhibit some of the greatest within-study variability. Constructed waterbodies often have lower net CO2 efflux, suggesting that these systems more of- ten switch between net autotrophy and heterotrophy than do small natural systems. Small artificial waterbodies have dis- proportionately higher CO2 and CH4 emissions than other natural waterbodies due to the direct impact of agricultural and urban land use (Wang et al., 2017). However, analysis of the limited literature shows that is not the case. We suggest that the lack of a clear distinction between constructed and naturally occurring small water bodies arises because of ge- ographical variation in the relative importance of the diverse factors regulating carbon metabolism (Figs. 3, 4). 4.2 Environmental drivers of CH4 concentrations The GAM suggested that CH4 concentrations were primarily related to internal biogeochemical processes and the influ- ence of the hydrological regime. For example, factors related to water-column productivity (DO, sediment C/N, DIN, con- www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 4220 4.3 Emissions from farm reservoirs compared to other small waterbodies If no mean value was reported, then the midpoint was inferred as the middle of range (dashed lines). Solid black line distinguished between positive and negative fluxes. All data are from the published literature and references can be found in the Table S6. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4221 Figure 5. Range of CO2 and CH4 (diffusive) fluxes observed in natural and constructed small (< 0.01 km2) waterbodies, including this study (farm reservoirs). Dots represent the mean reported in each study and error bars the range. If no mean value was reported, then the midpoint was inferred as the middle of range (dashed lines). Solid black line distinguished between positive and negative fluxes. All data are from the published literature and references can be found in the Table S6. Figure 6. Total average CO2 equivalent fluxes of CO2 and CH4 (diffusive) measured in natural and artificial small waterbodies (< 0.01 km2). CO2-e fluxes were calculated based on 100-year sustained-flux global warming potentials in Neubauer and Megoni- gal (2015). Relative proportions of each gas are indicated by shad- ing, and waterbody type is given by colour. All data are from the published literature and references can be found in the Table S6. ation represents monthly sampling between the periods of ice melt and ice formation on water bodies in Saskatchewan. Applying the average observed seasonal variation of 78 % and 93 % to our current spatial dataset suggests that CO2- e emissions from farm reservoirs may vary between −1.7 and 150 g CO2 m−2 d−1, or 0 % to 44 %, as acting net CO2- e sinks. Further study into the consistency of potential farm reservoir CO2 sinks on the temporal scale is required to bet- ter assess the overall GHG impact. Small natural ponds and wetlands have some of the high- est CO2-e emission rates, with particular importance of con- tributions from CH4 (Fig. 6). On average our farm reser- voirs had one of the highest CH4 contribution to CO2-e fluxes (74 %), in agreement with the one other farm reservoir study (83 %) of CH4 contribution (Ollivier et al., 2019). This large contribution from CH4 is similar to patterns recorded from lakes and impoundments globally, where large fresh- water bodies contribute to 75 % of all CO2-e efflux (DelSon- tro et al., 2018). 4.3 Emissions from farm reservoirs compared to other small waterbodies To date, small waterbodies on farms have been shown to be large emitters of both CO2 and CH4 (Fig. 5). However, in our study we show that this is not always the case. Diffu- sive fluxes varied −21 to 466 and 0.14 to 92 mmol m−2 d−1 for CO2 and CH4, respectively. These findings are consistent with other small artificial waterbodies which are strong CH4 sources that exhibit a large range of variability from 0.02 to 33 mmol m−2 d−1 (Grinham et al., 2018a; Ollivier et al., 2019). Average CH4 fluxes from our farm reservoirs corre- spond to 417 kg CH4 ha−1 yr−1, which is greater than the cur- rent IPCC emission factor estimate of 183 kg CH4 ha−1 yr−1 (IPCC, 2019). Considering the skewness of our CH4 data, our median value of 184 kg CH4 ha−1 yr−1 agrees with the emission factor of other artificial ponds. When assessing the GHG impact of constructed water- bodies, it is important to consider the relative contribution to CO2-equivalent (CO2-e) fluxes between CO2 and CH4. Here, CH4 fluxes were converted to CO2-e fluxes using the sustained-flux global warming potential over 100 years (Neubauer and Megonigal, 2015). On average, 8 % of farm reservoirs were acting as CO2-e sinks on the range of −0.6 to 79 g CO2 m−2 d−1 during the time of sampling. This num- ber offers a snapshot of the potential for farm reservoirs to act as a net CO2-e sink, and it is important to consider how sea- sonal variation influences the GHG sink/source status. Pre- liminary data on seasonal variation in CO2 and CH4 concen- trations from a smaller number of farm reservoirs indicate variation (represented as the standard deviation related to the mean) ranging between 20 % and 200 % and between 40 % and 200 % for CO2 and CH4, respectively. Here, this vari- The negative fluxes observed in our farm dams repre- sents one of the few studied small waterbodies that exhibit www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4221 Figure 5. Range of CO2 and CH4 (diffusive) fluxes observed in natural and constructed small (< 0.01 km2) waterbodies, including this study (farm reservoirs). Dots represent the mean reported in each study and error bars the range. 4.3 Emissions from farm reservoirs compared to other small waterbodies Fortunately, because the factors that regu- late CH4 emissions are becoming better identified (Fig. 4), there exists the possibility that artificial wetlands can be con- structed to minimize CH4-related CO2-e emissions and mit- igate the overall large rate of CO2-e emissions from agricul- ture (Robertson et al., 2000). Figure 6. Total average CO2 equivalent fluxes of CO2 and CH4 (diffusive) measured in natural and artificial small waterbodies (< 0.01 km2). CO2-e fluxes were calculated based on 100-year sustained-flux global warming potentials in Neubauer and Megoni- gal (2015). Relative proportions of each gas are indicated by shad- ing, and waterbody type is given by colour. All data are from the published literature and references can be found in the Table S6. gest that key variables including the degree of water-column stratification (buoyancy frequency), WRT, water source, land use, and elevation are all suitable parameters for manage- ment – for example, strategizing landscape positioning to favour groundwater influx of sulfate to reduce methano- genesis. Increasing WRT by creating deeper reservoirs may promote primary production through increased water clarity (Dirnberger and Weinberger, 2005), facilitate CH4 oxidation 5 Conclusion Until recently, carbon emissions from small farm reservoirs have been an overlooked yet potentially important source of CO2 and CH4 emissions within agricultural carbon bud- gets. To date, development of management strategies to re- duce GHG emissions from waterbodies has been limited by the lack of knowledge about the mechanisms regulating CO2 and CH4 production in these systems. By utilizing adaptive modelling techniques across a broad range of environmen- tal variables (abiotic, biotic, hydromorphological, landscape properties), we were able to explain a high degree of de- viance in reservoir CO2 and CH4 concentrations. We found that in situ water chemistry and local hydrological regime had the strongest impact on CO2 and CH4 concentrations. In agreement with previous studies, CH4 fluxes were the largest contributor to CO2-e emissions. However, in 19 reservoirs the net CO2-e emissions were found to be sinks. We suggest that, with optimal reservoir design and management, the cli- matic impact of farm reservoir C-emissions has the potential to be a carbon net sink. To further develop farm reservoir management practices that are locally effective, we express a need for more widespread farm waterbody GHG measure- ments across the globe to cover other continents and land uses. Nitrogen loading can also have a direct influence on ni- trous oxide (N2O), the third most potent greenhouse gas that can contribute substantially to CO2-e emissions in farm sys- tems (Robertson et al., 2000). The flux of N2O was con- strained in our earlier study (Webb et al., 2019), which found a small CO2-e sink (−89 to −3 mg CO2 m−2 d−1) for the ma- jority of these farm reservoirs despite high N concentrations. Similar to our CO2 model, stratification and primary produc- tion were important regulators in driving N2O uptake (Webb et al., 2019). Therefore, the potential to achieve net GHG sinks weighs mostly on the ability to reduce CH4 emissions in these systems. Studies have also shown the importance of emergent veg- etation plant species in sequestering carbon in sediments. Emergent vegetation was found to contribute significantly to the soil carbon pool of stormwater ponds compared to al- lochthonous sources (Moore and Hunt, 2012). However, in our CH4 model, the significant effect of sediment C : N ra- tios suggested that an autochthonous organic matter source from either phytoplankton or submerged macrophytes sup- ports greater CH4 production in farm reservoirs. J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs through the water column (Bastviken et al., 2008), and re- duce the impact of watershed-derived solutes, terrestrial OM, and benthic respiration. Additionally, deeper and larger arti- ficial waterbodies tend to have lower nutrient concentrations due to longer processing times (Chiandet and Xenopoulos, 2016). Finally, modest increases in pH may further enhance CO2 capture (Supplement), while having limited effect on CH4 fluxes (Fig. 4). (Joyce and Jewell, 2003; DelSontro et al., 2016). This rein- forces that design and management strategies that focus on reducing all pathways of CH4 emissions will be most ef- fective in curbing total CO2-e emissions. Deeper farm dams with steep side slopes will likely be effective in reducing ebullition events due to a limited macrophytes, reduced bot- tom water temperature in summer, and suppressed bubble release with higher water pressure (Joyce and Jewell, 2003; Natchimuthu et al., 2014; Grinham et al., 2018b). Agricultural and urban waterbodies are highly susceptible to nutrient enrichment due to their direct proximity to intensi- fied land uses. Reducing nutrient loading from the landscape will likely have one of the greatest impacts in minimizing C emissions from farm dams given that both CO2 and CH4 were strongly predicted by inorganic N species. In Australian farm reservoirs, for example, a 25 % reduction of nitrates can reduce CO2-e emissions by 50 % (Ollivier et al., 2019). Sim- ilarly, removing direct livestock access to farm waterbodies will improve water quality overall through reducing direct DIN inputs and dam infilling. 5 Conclusion The abil- ity of farm reservoirs to have a negative climate forcing will rely on the balance between GHG fluxes and sediment carbon accumulation. The effect different plant species and other aquatic primary producers have on both these processes needs to be evaluated in future studies as the current design of farm dams within the study area minimizes growth of emer- gent vegetation through steep sides and slopes. Data availability. All data used in the models are freely available on GitHub (https://github.com/JackieRWebb/Dugouts-CO2-CH4, last access: 31 October 2019) and Zenodo (https://doi.org/10.5281/ zenodo.3475628; Webb and Simpson, 2019). Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/bg-16-4211-2019-supplement. Author contributions. JRW, GLS, PRL, HMB, and KF designed the research; JRW performed the research and wrote the paper; HMB contributed the new reagents/analytic tools and managed gas anal- yses; HAH, PRL, GLS, and KF contributed towards ideas and data analysis; KRH performed the GIS analysis; and GLS developed the models. All authors edited and approved the paper. It is important to note that the CH4 contribution to CO2-e emissions is likely underestimated here as ebullition emis- sions were not measured. In farm reservoirs, ebullition flux can contribute > 90 % of total CH4 emissions and is often highest in the smallest size classes (Grinham et al., 2018a). However, the sporadic nature of this pathway remains diffi- cult to constrain for one single type of waterbody and may be a minor contributor in reservoirs and ponds > 3–5 m deep 4.4 Minimizing emissions: potential management solutions A combination of factors, including landscape position, con- struction, and management, could optimize features to min- imize carbon emissions from reservoirs and potentially en- hance the carbon storage on farms. From our models, we sug- www.biogeosciences.net/16/4211/2019/ Biogeosciences, 16, 4211–4227, 2019 J. R. Webb et al.: Regulation of carbon dioxide and methane in small agricultural reservoirs 4222 References Clifford, C. and Heffernan, J.: Artificial Aquatic Ecosystems, Water, 10, 1096, https://doi.org/10.3390/w10081096, 2018. Abril, G., Martinez, J.-M., Artigas, L. F., Moreira-Turcq, P., Benedetti, M. F., Vidal, L., Meziane, T., Kim, J.-H., Bernardes, M. 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SNX10 and PTGDS are Associated with the Progression and Prognosis of Cervical Squamous Cell Carcinoma

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SNX10 and PTGDS are Associated with the Progression and Prognosis of Cervical Squamous Cell Carcinoma Pinping Jiang  Nanjing Medical University Ying Cao  Nanjing Medical University Feng Gao  Nanjing Medical University Wei Sun  Nanjing Medical University Jinhui Liu  Nanjing Medical University Ziyan Ma  University of New South Wales Manxin Xie  Nanjing Medical University Shilong Fu  (  [email protected] ) Jiangsu Province Hospital and Nanjing Med https://orcid.org/0000-0001-7416-3070 Abstract Background: Cervical cancer (CC) is the primary cause of death in women. This study sought to investigate the potential mechanism and prognostic genes of CC. Methods: We downloaded four gene expression profiles from GEO. The RRA method was used to integrate and screen differentially expressed genes (DEGs) between CC and normal samples. Functional analysis was performed by clusterprofiler. We built PPI network by Search Tool for the Retrieval of Interacting Genes Database (STRING) and selected hub modules via Molecular COmplex Detection (MCODE). CMap database was used to find molecules with therapeutic potential for CC. The hub genes were validated in GEO datasets, Gene Expession Profiling Interactive Analysis (GEPIA), immunohistochemistry, Cox regression analysis, TCGA methylation analysis and ONCOMINE were carried out. ROC curve analysis and GSEA were also performed to describe the prognostic significance of hub genes. Methods: We downloaded four gene expression profiles from GEO. The RRA method was used to integrate and screen differentially expressed genes (DEGs) between CC and normal samples. Functional analysis was performed by clusterprofiler. We built PPI network by Search Tool for the Retrieval of Interacting Genes Database (STRING) and selected hub modules via Molecular COmplex Detection (MCODE). CMap database was used to find molecules with therapeutic potential for CC. The hub genes were validated in GEO datasets, Gene Expession Profiling Interactive Analysis (GEPIA), immunohistochemistry, Cox regression analysis, TCGA methylation analysis and ONCOMINE were carried out. ROC curve analysis and GSEA were also performed to describe the prognostic significance of hub genes Results: Functional analysis revealed that 147 DEGs were significantly enriched in binding, cell proliferation, transcriptional activity and cell cycle regulation. PPI network screened 30 hub genes, with CDK1 having the strongest connectivity with CC. Cmap showed that apigenin, thioguanine and trichostatin A might be used to treat CC(P<0.05). Eight genes (APOD, CXCL8, MMP1, MMP3, PLOD2, PTGDS, SNX10 and SPP1) were screened out through GEPIA. Of them, only PTGDS and SNX10 had not appeared in previous studies about CC. The validation in GEO showed that PTGDS showed low expression in tumor tissues while SNX10 showed high expression in tumor tissues. Their expression profiles were consistent with the results in immunohistochemistry. ROC curve analysis indicated that the model had a good diagnostic efficiency(AUC=0.738). GSEA showed that the two genes were associated with the chemokine signaling pathway(P<0.05). Abstract TCGA methylation analysis showed that patients with lowly-expressed and highly-methylated PTGDS had a worse prognosis than those with highly-expressed and lowly-methylated PTGDS (p=0.037). Cox regression analysis showed that SNX10 and PTGDS were independent prognostic indicators for OS among CC patients(P=0.007 and 0.003). Conclusions: PTGDS and SNX10 showed abnormal expression and methylation in CC. Both genes might have high prognostic value of CC patients. Research article Keywords: Cervical squamous cell carcinoma, methylation, prognosis, risk model, SNX10, PTGDS Posted Date: January 12th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-50778/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: January 12th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/26 Page 1/26 2.3 PPI network integration Search Tool for the Retrieval of Interacting Genes Database (STRING)[11] (http://www.string-db.org/) was used to assess PPI complex between identified and predicted proteins. In addition, the plug-in MCODE [12] of Cytoscape was conducted to select and visualize hub modules in PPI complex. 1. Background An annual death toll of 265,700 makes cervical cancer (CC) the second deadliest malignancy in females [1]. Despite pre-cancerous screening and emerging treatments, CC remains the primary cause of death in women in developing countries [2]. When CC metastasizes and recurs, the prognosis gets even worse. Therefore, it is of great significance to create new treatments of CC based on its to-be-clarified molecular mechanism. Gene expression microarray, as an efficient means of acquiring large-scale genetic data, is being widely used to study gene expression profiling in many human cancers. Upon microarray and databases, Page 2/26 effective analytic tools have been designed to explore tumor-associated genes, molecular mechanisms and target therapies . The integration of databases containing gene expression chips allows in-depth study of molecular mechanisms[3, 4]. effective analytic tools have been designed to explore tumor-associated genes, molecular mechanisms and target therapies . The integration of databases containing gene expression chips allows in-depth study of molecular mechanisms[3, 4]. Thousands of differentially expressed genes (DEGs) in CC have been discovered [5-7]. However, the results on some mRNAs are inconsistent. Here we use an unbiased approach to solve this problem. In our study, we screened DEGs from four profiles downloaded from GEO. PPI network was built by STRING Database and hub modules selected via plug-in MCODE. CMap was used to find potential genes associated with CC. We also validated hub genes with GEO, GEPIA, immunohistochemistry and ONCOMINE. ROC curve analysis and GESA were also done to tease out the significance of hub genes. The flow chart of this research was displayed in Figure 1. 2.1 Screening DEGs Keywords “cervical cancer geo accession” were put in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) and the gene expression profiles of GSE6791, GSE63514, GSE39001 and GSE9750 were downloaded. The dataset information is shown in Table 1. We processed unqualified data by R package. The data is calibrated, standardized and log2-transformed. Gene expression analysis was performed using the limma[8] package in the Bioconductor package. Relevant codes were placed into R. We selected four microarray datasets and analyzed them with limma. The |log2fold change (FC)| > 2 and adjusted p < 0.05 were set as cutoffs. RRA package was download (http://cran.r-project.org/) [9] and R was used for running the instruction code. 2.2 Functional analysis based on DEGs The biological significance of DEGs was analyzed with DAVID (https://david.ncifcrf.gov/) database and clusterprofiler[10] (a package visualizing the biological profiles of genes). P < 0.05 was considered to be statistically significant. 2.6 Validation of key genes We used GEPIA[14] (Gene Expression Profiling Interactive Analysis) to analyze the expression and prognostic significance of DEGs. After reviewing literature, we screened real hub genes from DEGs. Subsequently the real hub genes were validated into GEO datasets, including GSE7803 and GSE29576, and ONCOMINE database (www.oncomine.org). GSE7803 included 21 CC tissue samples and 10 normal tissue samples. GSE29576 included 45 CC tissue samples and 17 normal tissue samples. ONCOMINE[15] dataset is a publicly accessible online cancer microarray database that enables online analysis on relations between a candidate gene and various tumors according to DNA and RNA sequence data. The Human Protein Atlas (HPA) (http://www.proteinatlas.org/) was also used to validate the expression of the real hub genes. ROC curve analysis was performed to distinguish normal and cancer tissues. We used GEPIA[14] (Gene Expression Profiling Interactive Analysis) to prognostic significance of DEGs. After reviewing literature, we screen Subsequently the real hub genes were validated into GEO datasets, in and ONCOMINE database (www.oncomine.org). GSE7803 included 2 tissue samples. GSE29576 included 45 CC tissue samples and 17 no samples. ONCOMINE[15] dataset is a publicly accessible online canc enables online analysis on relations between a candidate gene and v RNA sequence data. The Human Protein Atlas (HPA) (http://www.pro validate the expression of the real hub genes. ROC curve analysis wa and cancer tissues. 2.5 Construction of a prognostic signature Univariate Cox proportional hazard regression analysis was performed based on DEGs. The genes associated with prognosis were defined using the cutoff value of p < 0.05. Next, a multivariate Cox regression model was constructed with genes of p < 0.01. Cox regression with p < 0.05 was constructed to estimate the risk score of each patient on the expression of the DEGs. To further screen out prognosis- related genes of CC, we constructed a linear risk model. The prognostic score = expRNA1×βRNA1+expRNA2×βRNA2+expRNA3×βRNA3+...expRNAn×βRNAn (expRNA is the expression level of each methylation-driven gene, and βRNA is the regression coefficient calculated by the multivariate Cox regression analysis). The prognostic risk value of each sample was calculated according to the formula, and then the median of the index value was cut off. Patients were divided into a low- and high-risk group according to their mean scores of prognostic risks. Kaplan-Meier survival analysis was conducted based on the low- and high-risk group. We also performed ROC curve analysis using five years as the predicted time to assess the predictive value of the outcome. The areas under the ROC curve, sensitivity and specificity were used to describe predictive values. Univariate Cox proportional hazard regression analysis was performed based on DEGs. The genes associated with prognosis were defined using the cutoff value of p < 0.05. Next, a multivariate Cox regression model was constructed with genes of p < 0.01. Cox regression with p < 0.05 was constructed to estimate the risk score of each patient on the expression of the DEGs. To further screen out prognosis- related genes of CC, we constructed a linear risk model. The prognostic score = g p g expRNA1×βRNA1+expRNA2×βRNA2+expRNA3×βRNA3+...expRNAn×βRNAn (expRNA is the expression level of each methylation-driven gene, and βRNA is the regression coefficient calculated by the multivariate Cox regression analysis). The prognostic risk value of each sample was calculated according to the formula, and then the median of the index value was cut off. Patients were divided into a low- and high-risk group according to their mean scores of prognostic risks. Kaplan-Meier survival analysis was conducted based on the low- and high-risk group. We also performed ROC curve analysis using five years as the predicted time to assess the predictive value of the outcome. The areas under the ROC curve, sensitivity and specificity were used to describe predictive values. 2.4 Identification of potential drugs CMap[13] is a computer simulation method for predicting the potential drug that may induce or reverse a biological state encoded by the gene expression signature. The different probe components commonly between CC and normal samples were screened out with CMap database and divided into the up- and Page 3/26 down-regulated groups. An enrichment score representing similarity was calculated. The positive score illustrated that the drug could induce cancer in human; the negative score illustrated the drug function oppositely and had potential therapeutic value. 2.7 Survival analysis and mapping of methylation level Survival analysis on gene methylation and expression was conducted through R package to identify key prognosis-associated genes in CC. To explore the relation between aberrant methylation and expression of genes, we extracted key genes with methylated expression from the downloaded data on TCGA CESC methylation. Then we evaluated the association between the methylated expression and the gene expression. 2.8 Gene set enrichment analysis (GSEA) Page 4/26 Based on the hub gene expression level, the samples were divided into two groups. In order to study the potential function of the DEGs. GSEA[16] (http://software.broadinstitute.org/gsea/index.jsp) was used to research a series of prior defined biological pathways which might be enriched in the gene rank derived from hub gene between the two groups. Annotated gene set of c2.cp.kegg.v6.0.symbols.gmt in Molecular Signatures Database (MSigDB, http://software.broadinstitute.org/gsea/msigdb/index.jsp) was selected as the reference. Additionally, we used “Clusterprofiler” package in R to handle the datasets, and the “Enrichplot” package to tease out the enriched pathways of the key genes. The adjusted-P< 0.05 was set as significant. 3.1 Identification of DEGs in CC The CC expression microarray datasets (GSE6791, GSE9750, GSE39001 and GSE63514) were firstly standardized (Figure S1). With limma package, 256 DEGs were filtered from GSE6791 (60 downregulated and 196 upregulated), 236 DEGs from GSE9750 (136 downregulated and 100 upregulated), 98 DEGs from GSE39001 (38 upregulated and 60 downregulated), 489 DEGs from GSE63514 (177 upregulated and 312 downregulated). DEGs from the 4 microarray datasets were exhibited in volcano maps (Figure S2A-D) and heatmaps (Figure S3A-D). We analyzed the four microarray datasets via the limma package and then with RRA method according to their log-folding variation values ((|log2fold change (FC)| > 1 and adj. p <0.05). The RRA method was based on the theory that genes in each experiment were randomly ordered. For the genes ranking higher in the experiment, the possibility of differential expression is inversely proportional to the value of P. Through analytic hierarchy analysis, we sorted out 74 up- regulated genes, and 73 down-regulated genes (Table 2). Finally, the R-heatmap software was performed to plot the top 40 up- and down-regulated genes (Figure 2). The CC expression microarray datasets (GSE6791, GSE9750, GSE39 standardized (Figure S1). With limma package, 256 DEGs were filtere and 196 upregulated), 236 DEGs from GSE9750 (136 downregulated from GSE39001 (38 upregulated and 60 downregulated), 489 DEGs f and 312 downregulated). DEGs from the 4 microarray datasets were S2A-D) and heatmaps (Figure S3A-D). We analyzed the four microar and then with RRA method according to their log-folding variation va adj. p <0.05). The RRA method was based on the theory that genes in ordered. For the genes ranking higher in the experiment, the possibilit inversely proportional to the value of P. Through analytic hierarchy a regulated genes, and 73 down-regulated genes (Table 2). Finally, the to plot the top 40 up- and down-regulated genes (Figure 2). 3.2 Functional analysis of DEGs The biological annotations of DEGs in CC were obtained with an online analysis tool named DAVID, which had GO analysis of up- and down-regulated genes (P＜0.05). The GO analysis of DEGs covered three aspects: molecular function, biological processes and cellular composition (Figure S4A). The upregulated genes were significantly enriched in microtubule binding, tubulin binding and ATPase activity (Figure 3A), and the down-regulated genes in serine-type peptidase activity, serine hydrolase activity and serine-type endopeptidase (Figure 3B). These results indicated that most DEGs were prominently enriched in structural molecule activity, midbody, kinesin complex and microtubule motor activity. (Figure S4 B-C and Figure S5A). Clusterprofile was performed to analyze the DEGs. The result showed that the upregulated genes were mostly enriched in DNA replication, Oocyte meiosis and Cell cycle. (Figure 3C), and the down- regulated genes in Arachidonic acid metabolism, prostate cancer and signaling pathways regulating pluripotency of stem cells (Figure 3D). The pathway-gene network (Figure S5B) suggested that the cell cycle was the most important term in the biological processes of CC. 3.3 PPI network construction and modules selection 3.3 PPI network construction and modules selection Page 5/26 The PPI network of DEGs was constructed, including 147 nodes (74 up-regulated genes and 73 down- regulated genes) and 562 edges (Figure 4A). Degrees ≥30 was set as the cutoff. A total of 16 genes, such as CDK1, TOP2A, NCAPG, and KIF11, were showed the most significant difference in expression (Figure 4B). A significant module was selected with plug-in MCODE, including 27 nodes and 343 edges (Figure S6A). GO and KEGG pathway enrichment analysis indicated that the genes in this module were mainly related to microtubule binding, tubulin binding, cell cycle and oocyte mitosis (Figure S6B and 6C). The PPI network of DEGs was constructed, including 147 nodes (74 up-regulated genes and 73 down- regulated genes) and 562 edges (Figure 4A). Degrees ≥30 was set as the cutoff. A total of 16 genes, such as CDK1, TOP2A, NCAPG, and KIF11, were showed the most significant difference in expression (Figure 4B). A significant module was selected with plug-in MCODE, including 27 nodes and 343 edges (Figure S6A). GO and KEGG pathway enrichment analysis indicated that the genes in this module were mainly related to microtubule binding, tubulin binding, cell cycle and oocyte mitosis (Figure S6B and 6C). 3.4 Small molecule drugs screening CMap network was used to analyze 147 differentially expressed genes into two groups (74 in up- regulated group and 73 in down-regulated group). After the signature query, the three compounds with the highest negative enrichment score (apigenin, thioguanine, and trichostatin A) were identified as potential therapeutic agents for CC (Table 3). The three-dimensional chemical structure of these three compounds is shown in Figure 5. 3.5 Validation of hub genes We validated DEGs at GEPIA website, including survival analysis and tissue sample expression analysis (Figure S7 and Figure S8). Eight genes (APOD, CXCL8, MMP1, MMP3, PLOD2, PTGDS, SNX10 and SPP1) had the same trend in both the above analysis. We literature-reviewed these eight genes, finding that only PTGDS and SNX10 had not been reported to be associated with CC. Therefore, we used GSE7803 and GSE29576 to validate PTGDS and SNX10 (Figure S9). The results showed that PTGDS had high expression levels in normal tissues and low expression levels in tumor tissues, while SNX10 showed an opposite profile. We further validated the two genes using immunohistochemistry (Figure 6A-B) and ONCOMINE, obtaining the results consistent with those from the GEO database (Figure 6C-D). The area under the curve of PTGDS was 0.919 and that of PTGDS was 0.905, suggesting that both can distinguish CC and normal tissue and have a good diagnostic efficiency (Figure 7A). GSEA was performed to search KEGG pathways enriched in the TCGA samples. The top ten most enriched pathways included “hematopoietic cell lineage”, “adhesion molecules cams”, “vascular smooth muscle contraction”, “systemic lupus erythematosus”, “chemokine signaling pathway”, “t cell receptor signaling pathway”, “cytokine cytokine receptor interaction”, “calcium signaling pathway”, “neuroactive ligand receptor interaction” and “leukocyte transendothelial migration” (Figure 7B) (adj.p < 0.05). In addition, the univariate and multivariate Cox proportional hazards regression analyses showed that SNX10 and PTGDS were independent prognostic indicators for OS among CESC patients (P=0.007 and 0.003)(Table 4). To find out the mechanism of abnormal gene expression, we analyzed the gene expression level and methylation level from the Illumina Human Methylation 450 platform based on TCGA data. The association between the methylated expression and the gene expression of the two key driving genes were shown in Figure7C-D. The survival analysis showed that the patients with low-expressed and hyper- methylated PTGDS had a worse prognosis than those with high-expressed and hypo-methylated Page 6/26 PTGDS(P=0.037) (Figure 7E). However, SNX10 methylation has no statistical significance in survival analysis. 3.6 Establishment of Cox regression model Univariate cox regression analysis screened out seven genes, including APOD, CXCL8, MMP1, MMP3, PLOD2, PTGDS and SPP1 (Figure S10). Multivariate Cox regression analysis screened five genes, including SPP1, CXCL8, PTGDS, PLOD2 and MMP3 (Figure S11). The score for predicting overall survival risk was calculated as followed: Risk score= 0.143* SPP1+0.136* CXCL8-0.093* PTGDS+0.206* PLOD2+0.067* MMP3. Based on the risk score, CC patients were divided into low- and high-risk groups. Kaplan-Meier survival analysis suggested that low-risk patients had better overall survival than high-risk patients in the TCGA cohort (Figure 8A). ROC curve analysis was also completed according to the 5-year survival of the area under the receiver operating characteristic curve (AUC) value. The specificity and sensitivity were both highest when the risk score was 0.738 (Figure 8B). The distribution of risk score, survival status, and the expression levels of five genes was also analyzed (Figure 8C-F). The expression levels of five genes in low- and high-risk groups were shown in Figure S12A. The univariate and multivariate Cox proportional hazards regression analyses showed that only the risk score based on five genes was independent prognostic indictor of CC (Figure S12B-C). The Heatmap showed the expression levels of the five genes in high- and low-risk patients in the TCGA dataset. We observed significant difference in survival state (P < 0.001) and stage (P < 0.05) (Figure S 12D). 4. Discussion CC brings on more than 265,700 deaths per year, making it the second deadlist malignancy in women. Nowadays, microarray and high-throughput sequencing technology are used to identify the potential targets in CC treatment. Previous studies often establish a single group or have a small-size, which restricts their reliability. This study analyzed the expression profiles of four genes using R software and bioinformatics tools. A total of 147 differential genes were identified using RRA analysis, including 73 downregulated and 74 upregulated. GO analysis indicated that the upregulated DEGs were primarily associated with microtubule binding and the downregulated DEGs with serine-type peptidase activity. KEGG pathway analysis indicated that these DEGs were primarily enriched in the cell cycle. Our findings echo with the previous studies. It has been reported that microtubule binding and cell cycle have an effect on CC[17]. Other studies showed that microtubule binding played a role in the biology of acute myeloid leukemia cells and colorectal cancer cells[18, 19]. Cell cycle also decides the abnormal proliferation of many tumor cells[20, 21]. Page 7/26 Page 7/26 PPI network displayed 30 hub genes associated with CC associated proteins. Next, we found CDK1 was in the center of PPI network of CESC. CDK1 was harbored by top module 1, suggesting that the top module 1 plays a crucial role in CC pathogenesis. Functional analysis indicated that the genes in this module were mainly enriched in microtubule binding, tubulin binding and cell cycle. CDK1 is a serine/threonine kinases that regulates the cell cycle by interacting with specific cyclins[22]. It has been reported that CDK1 regulated the development of CC and many other tumors[23]. Y. Zeng et al. found that Cyclin-dependent kinase 1 (CDK1)-mediated mitotic phosphorylation of the transcriptional co-repressor Vgll4 was mediated by CDK1 to its tumor-suppressing activity. K. Bednarek et al. found that CDK1 was involved in the processes of laryngeal squamous cell carcinoma [24, 25]. Cmap showed that apigenin, thioguanine and trichostatin A could be used to treat CC. Apigenin and trichostatin A can inhibit breast cancer growth[26, 27]. 6-thioguanine has also potential therapeutic effects on tumors[28]. Our findings may help create the appropriate drugs for CC treatment. Eight genes (APOD, CXCL8, MMP1, MMP3, PLOD2, PTGDS, SNX10 and SPP1) were screened out of DEGs through GEPIA. Of them, only PTGDS and SNX10 had not been reported in CC research. 4. Discussion According to GEO validation results, PTGDS was lowly expressed and SNX10 highly expressed in tumor tissues, which was consistent with the results from immunohistochemistry. TCGA methylation analysis showed that the patients with lowly-expressed and highly-methylated PTGDS had a worse prognosis than those with highly-expressed and lowly-methylated PTGDS. Cox regression analysis showed that SNX10 and PTGDS were independent prognostic indicators for OS among CC patients. GSEA showed that the two genes were associated with the chemokine signaling pathway. Zhong G et al. suggested that chemokine signaling is involved in the invasion and migration of lung cancer cells[29]. Chemokine signaling has also been reported to maneuver the progression of breast and hepatobiliary cancer[30, 31]. In addition, the prognostic signature was constructed based on the eight hub genes. Of them, five genes (SPP1, CXCL8, PTGDS, PLOD2, and MMP3) exhibited significant prognostic value. The Cox regression analysis showed that only the risk score of the five genes was an independent prognostic indicator of CC. Interestingly, all of the above genes are associated with cervical cancer. Yan R et al. found that CXCL8 had prognostic value in cervical carcinoma patients[32]. Tian R et al. identified the role of MMP1 in cervical cancer[33]. Xie B et al. defined that genetic polymorphisms in MMP 2, 3, 7, and 9 genes connected with the susceptibility and clinical outcome of cervical cancer in a Chinese Han population [34]. Xu F et al. found that hypoxia and TGF-β1 increased PLOD2 expression to facilitate epithelial-to-mesenchymal transition (EMT) and focal adhesion formation, thus promoting the migration and invasion of cervical cancer cells[35]. Chen X et al. found SPP1 inhibition enhanced the chemosensitivity of cervical cancer cell lines to cisplatin[36]. Using microarray analysis, Song JY et al. found that APOD played in the invasion of cervical cancer[37]. These genes play different roles in other tumors. For example, Allina DO et al. demonstrated the diagnostic significance of APOD for prostatic neoplasms[38]. Shen T et al. held that CXCL8 induced EMT in colon cancer [39]. Wang Y et al. found that CXCL8 regulated the development of breast cancer[40]. Ha H et al. found that CXCL8 was also involved in inflammatory diseases in addition to tumors[41]. Liu M et al. argued that MMP1 promoted the growth and Page 8/26 Page 8/26 metastasis of esophageal squamous cell carcinoma[42]. MMP1 also participated in breast and ovarian cancer[43, 44]. Banik D et al. demonstrated that MMP3 regulated tumor progression[45]. Conclusion Our study indicated that two novel genes PTGDS and SNX10 showed abnormal expression and methylation associated with CC development and explored their prognostic value. However, biofunctions of two genes remained to be unveiled with more in-depth research. The two genes might serve as potential prognostic biomarkers and therapeutic targets in the treatment of CC. Abbreviations Cervical cancer (CC); Differentially expressed gene (DEGs); Gene Expression Omnibus (GEO) ; RobustRankAggreg（RRA）; Gene Ontology (GO); Kyoto Encyclopedia of Genes and Genomes (KEGG); Connectivity Map (Cmap); protein–protein interaction (PPI) ; Search Tool for the Retrieval of Interacting Genes Database (STRING) ; Molecular Complex Detection (MCODE); Gene Expression Profiling Interactive Analysis (GEPIA) ; Receiver operating characteristic (ROC) ; Gene set enrichment analysis (GSEA) 4. Discussion Ji Y et al. demonstrated that C/EBPβ promoted tumor cell invasion and metastasis of colorectal cancer[46]. PLOD2 is implicated in cervical cancer[35] and renal cell carcinoma[47]. SPP1 linked with lung adenocarcinoma, gastric cancer and colorectal cancer [48-50]. Sorting nexin 10 (SNX10) can suppress the progression of colorectal cancer, liver cancer and stomach cancer[51, 52]. Cervantes-Anaya N et al. demonstrated SNX10/V-ATPase pathway regulated ciliogenesis in vitro and in vivo[52]. Prostaglandin D synthase (PTGDS), which catalyzes the conversion of prostaglandin H2 into prostaglandin D2 (PGD2), has been intensely studied [53]. Omori K et al. demonstrated that PTGDS attenuated the malignance of tumor endothelial cells and regulated the processes in gastric cancer and non-small cell lung cancer[54, 55]. The present study is the first to report the expression and prognostic calue of these two genes in CC. Their methylation is associated with CC prognosis, a finding that has never been reported before. This study has some limitations. First, the analysis is entirely based on open databases, so its results should be validated with functional experiments. Second, the five genetic profiles are based on a single cohort with relatively small sample size. Future studies should involve larger independent cohorts. Ethics approval and consent to participate This study does not involve the use of any animal or human data or tissue.The approval or the consent were not applicable. Consent to publication Page 9/26 Page 9/26 Not applicable. Fundings This research did not receive any specific grant from funding agencies in the public, commercial, or not- for-profit sectors. Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements Not applicable. Authors' Information 1Department of Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China 2 Department of Obstetrics and Gynecology, Changzhou Second People's Hospital,Changzhou 213000, Jiangsu, China 2 Department of Obstetrics and Gynecology, Changzhou Second People's Hospital,Changzhou 213000, Jiangsu, China 3 Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu, China 3 Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu, China Authors' contributions Authors SLF, MXX and PPJ designed the project. Authors YC and FG contributed on data analysis, PPJ, JHL and WS prepared the main manuscript. ZYM made revisions in language and finalization of the manuscript. All authors have reviewed and approved the manuscript. Competing interests The authors declare that they have no competing interests. References 1. 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Variables Univariate analysis 　 Multivariate analysis HR 95%CI      p 　 HR 95%CI   p Stage （  I & II） vs  Stage （III & IV） 2.338  1.364- 4.004 0.002  　 3.078  1.329- 7.129 0.009  Grade(G1 & G2）vs  Grade（G3) 1.221  0.947- 1.574 0.124    0.837  0.565- 1.240 0.375  Age（≤50） vs Age（>50) 1.263  0.755- 2.112 0.373    1.197  0.711- 2.011 0.498  SNX10 (high expression) vs SNX10 (low expression)  1.202  0.957- 1.509 0.113    1.424  1.103- 1.838 0.007  PTGDS (high expression) vs PTGDS (low expression)  0.838  0.729- 0.962 0.012  　 0.802  0.693- 0.928 0.003 References T bl Table 1 Details for GEO cervical cancer data Reference Sample GEO Platform Normal Tumor Pyeon D,et al(2007) Cervix GSE6791 GPL570 8 20 Scotto L,et al(2007) Cervix GSE9750 GPL96 24 33 Espinosa AM,et al(2012) Cervix GSE39001 GPL201 GPL6244 12 43 den Boon JA,et al(2014) Cervix GSE63514 GPL570 24 28 Table 1 Details for GEO cervical cancer data Table 2 Screening DEGs in cervical cancer by integrated microarray DEGs Gene name Upregulated MMP1 IFI44L MMP12 PLOD2 CXCL11 RFC4 HOXC6 TOP2A ISG15 SPP1 PRC1 RAD51AP1 SYCP2 DTL APOBEC3B MLF1 TTK CDKN2A INHBA NDC80 EZH2 CXCL8 KIF23 CTHRC1 MCM2 KIF20A KIF4A CDK1 MICB CENPE LAMP3 IFI44 CXCL13 CDC25B TOPBP1 CDC7 LMNB1 RRM2 CDC6 HLTF SYNGR3 NCAPG RYR1 ENO2 SMC4 NEK2 CXCL1 MCM3 C1QB SNX10 PPAP2C KIF11 MCM5 AIM2 AURKA MAD2L1 PBK CENPF KIF15 KNTC1 NTS FBXO5 STIL SPAG5 TRIP13 EPCAM MELK MMP3 KIF14 GZMB CDC20 CEP55 BUB1B NEFH CRNN MAL CRISP3 CRCT1 SPINK5 ALOX12 KRT13 SPRR3 PPP1R3C KRT1 SPRR1B APOD SPRR1A CFD IVL CXCL14 RHCG SPRR2B ENDOU EDN3 CRYAB TMPRSS11D CLIC3 HPGD UPK1A TST KLK11 BBOX1 EMP1 CLCA4 KLK12 SCNN1B NSG1 SLURP1 SOSTDC1 IL1R2 KRT4 KLF4 DSG1 PPL DEFB1 SULT2B1 GPX3 TGM3 ALOX12B ECM1 NDN ISL1 CRABP2 FCGBP PTGDS TMPRSS11B CCND1 FOSB GYS2 TGFBR3 LDOC1 S100A7 KRT2 FGFBP1 PRSS3 ID4 ADRB2 VAT1 SLIT2 CLDN8 KLK10 PTK6 SPINK2 AR PDGFD AKR1B10 EREG Downregulated Table 2 Screening DEGs in cervical cancer by integrated microarray DEGs Gene name Upregulated MMP1 IFI44L MMP12 PLOD2 CXCL11 RFC4 HOXC6 TOP2A ISG15 SPP1 PRC1 RAD51AP1 SYCP2 DTL APOBEC3B MLF1 TTK CDKN2A INHBA NDC80 EZH2 CXCL8 KIF23 CTHRC1 MCM2 KIF20A KIF4A CDK1 MICB CENPE LAMP3 IFI44 CXCL13 CDC25B TOPBP1 CDC7 LMNB1 RRM2 CDC6 HLTF SYNGR3 NCAPG RYR1 ENO2 SMC4 NEK2 CXCL1 MCM3 C1QB SNX10 PPAP2C KIF11 MCM5 AIM2 AURKA MAD2L1 PBK CENPF KIF15 KNTC1 NTS FBXO5 STIL SPAG5 TRIP13 EPCAM MELK MMP3 KIF14 GZMB CDC20 CEP55 BUB1B NEFH CRNN MAL CRISP3 CRCT1 SPINK5 ALOX12 KRT13 SPRR3 PPP1R3C KRT1 SPRR1B APOD SPRR1A CFD IVL CXCL14 RHCG SPRR2B ENDOU EDN3 CRYAB TMPRSS11D CLIC3 HPGD UPK1A TST KLK11 BBOX1 EMP1 CLCA4 KLK12 SCNN1B NSG1 SLURP1 SOSTDC1 IL1R2 KRT4 KLF4 DSG1 PPL DEFB1 SULT2B1 GPX3 TGM3 ALOX12B ECM1 NDN ISL1 CRABP2 FCGBP PTGDS TMPRSS11B CCND1 FOSB GYS2 TGFBR3 LDOC1 S100A7 KRT2 FGFBP1 PRSS3 ID4 ADRB2 VAT1 SLIT2 CLDN8 KLK10 PTK6 SPINK2 AR PDGFD AKR1B10 EREG Downregulated Page 15/26 Table 3 Results of CMap analysis Rank CMap name Mean N Enrichment P-value 1 apigenin -0.848 4 -0.973 0 2 thioguanosine -0.799 4 -0.96 0 3 trichostatin A -0.386 182 -0.261 0 4 viomycin 0.751 4 0.924 0.00004 5 adiphenine 0.779 5 0.907 0.00004 6 atractyloside 0.651 5 0.839 0.00024 7 chrysin -0.745 3 -0.939 0.00032 8 isoflupredone 0.768 3 0.937 0.00044 9 nadolol 0.649 4 0.866 0.00044 Table 3 Results of CMap analysis Page 16/26 Table 4. Supplementary Information (A) APOD, (B) CXCL8, (C) MMP1, (D) MMP3, (E) PLOD2, (F) PTGDS, (G) SNX10, (H) SPP1. Figure S7. Validation of genes expression in GEPIA. (A) APOD, (B) CXCL8, (C) MMP1, (D) MMP3, (E) PLOD2, (F) PTGDS, (G) SNX10, (H) SPP1. Figure S7. Validation of genes expression in GEPIA. (A) APOD, (B) CXCL8, (C) MMP1, (D) MMP3, (E) PLOD2, (F) PTGDS, (G) SNX10, (H) SPP1. Figure S8. Survival analysis of genes in GEPIA. (A) APOD, (B) CXCL8, (C) MMP1, (D) MMP3, (E) PLOD2, (F) PTGDS, (G) SNX10, (H) SPP1. Figure S8. Survival analysis of genes in GEPIA. (A) APOD, (B) CXCL8, (C) MMP1, (D) MMP3, (E) PLOD2, (F) PTGDS, (G) SNX10, (H) SPP1. Figure S8. Survival analysis of genes in GEPIA. (A) APOD, (B) CXCL8, (C) MMP1, (D) MMP3, (E) PLOD2, (F) PTGDS, (G) SNX10, (H) SPP1. Figure S9. Gene expression in GSE7803 and GSE29570. (A) PTGDS in GSE7803. (B) SNX10 in GSE7803. (C) PTGDS in GSE29570. (D) SNX10 in GSE29570. Figure S9. Gene expression in GSE7803 and GSE29570. (A) PTGDS in GSE7803. (B) SNX10 in GSE7803. (C) PTGDS in GSE29570. (D) SNX10 in GSE29570. Figure S10. Seven genes were screened by Univariate cox regression analysis. Figure S10. Seven genes were screened by Univariate cox regression analysis. Figure S11. Five genes were screened by Multivariate cox regression analysis Figure S11. Five genes were screened by Multivariate cox regression analysis Figure S12. Regression analysis of the 5 genes identified. (A) The expression level of the 5 genes in low- and high-risk groups. (B) The univariate Cox proportional hazards regression. (C) The multivariate Cox proportional hazards regression. (D) The heatmap of the 5 genes in high- and low-risk patients in TCGA dataset. Figure S12. Regression analysis of the 5 genes identified. (A) The expression level of the 5 genes in low- and high-risk groups. (B) The univariate Cox proportional hazards regression. (C) The multivariate Cox proportional hazards regression. (D) The heatmap of the 5 genes in high- and low-risk patients in TCGA dataset. Supplementary Information Figure S1. Standardization of gene expression. The blue bar represents the data before normalization, and the red bar represents the normalized data.(A) The standardization of GSE6791 data, (B) the standardization of GSE9750 data, (C) the standardization of GSE39001 data (D) the standardization of GSE63514 data. Figure S2. Differential expression of data between two sets of samples. The red points represent upregulated genes screened on the basis of |fold change| .2.0 and a corrected P-value of ,0.05. The green Page 17/26 points represent downregulation of the expression of genes screened on the basis of |fold change| .2.0 and a corrected P-value of ,0.05. The black points represent genes with no significant difference. FC is the fold change.(A)GSE6791 data, (B) GSE9750 data, (C) GSE39001 data and (D) GSE63514 data. points represent downregulation of the expression of genes screened on the basis of |fold change| .2.0 and a corrected P-value of ,0.05. The black points represent genes with no significant difference. FC is the fold change.(A)GSE6791 data, (B) GSE9750 data, (C) GSE39001 data and (D) GSE63514 data. Figure S3. Hierarchical clustering heatmap of DEGs screened on the basis of |fold change| .2.0 and a corrected P-value ,0.05. Red indicates that the expression of genes is relatively upregulated, green indicates that the expression of genes is relatively downregulated, and black indicates no significant changes in gene expression; gray indicates that the signal strength of genes was not high enough to be detected. (A) GSE6791 data, (B) GSE9750 data, (C) GSE39001 data and (D) GSE63514 data. Figure S4. GO enrichment analysis of DEGs in cervical cancer. (A) GO analysis divided DEGs into three functional groups: molecular function, biological processes, and cell composition. (B) GO enrichment significance items of DEGs in different functional groups.(C) Distribution of DEGs in cervical cancer for different GO-enriched functions. Figure S5. (A) Significant function enrichment of DEGs. Blue represents the GO groups, green represents downregulated genes and red represents upregulated genes. (B) Significant pathway enrichment of DEGs. Blue represents signaling pathway, green represents downregulated genes and red represents upregulated genes. Figure S6. (A) A significant module selected from protein–protein interaction network. (B)GO analysis of these significant molecule.(C) KEGG analysis of these significant molecule. Figure S6. (A) A significant module selected from protein–protein interaction network. (B)GO analysis of these significant molecule.(C) KEGG analysis of these significant molecule. Figure S7. Validation of genes expression in GEPIA. Figures Page 18/26 Figure 1 Flow chart of the present study. Page 19/26 Figure 2 LogFC heatmap of the image data of each expression microarray. Notes: The Figure 2 LogFC heatmap of the image data of each expression microarray. Notes: The abscissa is the geo ID, and the ordinate is the gene name. red represents logFC＞0, green represents logFC＜0, and the values in the box represent the logFC values. Page 20/26 Page 20/26 Figure 3 GO and KEGG analysis of the DEGs. (A) GO analysis of upregulated genes associated with cervic cancer. (B) GO analysis of downregulated genes associated with cervical cancer. (C) KEGG analy upregulated genes associated with cervical cancer. (D) KEGG analysis of downregulated genes Figure 3 GO and KEGG analysis of the DEGs. (A) GO analysis of upregulated genes associated with cervical cancer. (B) GO analysis of downregulated genes associated with cervical cancer. (C) KEGG analysis of upregulated genes associated with cervical cancer. (D) KEGG analysis of downregulated genes associated with cervical cancer. Figure 3 Figure 3 GO and KEGG analysis of the DEGs. (A) GO analysis of upregulated genes associated with cervical cancer. (B) GO analysis of downregulated genes associated with cervical cancer. (C) KEGG analysis of upregulated genes associated with cervical cancer. (D) KEGG analysis of downregulated genes associated with cervical cancer. Page 21/26 Page 21/26 Figure 4 PPI network analysis. (A) Using the STRING online database, a total of 147 DEG DEGs PPI network. (B) Degree of the top 30 nodes in the PPI network. All these n genes. Figure 4 PPI network analysis. (A) Using the STRING online database, a total of 147 DEGs were filtered into the DEGs PPI network. (B) Degree of the top 30 nodes in the PPI network. All these nodes are upregulated genes. Fi 4 Figure 4 PPI network analysis. (A) Using the STRING online database, a total of 147 DEGs were filtered into the DEGs PPI network. (B) Degree of the top 30 nodes in the PPI network. All these nodes are upregulated genes. Page 22/26 Figure 5 Figure 5 Figure 5 Figure 5 Three-dimensional diagram of the three most significant drugs. (A)Apigenin (B) Thioguanosine (C) Trichostatin A Three-dimensional diagram of the three most significant drugs. (A)Apigenin (B) Thioguanosine (C) Trichostatin A Three-dimensional diagram of the three most significant drugs. (A)Apigenin (B) Thioguanosine (C) Trichostatin A Trichostatin A Trichostatin A Figure 6 Validation of PTGDS and SNX10. (A) Immunohistochemistry of PTGDS. (B) Immunohistochemistry of SNX10. (C) Expression of PTGDS in ONCOMINE. (D) Expression of SNX10 in ONCOMINE. Page 23/26 Page 23/26 Figure 7 Validation of PTGDS and SNX10. (A) ROC curve analysis of the two genes. (B) GSEA of PTG SNX10. (C) The methylated expression and gene expression of PTGDS. (D) The methylated e and gene expression of SNX10. (E) Survival analysis of patients with methylated PTGDS exp Figure 7 Validation of PTGDS and SNX10. (A) ROC curve analysis of the two genes. (B) GSEA of PTGDS and SNX10. (C) The methylated expression and gene expression of PTGDS. (D) The methylated expression and gene expression of SNX10. (E) Survival analysis of patients with methylated PTGDS expression. Page 24/26 Page 24/26 Page 24/26 Figure 8 Survival prognosis model of the 5 hub genes. (A) Survival analysis showed that the patients in the high risk group had statistically significantly worse overall survival than those in low risk group in TCGA cohort. (B) ROC analysis was performed to find out the most optimal cutoff value to divide the CC patients into high risk and low risk group. (C-D) The risk scores for all patients in TCGA cohort are plotted in ascending order and marked as low risk (blue) or high risk (red), as divided by the threshold (vertical Figure 8 Survival prognosis model of the 5 hub genes. (A) Survival analysis showed that the patients in the high risk group had statistically significantly worse overall survival than those in low risk group in TCGA cohort. (B) ROC analysis was performed to find out the most optimal cutoff value to divide the CC patients into high risk and low risk group. (C-D) The risk scores for all patients in TCGA cohort are plotted in ascending order and marked as low risk (blue) or high risk (red), as divided by the threshold (vertical Page 25/26 Page 25/26 black line). (E) Eight expression and risk score distribution in TCGA cohort by z-score, with red indicating higher expression and light blue indicating lower expression. black line). (E) Eight expression and risk score distribution in TCGA cohort by z-score, with red indicating higher expression and light blue indicating lower expression. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. FigureSupp.docx FigureSupp.docx SupplementaryFigure12.tif SupplementaryFigure11.tiff SupplementaryFigure10.tif SupplementaryFigure9.tif SupplementaryFigure8.tif SupplementaryFigure7.tif SupplementaryFigure6.tif SupplementaryFigure5.tif SupplementaryFigure4.tif SupplementaryFigure3.tif SupplementaryFigure2.tif SupplementaryFigure1.tif FigureSupp.docx SupplementaryFigure12.tif SupplementaryFigure11.tiff SupplementaryFigure10.tif SupplementaryFigure9.tif SupplementaryFigure8.tif SupplementaryFigure7.tif SupplementaryFigure6.tif SupplementaryFigure5.tif SupplementaryFigure4.tif SupplementaryFigure3.tif SupplementaryFigure2.tif SupplementaryFigure1.tif SupplementaryFigure12.tif SupplementaryFigure11.tiff SupplementaryFigure10.tif SupplementaryFigure9.tif SupplementaryFigure8.tif SupplementaryFigure7.tif Page 26/26

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Early Life Nutrition and the Role of Complementary Feeding on Later Adherence to the Mediterranean Diet in Children up to 3 Years of Age

Nutrients

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 Citation: Gómez-Martín, M.; Herrero-Morín, D.; Arboleya, S.; Gueimonde, M.; González, S. Early Life Nutrition and the Role of Complementary Feeding on Later Adherence to the Mediterranean Diet in Children up to 3 Years of Age. Nutrients 2022, 14, 1664. https:// doi.org/10.3390/nu14081664 Keywords: infant diet; Mediterranean diet; weaning; complementary feeding; breastfeeding Academic Editor: Maria Immacolata Spagnuolo Article Early Life Nutrition and the Role of Complementary Feeding on Later Adherence to the Mediterranean Diet in Children up to 3 Years of Age Martín 1,2 , David Herrero-Morín 3 , Silvia Arboleya 2,4 , Miguel Gueimonde 2,4,* ál 1 2 * María Gómez-Martín 1,2 , David Herrero-Morín 3 , Silvia Arboleya 2,4 , Miguel Gueim and Sonia González 1,2,* 1 Department of Functional Biology, University of Oviedo, 33006 Oviedo, Spain; [email protected] 2 Diet, Microbiota and Health Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 1 Department of Functional Biology, University of Oviedo, 33006 Oviedo, Spain; gomezmarmaria@uniovi 1 Department of Functional Biology, University of Oviedo, 33006 Oviedo, Spain; [email protected] 2 Diet, Microbiota and Health Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain; [email protected] ( ) p * Correspondence: [email protected] (M.G.); [email protected] (S.G.); Tel.: +34-985-892-131 (M.G.); +34-985-104-209 (S.G.) Abstract: The first years of life represent a window of opportunity to establish proper dietary patterns and to maintain them over time. Our aim was to describe the diet of a cohort of Spanish children, from 2 to 36 months, and to identify the components that could influence the quality of the diet at 24 and 36 months of age. This was a longitudinal prospective study analyzing information from administered questionnaires about general characteristics and food frequency consumption in 97 full- term babies. At 2–3 months of age, only 53.6% of infants were observed to be breastfed. The intake of animal foodstuffs from 12 to 36 months was higher than national recommendations, and the contrary was true for fruits and vegetables. The intake of vitamin D was below European Food Safety Authority recommendations. Moreover, energy intake at 6 months was inversely associated with Mediterranean Diet Score (MDS) at 24 months, whereas vegetables intake was positively associated with MDS at 36 months. These results could be useful in the creation of future guidelines focused on the promotion of breastfeeding and healthy early-life food habits. nutrients nutrients nutrients nutrients nutrients 1. Introduction Received: 14 March 2022 Accepted: 14 April 2022 Published: 16 April 2022 The period from conception to the age of two years has been described as a window of opportunity to promote long-term effects contributing to health [1]. Following a high- quality diet starting from early life, characterized by a wide variety of nutritious foods and a balanced intake of macro- and micronutrients for energy, is essential to lower the later risk of diet-related non-communicable diseases [2,3]. Until the age of 6 months, breastfeeding provides all the nutritional requirements for a newborn [4]. However, from this age onwards, there is controversy about the ideal pattern for the introduction of complementary feeding. At this stage, children should begin to be introduced progressively to safe and adequate foods until they reach a diet similar to that of an adult, at approximately two years of age [4]. With independence of the nutritional composition, the consumption of certain food groups, such as fruits and vegetables, provides a wide repertoire of flavors, aromas and textures that will shape the future dietary preferences of the infant towards a more varied and nutrient-dense diet [5]. The Avon Longitudinal Study of Parents and Children (ALSPAC) calculated a complementary feeding utility index based on recommendations [6]. Higher index scores were linked to longer breastfeeding and higher intake of fruits and vegetables and a lower presence of ready-prepared baby foods. In terms of health, higher scores were positively related to intelligence quotient and ‘healthy’ dietary patterns in Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/nutrients Nutrients 2022, 14, 1664. https://doi.org/10.3390/nu14081664 Nutrients 2022, 14, 1664 2 of 14 childhood [6]. In accordance with this, some longitudinal studies demonstrated that a greater proportion of total energy from fruits and vegetables in children was associated with a reduction in the risk of cardiovascular mortality in adulthood, contrary to what occurs for meat, dairy products and sugar supply [7]. 2.1. Sample Recruitment and Study Design The cohort was composed of 97 full-term babies (37–40 gestational weeks) at the lactation period (2–3 months of life), 93 at the weaning period (6 months) and 90 subjects at the transition diet (12 months); meanwhile, the family diet (24 and 36 months) was composed of 76 and 64 subjects, respectively (Figure 1). Participants were recruited through the Primary Care Pediatrics Service in Asturias, on the north coast of Spain, at the first medical consultation. When the legal tutors or caregivers of all participants were informed of the objectives of the study, they signed their written consent. The Regional Ethics Committee of Clinical Research of Asturias (Reference no. 12/16, 03/02/2016) and the Committee on Bioethics of CSIC (Reference no. PCIN-2015-233) evaluated and approved the study procedures. All protocols were performed in line with the principles stated in the Declaration of Helsinki, the Bioethics Convention of University of Oviedo, the Council of Europe’s Convention on Human Rights and Biomedicine and in Spanish legislation on bioethics. The Directive 95/46/EC of the European Parliament and the Council of 24 October 1995 on the protection of individuals regarding the processing of personal data and on the free movement of such data was strictly followed. 1. Introduction Although most previous works in the literature focused on the importance of isolated food groups or particular nutrients, this may hide the fact that different foods are combined as part of the diet, and this implies a high degree of correlation among them. Despite the fact that at present, no validated index has been found to evaluate the quality of diet in children, one of the most recognized beneficial dietary patterns worldwide is the Mediterranean Diet (MD) [8]. Adherence to this pattern, represented by a high content of fruits and vegetables, a low proportion of meat and dairy products and the use of olive oil as culinary lipids [9], from an early age could bring numerous benefits [10,11]. A study that investigated the association between parents’ lifestyle determinants and children’s dietary habits and physical activity levels showed that higher levels of maternal educational and physical activity were positively associated with children’s MD [12]. Based on the above evidence, it seems reasonable to hypothesize that the choice of breastfeeding during the first months of life and, subsequently, a healthy dietary pattern with a high repertoire of foods with high nutritional density, may condition the achievement of a healthier diet later in life. For all these reasons, research on the eating habits of children in this age range is of great relevance for the establishment of health promotion strategies in the early years of life that will improve later health [13]. In this setting, the aim of this study was to assess the diet of a cohort of Spanish children and to identify the components of weaning and complementary feeding that could influence the dietary quality at 24 and 36 months of age. 2. Materials and Methods 2.1. Sample Recruitment and Study Design 2 G l Ch t i ti 2.3. Nutritional Assessment .2. General Characteristics At baseline, data were collected on the characteristics of the infants (i.e., sex and age nd the type of delivery. In addition, several characteristics related to dietary habits, such s type of breastfeeding, consumption of vitamin and mineral supplements, diet textur r prescription of a therapeutic diet, were assessed at the different sampling times. Th eight (cm) and weight (kg) of the children to the nearest 0.1 cm and 0.1 kg, respectively were measured with calibrated and suitable equipment by pediatric nurses. Dietary questionnaires were collected at the time of recruitment and at 2–3, 6, 12, 2 nd 36 months (Figure 1). .3. Nutritional Assessment The children’s diets were registered using a self-administered food propensity ques onnaire for the previous week based on the Pilot Study for the Assessment of Nutrien ntake and Food Consumption among Children in Europe (PANCAKE) [14] and adapted or Spanish children and local culinary customs and recipes. Furthermore, food dairie were administered using an online tool. Foods were categorized into 12 food groups ac ording to the European Prospective Investigation into Cancer (EPIC) criteria [15], incor orating another 2 concerning infant products: breast milk and processed infant products The children’s diets were registered using a self-administered food propensity ques- tionnaire for the previous week based on the Pilot Study for the Assessment of Nutrient Intake and Food Consumption among Children in Europe (PANCAKE) [14] and adapted for Spanish children and local culinary customs and recipes. Furthermore, food dairies were administered using an online tool. Foods were categorized into 12 food groups according to the European Prospective Investigation into Cancer (EPIC) criteria [15], incorporating another 2 concerning infant products: breast milk and processed infant products. 2 G l Ch t i ti 2.3. Nutritional Assessment Food groups included: fats (vegetable oils and solid fats); vegetables (bulbs, mushrooms, roots, inflorescences and stem and leaf vegetables); legumes (lentils, chickpeas, soy, beans and peas); fruits (fresh, dried and canned fruits); potatoes and tubers (potato and sweet potato); cereals and cereal products (bread, pasta, flours and grains); meat and meat products; fish (fish and fish products, crustaceans and mollusks); eggs; milk and dairy products (milk, yogurt, dairy dessert, milkshake and fresh, mature and processed cheeses), sweet and desserts (sweets, cake, biscuits, chocolate, honey and others), sweetened beverages (natural and non-natural fruit juices, soft drinks and soy-based beverages) and human breast milk and processed infant products (infant formulas: starter formulas, special starter formulas, follow-up formulas, special follow-up formulas, growing-up milk; infants cereals and infant purees: fruits, fruit and cereals, vegetables, legumes and pasta, meat, fish and others) [16]. porating another 2 concerning infant products: breast milk and processed infant products Food groups included: fats (vegetable oils and solid fats); vegetables (bulbs, mushrooms oots, inflorescences and stem and leaf vegetables); legumes (lentils, chickpeas, soy, bean nd peas); fruits (fresh, dried and canned fruits); potatoes and tubers (potato and swee potato); cereals and cereal products (bread, pasta, flours and grains); meat and meat prod ucts; fish (fish and fish products, crustaceans and mollusks); eggs; milk and dairy prod ucts (milk, yogurt, dairy dessert, milkshake and fresh, mature and processed cheeses) The questionnaires were completed by the mother, father or caregiver of the child, who received them by e-mail or mobile phone. Specific instructions to complete the questionnaire were detailed at the start of each section and the validated photo album developed by the PANCAKE was used for serving size estimation, taking into account the EU-Menu guidelines [17]. As dietary information was collected over a prolonged period, at different times from birth, an adapted different version of the same questionnaire was used at the lactation period due to the absence of complementary feeding. weet and desserts (sweets, cake, biscuits, chocolate, honey and others), sweetened bev rages (natural and non-natural fruit juices, soft drinks and soy-based beverages) and hu man breast milk and processed infant products (infant formulas: starter formulas, specia tarter formulas, follow-up formulas, special follow-up formulas, growing-up milk; in ants cereals and infant purees: fruits, fruit and cereals, vegetables, legumes and pasta meat, fish and others) [16]. 2.2. General Characteristics At baseline, data were collected on the characteristics of the infants (i.e., sex and age) and the type of delivery. In addition, several characteristics related to dietary habits, such as type of breastfeeding, consumption of vitamin and mineral supplements, diet texture or prescription of a therapeutic diet, were assessed at the different sampling times. The height (cm) and weight (kg) of the children to the nearest 0.1 cm and 0.1 kg, respectively, were measured with calibrated and suitable equipment by pediatric nurses. Dietary questionnaires were collected at the time of recruitment and at 2–3, 6, 12, 24 and 36 months (Figure 1). Nutrients 2022, 14, 1664 trients 2022, 14, x FOR PE 3 of 14 3 of 1 Figure 1. Study cohort and sampling points.X, data available. Figure 1. Study cohort and sampling points.X, data available. igure 1. Study cohort and sampling points X data available Figure 1. Study cohort and sampling points.X, data available. igure 1. Study cohort and sampling points.X, data available. Figure 1. Study cohort and sampling points.X, data available. 2 G l Ch t i ti 2.3. Nutritional Assessment The questionnaires were completed by the mother, father or caregiver of the child who received them by e-mail or mobile phone. Specific instructions to complete the ques ionnaire were detailed at the start of each section and the validated photo album devel Breastfeeding was assessed in each time period. To calculate the categorical variable, the type of breastfeeding was classified as breastfeeding (including mixed) or infant formula. Regarding the quantitative variable, the volume of breast milk received was estimated by using the mean values reported in the previous studies for each stage of age (780 mL for infants up to 6 months and 600 mL for infants from 7 to 12 months, in the cases of exclusive breastfeeding) [18,19]. For the infant formulas, the volume reported by the parents was used, assuming that the manufacturer’s prescriptions regarding the weight of powdered milk to be dissolved per volume were respected. In mixed-fed infants, based on existing literature, the amount of formula consumed per day was measured, and the remaining Nutrients 2022, 14, 1664 4 of 14 volume of formula consumed per day was assumed to be breast milk up to 780 mL from start to 6 months and 600 mL at 12 months [18]. volume of formula consumed per day was assumed to be breast milk up to 780 mL from start to 6 months and 600 mL at 12 months [18]. The energy content and nutritional composition was calculated using the food com- position tables developed by the Centro de Enseñanza Superior de Nutrición Humana y Dietética (CESNID) [20]. The nutritional composition of maternal breast milk [21], infant formula, cereal products and infant purees was obtained from the processed baby foods composition table [16]. p Furthermore, detailed information regarding the type of protein or carbohydrate consumed was extended from the food composition tables published by the United States Department of Agriculture (USDA) [22]. p g A Mediterranean Diet Score (MDS) was created based on previous studies and adapted [23] for the dietary data obtained at 24 and 36 months of age. 2.4. Statistical Analyses The results were analyzed using the IBM SPSS software version 25.0 (IBM SPSS, Inc., Chicago, IL, USA). Goodness of fit to the normal distribution was determined by means of the Kolmogorov–Smirnov test. When normality of the variables was not achieved, nonpara- metric tests were used. In general, categorical variables were summarized as percentages and continuous variables using the medians and interquartile ranges (percentile 25 and percentile 75) or the means and standard deviations for descriptive purposes. The Student’s t-test and Kruskal–Wallis test were used to evaluate differences in continuous variables and the Z-test, Fisher’s test and chi-square test for categories variables. Adherence to dietary reference values (DRVs) was calculated using the European Food Safety Authority’s (EFSA) recommendations for children aged 1–3 years [24]. The parameters used were adequate intake (AI) and average requirement (AR). The AI can also be used to determine the proportion of individuals with adequate nutrient intake, while the proportion of the population with usual intakes below the AR provides an estimate of the proportion of the group whose intake does not meet nutrient requirements. In order to analyze the impact of the consumption of the food groups, accounting for 80% of the energy intake at 6 months, on the Mediterranean Diet index score at 24 and 36 months, a stepwise linear regression model was conducted. The adequacy of the major food groups to the national recommendations for this age group was calculated [25]. For this purpose, the lowest portions recommended for vegetables, legumes and milk and dairy products, together with the large size for fish and meat, were established as adequacy criteria. GraphPad Prism 8 and BioRender were used for graphical representations. 3. Results 3.1. Description of the Sample and Mediterranean Diet Score Calculation 3.1. Description of the Sample and Mediterranean Diet Score Calculation 24 Months 36 Months Boys (n = 44) Girls (n = 32) Boys (n = 37) Girls (n = 27) Mean ± SD Median Mean ± SD Median Mean ± SD Median Mean ± SD Median Score MUFA/SFA ratio a 1.19 ± 0.35 <1.07 ≥1.08 1.26 ± 0.40 <1.13 ≥1.14 1.23 ± 0.30 <1.19 ≥1.20 1.48 ± 0.45 <1.35 ≥1.36 0 1 Legumes (g/day) 34.85 ± 21.35 <29.93 ≥29.94 27.34 ± 16.13 <24.28 ≥24.29 36.97 ± 17.73 <35.70 ≥35.71 37.50 ± 28.02 <34.28 ≥34.29 0 1 Cereals and potatoes (g/day) 121.07 ± 55.94 <115.34 ≥115.35 105.12 ± 56.91 <98.47 ≥98.48 147.85 ± 52.66 <132.12 ≥132.13 130.24 ± 71.61 <116.42 ≥116.43 0 1 Vegetables (g/day) 101.24 ± 75.13 <85.60 ≥85.61 107.38 ± 98.27 <89.04 ≥89.05 106.83 ± 91.60 <84.85 ≥84.86 114.40 ± 143.73 <64.28 ≥64.29 0 1 Fruits (g/day) 173.95 ± 86.96 <167.23 ≥167.24 186.63 ± 121.79 <140.16 ≥140.17 212.68 ± 99.61 <202.28 ≥202.29 171.52 ± 104.47 <145.07 ≥145.08 0 1 Dairy products b 438.48 ± 206.20 ≥465.63 <465.62 397.31 ± 208.15 ≥377.94 <377.93 470.34 ± 224.86 ≥462.50 <462.49 274.05 ± 170.06 ≥275.0 <274.9 0 1 Meat (g/day) 41.20 ± 23.95 ≥39.29 <39.28 39.30 ± 21.06 ≥39.64 <39.63 50.19 ± 35.13 ≥45.71 <45.70 48.00 ± 31.93 ≥38.57 <38.56 0 1 Values are presented as the mean ± standard deviation. a Monounsaturated fatty acids/saturated fatty acid; b dairy products included: milk (mL/day), milkshake (mL/day), yogurt and cheese (g/day). Table 1. Mediterranean Diet Score calculation by age group and gender. Values are presented as the mean ± standard deviation. a Monounsaturated fatty acids/saturated fatty acid; b dairy products included: milk (mL/day), milkshake (mL/day), yogurt and cheese (g/day). The general characteristics of the sample, according to the period studied, are pre- sented in Table 2. As expected, weight and height increased significantly across the follow up (from 5.82 to 15.22 kg and 59.98 to 96.27 cm, respectively). The percentage of breastfeed- ing decreased from lactation period (53.6%) up to 36 months (7.8%) as well as the percentage of mineral or vitamin supplementation (from 92.7% to 0%). In terms of food texture, there was a significant decrease in the proportion of children fed with puree between 6 and 12 months of age. No significant differences were found for the rest of the variables studied. Table 2. General characteristics of the cohort by period. Table 2. General characteristics of the cohort by period. Table 2. 3.1. Description of the Sample and Mediterranean Diet Score Calculation In terms of food texture, there was a significant decrease in the proportion of children fed with puree between 6 and 12 months of age. No significant differences were found for the rest of the variables studied. Table 2. General characteristics of the cohort by period. Lactation Period Weaning Period Transition Diet Family Diet 2–3 Months 6 Months 12 Months 24 Months 36 Months Subjects (n) 97 93 90 76 64 Gender Male 56 (57.7) 53 (57.0) 51 (56.7) 44 (57.9) 37 (57.8) Female 41 (42.3) 40 (43.0) 39 (43.3) 32 (42.1) 27 (42.2) Weight (kg) 5.82 ± 0.83 7.76 ± 0.84 * 9.97 ± 1.52 * 12.66 ± 1.76 * 15.22 ± 2.52 * Height (cm) 59.98 ± 2.88 67.73 ± 2.36 * 75.89 ± 3.37 * 88.46 ± 3.84 * 96.27 ± 4.58 * Lactation BF 52 (53.6) 39 (41.9) 23 (25.6) * 10 (13.3) 5 (7.8) IF 45 (46.4) 54 (58.1) 56 (62.2) 9 (12.0) * 4 (6.3) Other 0 0 11 (12.2) 56 (74.7) * 55 (85.9) Supplementation No 7 (7.3) 6 (6.5) 10 (11.1) 75 (98.7) * 64 (100) Yes 89 (92.7) 87 (93.5) 80 (88.9) 1 (1.3) * 0 Food texture Mashed food 0 80 (87.9) 8 (8.9) * 0 0 Semi-solid 0 9 (9.9) 60 (66.7) * 1 (1.3) * 1 (1.6) Regular 0 2 (2.2) 22 (24.4) * 75 (98.7) * 63 (98.4) Special diet No 0 88 (94.6) 87 (96.7) 73 (96.1) 63 (98.4) Yes 0 5 (5.4) 3 (3.3) 3 (3.9) 1 (1.6) Delivery type Vaginal 75 (77.3) 71 (76.3) 68 (75.6) 58 (76.3) 48 (75.0) C-section 22 (22.7) 22 (23.7) 22 (24.4) 18 (23.7) 16 (25.0) Data expressed as N (%) or the mean ± standard deviation. BF, breastfeeding; C-section, caesarean section; IF, infant formula. * Differences compared to previous category (p < 0.05). Table 1. Mediterranean Diet Score calculation by age group and gender. 3.1. Description of the Sample and Mediterranean Diet Score Calculation Mean and median intake of the MDS items at 24 and 36 months by gender is presented in Table 1. Higher consumption (above the median) of fruits, cereals (included potatoes), vegetables, legumes and the ratio of monounsaturated/saturated lipids and lower con- sumption of meat and dairy products each contributed one point to the total score. Thus, the total MDS ranged from 0 to 7. 5 of 14 Nutrients 2022, 14, 1664 Table 1. Mediterranean Diet Score calculation by age group and gender. 24 Months 36 Months Boys (n = 44) Girls (n = 32) Boys (n = 37) Girls (n = 27) Mean ± SD Median Mean ± SD Median Mean ± SD Median Mean ± SD Median Score MUFA/SFA ratio a 1.19 ± 0.35 <1.07 ≥1.08 1.26 ± 0.40 <1.13 ≥1.14 1.23 ± 0.30 <1.19 ≥1.20 1.48 ± 0.45 <1.35 ≥1.36 0 1 Legumes (g/day) 34.85 ± 21.35 <29.93 ≥29.94 27.34 ± 16.13 <24.28 ≥24.29 36.97 ± 17.73 <35.70 ≥35.71 37.50 ± 28.02 <34.28 ≥34.29 0 1 Cereals and potatoes (g/day) 121.07 ± 55.94 <115.34 ≥115.35 105.12 ± 56.91 <98.47 ≥98.48 147.85 ± 52.66 <132.12 ≥132.13 130.24 ± 71.61 <116.42 ≥116.43 0 1 Vegetables (g/day) 101.24 ± 75.13 <85.60 ≥85.61 107.38 ± 98.27 <89.04 ≥89.05 106.83 ± 91.60 <84.85 ≥84.86 114.40 ± 143.73 <64.28 ≥64.29 0 1 Fruits (g/day) 173.95 ± 86.96 <167.23 ≥167.24 186.63 ± 121.79 <140.16 ≥140.17 212.68 ± 99.61 <202.28 ≥202.29 171.52 ± 104.47 <145.07 ≥145.08 0 1 Dairy products b 438.48 ± 206.20 ≥465.63 <465.62 397.31 ± 208.15 ≥377.94 <377.93 470.34 ± 224.86 ≥462.50 <462.49 274.05 ± 170.06 ≥275.0 <274.9 0 1 Meat (g/day) 41.20 ± 23.95 ≥39.29 <39.28 39.30 ± 21.06 ≥39.64 <39.63 50.19 ± 35.13 ≥45.71 <45.70 48.00 ± 31.93 ≥38.57 <38.56 0 1 Values are presented as the mean ± standard deviation. a Monounsaturated fatty acids/saturated fatty acid; b dairy products included: milk (mL/day), milkshake (mL/day), yogurt and cheese (g/day). The general characteristics of the sample, according to the period studied, are pre- sented in Table 2. As expected, weight and height increased significantly across the follow up (from 5.82 to 15.22 kg and 59.98 to 96.27 cm, respectively). The percentage of breastfeed- ing decreased from lactation period (53.6%) up to 36 months (7.8%) as well as the percentage of mineral or vitamin supplementation (from 92.7% to 0%). Data expressed as N (%) or the mean ± standard deviation. BF, breastfeeding; C-section, caesarean section; IF, infant formula. * Differences compared to previous category (p < 0.05). 3.2. Dietary Intake 3.2. Dietary Intake 3.2. Dietary Intake 3.2. Dietary Intake As expected, during lactation either infant formula or breast milk were the major sources of energy, and at the weaning period, the diet started to become more varied (Figure 2). Fats, vegetables, tubers, fruits and processed baby foods were the first groups included into infants’ diet (Supplementary Materials Table S1). At the so-called transition diet, the contribution of infant formula and breastfeeding to energy intake decreased in favor of dairy products, fruits, infant cereals, fats and tubers, among others. From 12 to 24 months, the principal observed variation derived from the substitution of breast milk and infant formulas with cow’s milk. No differences were found between 24 and 36 months. In order to assess the degree of adherence to the dietary recommendations for children, the daily intake of the major food groups was compared with the recommendations. Intake of protein foods, such as meat and fish, were above recommendations, while fruits and vegetables were below at all ages (Supplementary Materials Figure S1). As expected, during lactation either infant formula or breast milk were the major sources of energy, and at the weaning period, the diet started to become more varied (Fig- ure 2). Fats, vegetables, tubers, fruits and processed baby foods were the first groups in- cluded into infants’ diet (Supplementary Materials Table S1). At the so-called transition diet, the contribution of infant formula and breastfeeding to energy intake decreased in favor of dairy products, fruits, infant cereals, fats and tubers, among others. From 12 to 24 months, the principal observed variation derived from the substitution of breast milk and infant formulas with cow’s milk. No differences were found between 24 and 36 months. In order to assess the degree of adherence to the dietary recommendations for children, the daily intake of the major food groups was compared with the recommendations. In- take of protein foods, such as meat and fish, were above recommendations, while fruits and vegetables were below at all ages (Supplementary Materials Figure S1). Figure 2. Change in the energy provided by food groups across the follow up. 3.1. Description of the Sample and Mediterranean Diet Score Calculation General characteristics of the cohort by period. Lactation Period Weaning Period Transition Diet Family Diet 2–3 Months 6 Months 12 Months 24 Months 36 Months Subjects (n) 97 93 90 76 64 Gender Male 56 (57.7) 53 (57.0) 51 (56.7) 44 (57.9) 37 (57.8) Female 41 (42.3) 40 (43.0) 39 (43.3) 32 (42.1) 27 (42.2) Weight (kg) 5.82 ± 0.83 7.76 ± 0.84 * 9.97 ± 1.52 * 12.66 ± 1.76 * 15.22 ± 2.52 * Height (cm) 59.98 ± 2.88 67.73 ± 2.36 * 75.89 ± 3.37 * 88.46 ± 3.84 * 96.27 ± 4.58 * Lactation BF 52 (53.6) 39 (41.9) 23 (25.6) * 10 (13.3) 5 (7.8) IF 45 (46.4) 54 (58.1) 56 (62.2) 9 (12.0) * 4 (6.3) Other 0 0 11 (12.2) 56 (74.7) * 55 (85.9) Supplementation No 7 (7.3) 6 (6.5) 10 (11.1) 75 (98.7) * 64 (100) Yes 89 (92.7) 87 (93.5) 80 (88.9) 1 (1.3) * 0 Food texture Mashed food 0 80 (87.9) 8 (8.9) * 0 0 Semi-solid 0 9 (9.9) 60 (66.7) * 1 (1.3) * 1 (1.6) Regular 0 2 (2.2) 22 (24.4) * 75 (98.7) * 63 (98.4) Special diet No 0 88 (94.6) 87 (96.7) 73 (96.1) 63 (98.4) Yes 0 5 (5.4) 3 (3.3) 3 (3.9) 1 (1.6) Delivery type Vaginal 75 (77.3) 71 (76.3) 68 (75.6) 58 (76.3) 48 (75.0) C-section 22 (22.7) 22 (23.7) 22 (24.4) 18 (23.7) 16 (25.0) Data expressed as N (%) or the mean ± standard deviation. BF, breastfeeding; C-section, caesarean section; IF, infant formula. * Differences compared to previous category (p < 0.05). Nutrients 2022, 14, 1664 6 of 14 sarean 3.2. Dietary Intake 3.2. Dietary Intake 0 50 100 2-3 months 6 months 12 months 24 months 36 months % Fruits Infant cereals Fats Tubers Infant purees Vegetables Cereals Meat and meat products Sweetened beverages Sweets and desserts Milk and dairy Legumes Human breast milk Infant formulas Fish and fish products Eggs Transition diet Weaning period Lactation period Family diet Figure 2. Change in the energy provided by food groups across the follow up. Family diet 24 months 12 months Transition diet 12 months Transition diet 12 months 2-3 months Lactation period Figure 2. Change in the energy provided by food groups across the follow up. Figure 2. Change in the energy provided by food groups across the follow up. 3.3. Nutritional Status 3.3. Nutritional Status Infant’s nutritional intakes at 12, 24 and 36 months, including a comparison with the recommended daily values by age, are presented in Table 3. The data revealed an increase in the intake of most nutrients at 24 and 36 months, in respect to 12 months, as expected. For vitamins, the intake of vitamin D in all periods studied; choline and copper at 12 months; vitamin E at 24 and 36 months were under the recommendations. In the case of minerals, the compliance of AI of magnesium increased from 12 to 24 months, but a de- creased was observed from 24 to 36 months (Table 3). Infant’s nutritional intakes at 12, 24 and 36 months, including a comparison with the recommended daily values by age, are presented in Table 3. The data revealed an increase in the intake of most nutrients at 24 and 36 months, in respect to 12 months, as expected. For vitamins, the intake of vitamin D in all periods studied; choline and copper at 12 months; vitamin E at 24 and 36 months were under the recommendations. In the case of minerals, the compliance of AI of magnesium increased from 12 to 24 months, but a decreased was observed from 24 to 36 months (Table 3). Infant’s nutritional intakes at 12, 24 and 36 months, including a comparison with the recommended daily values by age, are presented in Table 3. The data revealed an increase in the intake of most nutrients at 24 and 36 months, in respect to 12 months, as expected. For vitamins, the intake of vitamin D in all periods studied; choline and copper at 12 months; vitamin E at 24 and 36 months were under the recommendations. In the case of minerals, the compliance of AI of magnesium increased from 12 to 24 months, but a de- creased was observed from 24 to 36 months (Table 3). Infant’s nutritional intakes at 12, 24 and 36 months, including a comparison with the recommended daily values by age, are presented in Table 3. The data revealed an increase in the intake of most nutrients at 24 and 36 months, in respect to 12 months, as expected. For vitamins, the intake of vitamin D in all periods studied; choline and copper at 12 months; vitamin E at 24 and 36 months were under the recommendations. 3.3. Nutritional Status 3.3. Nutritional Status In the case of minerals, the compliance of AI of magnesium increased from 12 to 24 months, but a decreased was observed from 24 to 36 months (Table 3). Nutrients 2022, 14, 1664 7 of 14 7 of 14 Table 3. Energy and macro- and micronutrients intake compared with dietary reference values (DRVs) based on intakes from the EFSA (European Food Safety Authority) in the study’s cohort at 12, 24 and 36 months. 3.3. Nutritional Status 3.3. Nutritional Status DRV 12 Months n= 90 DRV Compliance 12 Months (%) 24 Months n = 76 DRV Compliance 24 Months (%) 36 Months n = 64 DRV Compliance 36 Months (%) AI AR Median (IR) >AI <AR Median (IR) >AI <AR Median (IR) >AI <AR Energy (kcal/day) - - 1019.0 (925.0–1146.1) a - - 1147.4 (941.9–1363.8) a - - 1253.0 (1016.8–1470.0) a - - Macronutrients - - - - - - Fat (g/day) - - 36.2 (32.5–40.6) a - - 41.1 (35.1–47.5) a - - 46.2 (36.0–54.8) a - - SFA (g/day) ALAP - 10.7 (7.4–12.8) a - - 15.2 (11.5–18.3) b - - 15.7 (12.0–19.6) b - - MUFA (g/day) - - 10.2 (9.0–12.4) a - - 16.5 (13.7–19.7) b - - 17.9 (14.5–26.1) b - - PUFA (g/day) - - 2.8 (2.2–3.4) a - - 4.3 (3.6–5.1) a,b - - 5.5 (4.1–6.7) b - - Carbohydrate (g/day) - - 130.1 (119.5–151.9) a - - 139.5 (109.0–168.4) a - - 148.5 (122.5–172.4) a - - Dietary fiber (g/day) 10 13.6 (11.4–16.3) a 83.3 - 14.3 (11.2–16.8) a 84.2 - 15.8 (12.1–18.8) a 79.7 - Protein (g/day) - - 36.8 (30.7–43.1) a 52.2 (44.1–63.0) a 55.7 (47.6–65.9) a - - Animal protein (g/day) - - 17.0 (12.4–23.6) a 32.2 (24.0–39.2) b 33.9 (28.4–40.9) b - - Vegetal protein (g/day) - - 11.8 (9.2–15.3) a 17.1 (13.2–22.0) a,b 20.8 (15.8–25.3) b - - Micronutrients - - Vitamin A (µg RAE/day) - 205 959.7 (737.7–1282.4) a - 0 564.8 (388.7–841.3) b - 6.6* 564.1 (364.1–861.0) b - 6.3 Thiamin (mg/day) - 0.072 1.0 (0.8–1.2) a - 0 0.9 (0.8–1.1) a - 0 1.1 (0.8–1.3) a - 0 Riboflavin (mg/day) - 0.5 1.4 (1.1–1.6) a - 1.1 1.4 (1.1–1.7) a - 2.6 1.5 (1.1–1.8) a - 0 Niacin (mg/day) - 1.3 9.3 (8.0–12.1) a - 0 10.8 (8.2–13.4) a - 0 11.4 (9.3–14.9) a - 0 Vitamin B-6 (mg/day) - 0.5 1.4 (1.2–1.7) a - 1.1 1.4 (1.1–1.8) a - 0 1.6 (1.2–1.9) a - 0 Folate (µg DFE/day) - 90 359.0 (249.2–462.7) a - 1.1 446.2 (342.4–570.3) a - 1.3 566.7 (413.9–676.2) a - 0 Vitamin B-12 (µg/day) 1.5 2.4 (1.8–3.4) a 88.9 - 3.0 (2.2–4.2) a 92.1 - 3.0 (2.3–3.8) a 92.2 - Vitamin C (mg/day) 15 162.3 (122.0–224.8) a - 0 93.5 (57.7–129.3) b - 1.3 88.5 (59.6–149.0) b - 1.6 Vitamin D (µg/day) 15 - 6.0 (3.8–7.3) a 0 - 2.8 (0.9–4.1) b 1.3 - 2.3 (1.1–4.0) b 0 - Vitamin E (mg/day) 6 $ - 8.7 (6.7–11.0) a 80.0 - 5.1 (4.0–6.7) b 38.2 * - 5.7 (4.5–7.5) b 12.5 * - Vitamin K (µg/day) 12 - 45.0 (27.4–82.1) a 94.4 - 31.7 (19.9–75.4) a 94.7 - 38.8 (21.1–116.5) a 90.6 - Choline (mg/day) 140 - 112.4 (88.2–155.8) a 27.8 - 234.8 (184.4–286.5) b 88.2 * - 266.3 (187.0–299.4) b 89.1 - Calcium (mg/day) - 390 643.3 (525.3–736.1) a - 8.9 730.0 (577.8–975.8) a - 7.9 650.1 (527.4–889.1) a - 7.8 Copper (mg/day) 0.7 ‡ - 0.6 (0.4–0.7) a 27.8 - 0.9 (0.7–1.0) a 73.7 * - 1.0 (0.7–1.2) a 48.4 * - Phosphorus (mg/day) 250 - 631.0 (551.8–782.9) a 100 - 930.0 (777.1–1121.5) a 100 - 948.9 (774.4–1115.4) a 100 - Table 3. 3.3. Nutritional Status 3.3. Nutritional Status On the other hand, a decreased was observed in the contribution of fats, from 49% at the lactation period to 33% at 36 months. The variations in the contribution of macronutrient to energy intake during the study is presented in Figure 3. The contribution of protein to energy intake during lactation was 7%, and it increased significantly until the age of 24 months (19%). On the other hand, a decreased was observed in the contribution of fats, from 49% at the lactation period to 33% at 36 months. Figure 3. Percentage of the total energy intake provided by each macronutrient across the follow up. Mean daily energy intake: lactation period 538.64 kcal; weaning period 726.74 kcal; transition diet 1046.94 kcal and family diet at 24 months 1147.50 kcal and 36 months 1282.36 kcal. * p-Value < 0.05 from Kruskal–Wallis test compared to the previous category. 2-3 months 6 months 12 months 24 months 36 months 0 50 100 % Carbohydrates % Proteins % Fats Transition diet Weaning period Lactation period * * * * * * Family diet Figure 3. Percentage of the total energy intake provided by each macronutrient across the follow up. Mean daily energy intake: lactation period 538.64 kcal; weaning period 726.74 kcal; transition diet 1046.94 kcal and family diet at 24 months 1147.50 kcal and 36 months 1282.36 kcal. * p-Value < 0.05 from Kruskal–Wallis test compared to the previous category. Figure 3. Percentage of the total energy intake provided by each macronutrient across the follow up. Mean daily energy intake: lactation period 538.64 kcal; weaning period 726.74 kcal; transition diet 1046.94 kcal and family diet at 24 months 1147.50 kcal and 36 months 1282.36 kcal. * p-Value < 0.05 from Kruskal–Wallis test compared to the previous category. Figure 3. Percentage of the total energy intake provided by each macronutrient across the follow up. Mean daily energy intake: lactation period 538.64 kcal; weaning period 726.74 kcal; transition diet 1046.94 kcal and family diet at 24 months 1147.50 kcal and 36 months 1282.36 kcal. * p-Value < 0.05 from Kruskal–Wallis test compared to the previous category. 3.3. Nutritional Status 3.3. Nutritional Status Energy and macro- and micronutrients intake compared with dietary reference values (DRVs) based on intakes from the EFSA (European Food Safety Authority) in the study’s cohort at 12, 24 and 36 months. Nutrients 2022, 14, 1664 8 of 14 Table 3. Cont. DRV 12 Months n= 90 DRV Compliance 12 Months (%) 24 Months n = 76 DRV Compliance 24 Months (%) 36 Months n = 64 DRV Compliance 36 Months (%) AI AR Median (IR) >AI <AR Median (IR) >AI <AR Median (IR) >AI <AR Potassium (mg/day) 800 - 2070.7 (1712.9–2542.4) a 98.9 - 2365.8 (1836.7–2657.4) a 98.7 - 2506.2 (1941.0–2739.1) a 100 - Iron (mg/day) - 5 9.4 (7.6–11.3) a - 2.2 7.6 (6.3–8.8) a - 13.2 * 8.3 (6.6–9.9) a,b - 6.4 Magnesium (mg/day) 170 & - 147.7 (123.0–186.9) a 35.6 - 194.8 (155.8–232.1) a 65.8* - 203.7 (166.6–240.1) a 28.1 * - Selenium (µg/day) 15 - 30.9 (21.5–46.1) a 95.6 - 66.4 (49.5–79.7) b 100 - 68.5 (50.1–81.5) b 100 - Zinc (mg/day) - 3.6 5.8 (4.5–6.8) a - 10.0 6.4 (5.3–7.9) a - 3.9 6.6 (5.6–7.8) a - 4.7 Values are presented as the median (interquartile range). $ 9 mg/day at 3 years; & 230 mg/day at 3 years; ‡ 1 mg/day at 3 years. AI, adequate intake; ALAP, as low as possible; AR, average requirement; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; SFAs, saturated fatty acids. Different letters indicate significant differences between infants’ ages from the Kruskal–Wallis test (p < 0.05). * Differences compared to the previous category from a chi-square test (p < 0.05). Values are presented as the median (interquartile range). $ 9 mg/day at 3 years; & 230 mg/day at 3 years; ‡ 1 mg/day at 3 years. AI, adequate intake; ALAP, as low as possible; AR, average requirement; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; SFAs, saturated fatty acids. Different letters indicate significant differences between infants’ ages from the Kruskal–Wallis test (p < 0.05). * Differences compared to the previous category from a chi-square test (p < 0.05). 9 of 14 Nutrients 2022, 14, 1664 9 of 14 The variations in the contribution of macronutrient to energy intake during the study is presented in Figure 3. The contribution of protein to energy intake during lactation was 7%, and it increased significantly until the age of 24 months (19%). 3.4. Dietary Quality 3.4. Dietary Quality With the aim of exploring the impact of diet during the weaning period on the ad- herence to MD at 24 and 36 months, stepwise regression was conducted (Table 4). Energy intake at the weaning period was inversely associated with MDS at 24 months, contrary to vegetables that were positively associated with MDS at 36 months. With the aim of exploring the impact of diet during the weaning period on the ad- herence to MD at 24 and 36 months, stepwise regression was conducted (Table 4). Energy intake at the weaning period was inversely associated with MDS at 24 months, contrary to vegetables that were positively associated with MDS at 36 months. Table 4. Linear association between diet at 6 months and the Mediterranean Diet Score (MDS) at 24 and 36 months. MDS Predictors R2 β p MDS at 24 months Model Energy 0.069 −0.286 0.013 MDS at 36 months Model Vegetables 0.105 0.365 0.006 Fruits 0.105 −0.265 0.044 Result from linear stepwise regression analyses including energy, fruit, vegetables, fats, tubers, infant products and human breast milk at 6 months as potential predictors, and the Mediterranean Diet Score as the dependent variable β standardized regression coefficient; R2 adjusted coefficient Table 4. Linear association between diet at 6 months and the Mediterranean Diet Score (MDS) at 24 and 36 months. MDS Predictors R2 β p MDS at 24 months Model Energy 0.069 −0.286 0.013 MDS at 36 months Model Vegetables 0.105 0.365 0.006 Fruits 0.105 −0.265 0.044 Result from linear stepwise regression analyses including energy, fruit, vegetables, fats, tubers, infant products and human breast milk at 6 months as potential predictors, and the Mediterranean Diet Score as the dependent variable. β, standardized regression coefficient; R2, adjusted coefficient of multiple determination; p, p-value. Table 4. Linear association between diet at 6 months and the Mediterranean Diet Score (MDS) at 24 and 36 months. Table 4. Linear association between diet at 6 months and the Mediterranean Diet Score (MDS) at 24 and 36 months. 4. Discussion Our results increase the existing knowledge about the evolution of diet from the lactation period to the family diet in a Mediterranean cohort, identifying dietary targets that could determine the adherence to a higher quality diet in later age. Considering the importance to health of the first stage of life, the knowledge obtained from these results could be useful in the creation of future guidelines focused on the promotion of healthy habits. Despite WHO recommendations for exclusive breastfeeding up to 6 months, our results were similar to others with only a 53.6% of the children in the sample breastfed at 3 months [26]. Over time, the duration of breastfeeding was relatively good with 7.8% of the sample being breastfed at 36 months compared to the 2–6% reported by other authors for the interval of ages between 23 and 48 months [27,28]. Therefore, it is still necessary to strengthen strategies to promote it in the first months of life. As it has been described, breastfed infants showed a lower weight than formula-fed, 5.63 vs. 6.04 kg at the lactation period [29,30], contrary to what occurs in infant formula-fed babies, who presented a higher weight gain than those breastfed from 2 to 12 months of age [31,32]. This finding could be related with the association between breastfeeding and appetite regulation [33] or by the differences in the amount of energy, macro- and micronutrients and bioactive components between breast milk and infant formulas. In this regard, it is of great interest that children who were breastfed during the lactation period scored better on the MDS at 24 months of age. Even though these results are at the limit of statistical significance, they agree with numerous data about the impact of breastfeeding on a better acceptance of fruits and vegetables later in life [34]. However, considering that at this age infants do not have the capacity to make independent food choices, it cannot be discarded that family dietary habits may be influencing the observed associations. The complementary feeding represents a crucial stage in which a balance must be reached in order to guarantee the energetic and nutritional requirements of the child, allow- ing him/her to have adequate development according to age and considering their limited digestive capacity. 3.4. Dietary Quality 3.4. Dietary Quality Result from linear stepwise regression analyses including energy, fruit, vegetables, fats, tubers, infant products and human breast milk at 6 months as potential predictors, and the Mediterranean Diet Score as the dependent variable β standardized regression coefficient; R2 adjusted coefficient Result from linear stepwise regression analyses including energy, fruit, vegetables, fats, tubers, infant products and human breast milk at 6 months as potential predictors, and the Mediterranean Diet Score as the dependent variable. β, standardized regression coefficient; R2, adjusted coefficient of multiple determination; p, p-value. of multiple determination; p, p-value. On the other hand, fruits were also negatively associated with MDS at this age (Table 4). It is also noteworthy that a higher percentage of breastfed infants in the lactation period scored better on the MDS at 24 months of age than formula-fed infants. However, these On the other hand, fruits were also negatively associated with MDS at this age (Table 4). It is also noteworthy that a higher percentage of breastfed infants in the lactation period scored better on the MDS at 24 months of age than formula-fed infants. However, these differences were not significant (67.6% vs. 45.0%, p-value = 0.051). 10 of 14 Nutrients 2022, 14, 1664 10 of 14 4. Discussion At 12 months of age, the diet was called a Nutrients 2022, 14, 1664 11 of 14 11 of 14 transitional diet because, although all the food groups are consumed, as has been described in previous studies [44], breastfeeding, infant products and a semi-solid consistency in preparations were still maintained. At 24 and 36 months, most of the sample had a regular family diet. Interestingly, at 36 months, a reduction in the intake of vegetables was observed in favor of meat and meat products, which is in line with other studies and representative of the diet of adults in Westernized countries [45,46]. At the nutritional level, the percentage of total energy intake provided by each macronutrient was similar to previous studies [47–49], showing a moderate increase in protein from 12 to 36 months (15, 19 and 18%, respectively). The requirements for most nutrients were met at all ages studied. Some differences with other studies conducted in the pediatric population were observed. The median energy intake of our children compared to the DONALD study (German population) [50] was higher at all ages; however, compared to the ALSALMA study in Spanish children [48], it was only higher at 12 months. The US study, FITS, showed higher medians at all ages compared to our data [27]. As regards to nutrients, median protein intake at 24 months was higher in our study than in the ALSALMA and FITS (52.2 vs. 46.3 and 50 g/day, respectively) [27,48]. In addition, the median fiber intake in our study was higher for all of the studied times compared with the other studies [27,47,48]. These differences could be due to the presence of several factors including different methodologies, a wider range of age compared, the size of the sample and the different dietary habits among the populations studied. Regarding micronutrients, vitamin D was the most compromised in the study, in accordance with previous studies [49,51]. The determination of vitamin D intake depends on information from food composition tables and is subjected to seasonal variation. In Spain, children receive vitamin D supplements up to 12 months of age [52] and, as it is a sunny country, the requirements are likely to be covered [51]. However, a previous study in this area of the country suggested that sun exposure may not be enough to cover these deficiencies [53]. 4. Discussion In this regard, the European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) defines exclusive breastfeeding for around six months and establishes that the introduction of complementary foods should not occur before 17 weeks but should not be delayed beyond 26 weeks [35]. Apart from infant formula and breast milk, fruits were one of the most consumed foods in the sample at 6 months of age, being the third source of energy followed by infant cereals. This pattern was similar to other Mediterranean countries, such as Italy [36], and different in respect to others, such as England, where baby rice is the starting food [37]. Some food groups considered most allergenic, such as cow’s milk protein (except whole cow’s milk), egg, soy, wheat, peanut, tree nuts, fish and shellfish, were probably introduced into the diet beyond 6 months of age [38–40] and, in consequence, their contribution to the total energy intake at this age was inestimable. With independence of food groups, it was observed that energy intake at 6 months was inversely related with MDS at 24 months, which may be in line with other studies showing that the early introduction of high density foods is a predictor of a less healthy diet in the future [41]. Regarding the recommended daily servings by food group, it was observed that there was no agreement at these ages [25,42,43]. When comparing our data with the recommendations of the Spanish Society of Community Nutrition (SENC) [42] for children from 1 to 6 years of age, it was revealed that at 1 year they did not reach the daily recommendations of any food groups, except for red and processed meats, which exceeded the recommendations. On the other hand, at 36 months, a better adherence was observed, with fruits, vegetables and oils being slightly below the recommendations and red and processed meats above them. These discrepancies were due to the different portion sizes for vegetables, fruits and protein groups used to highlight the need for harmonized and standardized portion sizes for this age group [25,42,43]. From 6 to 12 months, a progressive change in food texture was produced from 9.9% of a semi-solid diet to 66.7%, respectively. 4. Discussion Therefore, it would be necessary to clarify whether supplementation with this vitamin for a longer period is needed to cover the nutritional requirements. Concerning adherence to the Mediterranean Diet at 36 months, it was found that those children who ate vegetables at 6 months of life had better MDS at 36 months. The relationship between the vegetable group and increased adherence to the Mediterranean Diet is well documented [54–56]. However, our results also show an inverse association between fruit consumption and MDS, contrary to other works [56,57], the reason for which remains to be elucidated. Among the fruits most consumed in the study sample were banana (52.7%), apple (48.4%), pear (47.3%) and orange (26.9%) followed by plum and strawberry in smaller proportions (4.3% and 2.2%, respectively). This study has several limitations related to its observational nature and the collection of dietary information. In interpreting this information, it should be noted that the energy and nutrient contents of processed infant foods were considered, a factor that has hitherto been underestimated in the literature. Regarding the quantification of breast milk energy, it is necessary to mention a limitation of the study. Since it was not possible to record the exact volume of milk produced by the mother, an indirect estimation was made using the mean amounts established in the literature for each age range [18,19]. While the quality of the FFQ depends on the respondent’s memory, its ability to accurately classify energy and all nutrient intakes in children is enhanced by the fact that the questionnaires were adapted from the PANCAKE study and photographs made it easier to interpret. In addition, it allowed for comparison with other studies on the European infant population. 5. Conclusions Due to the low percentage of breastfeeding reported during the lactation period, it would be advisable to establish strategies to promote breastfeeding until at least 6 months of life. The diet of children evaluated at 12, 24 and 36 months was characterized by a low intake of fruits and vegetables and an excess of meat, showing a pattern similar to adults in Western countries. In addition, the results allow us to hypothesize that a lower energy Nutrients 2022, 14, 1664 12 of 14 12 of 14 intake and the introduction at 6 months to certain food groups, such as vegetables, are associated with better MDS scores at 24 and 36 months, respectively. Finally, given the large percentage of vitamin D deficiency, it could be suggested as a nutritional target for infants over 12 months of age. intake and the introduction at 6 months to certain food groups, such as vegetables, are associated with better MDS scores at 24 and 36 months, respectively. Finally, given the large percentage of vitamin D deficiency, it could be suggested as a nutritional target for infants over 12 months of age. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu14081664/s1. Table S1: Evolution of the intake of the major food groups with follow up; Figure S1: Adherence to daily food recommendations in the sample with follow up [25]. Author Contributions: S.G. and M.G. designed the study; D.H.-M. and S.A. recruited participants; M.G.-M. performed the nutritional assessment and statistical analyses; S.G. and M.G.-M. drafted the manuscript; M.G.-M., D.H.-M., S.A., M.G. and S.G. revised the final version of the paper. All authors have read and agreed to the published version of the manuscript. Funding: This work was financed by PCIN-2015-233 (Project EarlyMicroHealth from the EU Joint Program Initiative HDHL) (MINECO/FEDER, UE) and by the University of Oviedo (Diet, Rhythms and Early Acquisition Microbiota (DREAMS) Project; Reference no. 2017/00001/003/005/013). S.A. was the recipient of a postdoctoral Juan de la Cierva Contract (Reference no. IJCI-2017-32156) funded by MCIU/AEI/10.13039/501100011033. M.G-M. was the recipient of a pre-doctoral FPU contract (FPU18/03393) funded by the Spanish Ministry of Science, Innovation, and Universities. Institutional Review Board Statement: The study was evaluated and approved by the Regional Ethics Committee of Clinical Research of Asturias (Reference no. 12/16, 3 February 2016) and the Committee on Bioethics of CSIC (Reference no. PCIN-2015-233). Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank all the families involved in the EarlyMicroHealth study as well as the Primary Care Pediatric Nurses, Margot Calzón and Teresa López, for their contribution to volunteer recruitment. Conflicts of Interest: The authors declare no conflict of interest. reen, R.; Sutherland, J.; Dangour, A.D.; Shankar, B.; Webb, P. Global dietary quality, undernutrition and no sease: A longitudinal modelling study BMJ Open 2016 6 e009331 e009340 [CrossRef] [ ] ; Sutherland, J.; Dangour, A.D.; Shankar, B.; Webb, P. Global dietary quality, undernutrition and non-com g g y p 8. United Nations Educational Scientific and Cultural Organization (UNESCO). Evaluation of Nominations for Inscription in 2010 on the Representative List of the Intangible Cultural Heritage of Humanity. Available online: https://ich.unesco.org/en/ decisions/5.COM/6.41 (accessed on 10 February 2022). , ; , J ; g , ; , ; , y q y, e: A longitudinal modelling study. BMJ Open 2016, 6, e009331–e009340. [CrossRef] 5. Conclusions The procedures were performed in accordance with the fundamental principles set out in the Declaration of Helsinki, the Oviedo Bioethics Convention, the Council of Europe Convention on Human Rights and Biomedicine and in Spanish legislation on bioethics. 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Dynamic evolution of ceftazidime–avibactam resistance due to interchanges between blaKPC-2 and blaKPC-145 during treatment of Klebsiella pneumoniae infection

Frontiers in cellular and infection microbiology

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Dynamic evolution of ceftazidime–avibactam resistance due to interchanges between blaKPC-2 and blaKPC-145 during treatment of Klebsiella pneumoniae infection OPEN ACCESS EDITED BY Ronni Mol Joji, Arabian Gulf University, Bahrain REVIEWED BY Dafne Bongiorno, University of Catania, Italy Qiucheng Shi, Zhejiang University, China *CORRESPONDENCE Junqi Huang [email protected] Kang Liao [email protected] †These authors have contributed equally to this work RECEIVED 22 June 2023 ACCEPTED 31 July 2023 PUBLISHED 21 August 2023 CITATION Chen Y, Yang R, Guo P, Liu P, Deng J, Wu Z, Wu Q, Huang J and Liao K (2023) Dynamic evolution of ceftazidime– avibactam resistance due to interchanges between blaKPC-2 and blaKPC-145 during treatment of Klebsiella pneumoniae infection. Front. Cell. Infect. Microbiol. 13:1244511. doi: 10.3389/fcimb.2023.1244511 COPYRIGHT Yili Chen 1†, Runshi Yang 2†, Penghao Guo 1, Pingjuan Liu 1, Jiankai Deng 1, Zhongwen Wu 1, Qingping Wu 2, Junqi Huang 1,3,4,5* and Kang Liao 1* RECEIVED 22 June 2023 1Department of Laboratory Medicine, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, 2Guangdong Provincial Key Laboratory of Microbial Safety and Health, Key Laboratory of Agricultural Microbiomics and Precision Application, Ministry of Agriculture and Rural Affairs, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China, 3Organ Transplant Center, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, 4Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China, 5Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China CITATION Chen Y, Yang R, Guo P, Liu P, Deng J, Wu Z, Wu Q, Huang J and Liao K (2023) Dynamic evolution of ceftazidime– avibactam resistance due to interchanges between blaKPC-2 and blaKPC-145 during treatment of Klebsiella pneumoniae infection. Front. Cell. Infect. Microbiol. 13:1244511. doi: 10.3389/fcimb.2023.1244511 Background: The emergence of ceftazidime–avibactam (CZA) resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) is of major concern due to limited therapeutic options. © 2023 Chen, Yang, Guo, Liu, Deng, Wu, Wu, Huang and Liao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Methods: In this study, 10 CRKP strains were isolated from different samples of a patient with CRKP infection receiving CZA treatment. TYPE Original Research PUBLISHED 21 August 2023 DOI 10.3389/fcimb.2023.1244511 TYPE Original Research PUBLISHED 21 August 2023 DOI 10.3389/fcimb.2023.1244511 Frontiers in Cellular and Infection Microbiology Antimicrobial susceptibility testing Antimicrobial susceptibilities for the strains were detected by Gram-negative susceptibility (GNS) cards on the Vitek-2 system (bioMérieux, Marcy l’Etoile, France). MICs of tigecycline and CZA were determined by broth microdilution method. Susceptibility testing results were interpreted under the criteria recommended by the Clinical and Laboratory Standards Institute (CLSI, 2022). The quality control strain for susceptibility testing was Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603. Ceftazidime–avibactam (CZA) has been approved as an effective antibiotic for CRE treatment. However, the emergency of CZA resistance rapidly appeared after its widespread application for the treatment of CRE infections (Shields et al., 2016). Some sporadic reports of CZA resistance were observed after the use of CZA for the treatment of KPC in China, such as in Shanghai (Shi et al., 2020) and Henan (Li et al., 2021a). Nevertheless, there are currently no reports of CZA developing resistance after use in southern China. In this study, we reported the dynamic evolution of CZA resistance due to a mutation between plasmid-borne blaKPC-2 and blaKPC-145 by sequencing clinical isolates from a patient with CRKP infection undergoing CZA treatment. To our knowledge, this is the first report on KPC-145 producing CRKP, which harbored a new amino acid substitution site on the basis of KPC-33. Dynamic evolution of ceftazidime–avibactam resistance due to interchanges between blaKPC-2 and blaKPC-145 during treatment of Klebsiella pneumoniae infection Whole-genome sequencing (WGS) and conjugation experiments were performed to determine the transferability of the carbapenem resistance gene. Results: This infection began with a KPC-2-producing K. pneumoniae (CZA MIC = 2 mg/mL, imipenem MIC ≥16 mg/mL). After 20 days of CZA treatment, the strains switched to the amino acid substitution of T263A caused by a novel KPC- producing gene, blaKPC-145, which restored carbapenem susceptibility but showed CZA resistance (CZA MIC ≥256 mg/mL, imipenem MIC = 1 mg/mL). The blaKPC-145 gene was located on a 148,185-bp untransformable IncFII-type plasmid. The subsequent use of carbapenem against KPC-145-producing K. pneumoniae infection led to a reversion of KPC-2 production (CZA MIC = 2 mg/ mL, imipenem MIC ≥16 mg/mL). WGS analysis showed that all isolates belonged to ST11-KL47, and the number of SNPs was 14. This implied that these blaKPC- positive K. pneumoniae isolates might originate from a single clone and have been colonized for a long time during the 120-day treatment period. 01 Frontiers in Cellular and Infection Microbiology frontiersin.org Chen et al. 10.3389/fcimb.2023.1244511 10.3389/fcimb.2023.1244511 Conclusion: This is the first report of CZA resistance caused by blaKPC-145, which emerged during the treatment with CZA against blaKPC-2-positive K. pneumoniae-associated infection in China. These findings indicated that routine testing for antibiotic susceptibility and carbapenemase genotype is essential during CZA treatment. carbapenem resistance, Klebsiella pneumoniae, ceftazidime-avibactam resistance, KPC-2, KPC-145 Carbapenemase phenotype and genotype detection The modified carbapenem inactivation method (mCIM) was carried out according to the recommendations of the CLSI (M100- S29). A carbapenemase inhibitor enhancement testing kit was purchased from Zhuhai Deere Bioengineering Co. Ltd. (Zhuhai, Guangdong, China), and the assay was performed as previously described (Tsakris et al., 2010). Introduction around the liver and around the pancreas. The patient developed septic shock 20 days after admission. The first K. pneumoniae strain (CZHKP-01) was obtained from a blood culture before CZA treatment. The third strain (CZHKP-03) was isolated from stool specimens. The remaining eight strains were isolated from the drainage fluid of the abdominal cavity. Species identification was performed by matrix-assisted laser desorption ionization–time-of- flight mass spectrometry (MALDI-TOF MS) (bioMérieux, Marcy l’Etoile, France). With the extensive application of carbapenems, carbapenem- resistant Enterobacterales (CRE) infections have been a major threat to public healthcare, especially carbapenem-resistant Klebsiella pneumoniae (CRKP)-associated infections (Kim et al., 2017; Plazak et al., 2018). The China Antimicrobial Surveillance Network (CHINET) reported that there is an upward trend in carbapenem resistance in Klebsiella pneumoniae (Hu et al., 2019). CRE-related infections caused the deaths of 42.1% of patients with infections and 73% of severely infected patients who were in septic shock (Daikos et al., 2014; Xu et al., 2017). The production of Klebsiella pneumoniae carbapenemase (KPC), especially KPC-2, is dominant among CRKP ST11 strains in China (Yang et al., 2013; Zhang et al., 2017). Patients and methods Xpert Carba-R (Cepheid, Sunnyvale, CA, USA), NG-Test® CARBA 5 (NG Biotech, Guipry, France), and Goldstream Carbapenem-resistant K.N.I.V.O. Detection K-Set (Beijing Gold Mountain River Tech Development Co. Ltd., Beijing, China) were applied for rapid detection of 10 strains carbapenemase genotype according to the instructions. Targeted PCR was performed. The primers KPC1F (5′-GCTACACCTAGCTCCACCTTC-3′) and KPC1R (5′-GCATGGATTACCAACCACTGT-3′) were used to sequence the entire open reading frame (ORF) of the blaKPC gene (Smith Moland et al., 2003). Frontiers in Cellular and Infection Microbiology carbapenem resistance, Klebsiella pneumoniae, ceftazidime-avibactam resistance KPC-2, KPC-145 KEYWORDS Sample source and identification Ten strains of K. pneumoniae were isolated from a 32-year-old man who was diagnosed with severe acute pancreatitis and multiple organ failure on admission to the First Affiliated Hospital of Sun Yat-sen University, Guangzhou. Empiric treatment with tigecycline, imipenem, and caspofungin was adopted, and surgical drainage was performed gradually from 02 frontiersin.org Chen et al. 10.3389/fcimb.2023.1244511 Alterations in drug resistance during treatment In our case, after initial combination treatment with imipenem (2.0 g, IV, Q8h) and tigecycline (first dose of 100 mg, then 50 mg, IV DRIP, Q12h) for 20 days, blaKPC-2-positive K. pneumoniae strains (CZHKP-01 and CZHKP-02) were isolated from the blood culture and drainage fluid, showing resistance to imipenem and meropenem (Table 1). Subsequently, antibiotic therapy was adjusted to combination with CZA (2.5 g, IV DRIP, Q8h), polymyxin B (first dose of 1,000,000 U, then 500,000 U, IV, Q12h), tigecycline, and caspofungin, along with adequate surgical drainage (from perihepatic, lesser omental sac, right peripancreatic, and cystic duct). However, after using CZA for 20 days, the patient’s body temperature rose again. The first CZA resistance K. pneumoniae strain CZHKP-03 was isolated from fecal culture followed by CZHKP-05~CZHKP-09 (all isolated from abdominal drainage fluid). These strains restored carbapenem susceptibility but showed CZA resistance (CZA MIC ≥256 mg/mL, imipenem MIC = 1 mg/mL). Considering that long-term use of CZA may lead to drug-resistant strains, we stopped using CZA and switched to polymyxin B and tigecycline for anti-infection treatment. Results Plasmid DNA was extracted from parent strains, and primers targeting the entire ORF of the blaKPC-2 gene and blaKPC-145 gene, along with NcoI and BamHI restriction sites, were designed and used for PCR. The PCR products and the plasmid vector pET28a were digested with the restriction enzymes NcoI–BamHI (TaKaRa Biotechnology, Dalian, China) and then ligated at 16°C overnight. The recombinant plasmids were transformed into E. coli BL21 (DE3); all the transformants were confirmed by PCR and sequencing analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to verify the expression of the target proteins KPC-2 and KPC-145. Antimicrobial susceptibility testing for the BL21 transformants was performed as mentioned above. Transferability of carbapenem resistance gene characterize IncF plasmids, as described previously (Carattoli et al., 2005; Villa et al., 2010). Prophage analysis was conducted by PHAST (http://phaster.ca/). Multilocus sequence types (MLST) and antibiotic resistance genes (ARGs) were analyzed using ABRicate (https:// github.com/tseemann/abricate). Capsule serotype and virulence genes (yersiniabactin, colibactin, and other siderophore locus) were determined using Kleborate (Lam et al., 2021). Core genome alignment and calling of single nucleotide polymorphisms (SNPs) were carried out using the Snippy (https://github.com/tseemann/ snippy) against reference strain CZHKP-07 and followed by purging using Gubbins (Croucher et al., 2015). The heatmap showing the SNP matrix was generated by using the “pheatmap” package in RStudio; core-genome SNPs were used to construct a maximum-likelihood phylogenetic tree by RAxML (Stamatakis, 2014) and visualized by iTOL (Letunic and Bork, 2016). The transferability of the carbapenem resistance phenotype was determined by conjugation experiments (Lorenzo-Diaz and Espinosa, 2009) using the streptomycin-resistant E. coli C600 strain. Transconjugants were chosen on MacConkey agar plates (Huankai Co. Ltd., Guangzhou, China) supplemented with cefotaxime (1 mg/L) and streptomycin (1,500 mg/L). The electrotransformation experiments (Choi et al., 2006) were further performed for isolates that failed conjugation experiments. In brief, plasmid DNA was extracted using a QIAPrep Plasmid Midi Kit (QIAGEN, Hilden, Germany) and transformed into electro- competent E. coli DH5a (TaKaRa Biotechnology, Dalian, China). electrotransformants were selected on LB agar plates (Huankai Co. Ltd., Guangzhou, China) supplemented with cefotaxime (1 mg/L). We further confirmed the transconjugants or electrotransformants by PCR for the blaKPC-2 gene and tested for antimicrobial susceptibility as described above. Data availability The sequence of blaKPC-145 has been deposited in GenBank under Accession No. OP626310. Frontiers in Cellular and Infection Microbiology frontiersin.org Cloning of blaKPC-145 gene The recombinant plasmid carrying the blaKPC-145 gene in E. coli BL21, which conferred resistance to ceftazidime/avibactam (MIC = 2 mg/L) and displayed decreased carbapenem MICs (imipenem MIC = 0.125 mg/L and meropenem MIC = 0.25 mg/L) compared with blaKPC-2 in BL21. Moreover, the MICs of cefotaxime, ceftazidime, and aztreonam were 32-, 16-, and 16-fold higher than the original recipient E. coli BL21. However, compared with blaKPC-2, the MICs of blaKPC-145-carrying electrotransformants to carbapenems were 32- to 128-fold lower than those of blaKPC-2-carrying transformants. Whole-genome sequencing and bioinformatics analysis Detection K-Set GeneXpert Ceftazidime– avibactam Imipenem Meropenem Polymyxin B Tigecycline CZHKP-01 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC CZHKP-02 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC CZHKP-03 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-04 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC CZHKP-05 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-06 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-07 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-08 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-09 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-10 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC aPositive for Ambler class A carbapenemase F ti i C ll l d I f ti Mi bi l TABLE 1 Klebsiella pneumoniae drug susceptibility testing and carbapenemase testing results. Strain MIC (mg/mL) mCIM Carbapenemase inhibitor enhancement NG-test CARBA 5 K.N.I.V.O. Detection K-Set GeneXpert Ceftazidime– avibactam Imipenem Meropenem Polymyxin B Tigecycline CZHKP-01 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC CZHKP-02 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC CZHKP-03 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-04 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC CZHKP-05 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-06 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-07 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-08 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-09 ≥256 1 4 ≤0.5 2 Neg Neg Neg KPC KPC CZHKP-10 2 ≥16 ≥16 ≤0.5 2 Posa Posa KPC KPC KPC aPositive for Ambler class A carbapenemase. treatment on the 120th day of hospitalization. Clinical and microbiological details, timelines, and antibiotic therapies used are summarized in Figure 1. Detection of carbapenemase The performance comparison of different carbapenem enzyme detection methods among the 10 strains is shown in Table 1. Sanger sequencing results of the blaKPC gene revealed that four CZA- susceptible isolates (CZHKP-01, CZHKP-02, CZHKP-04, CZHKP- 10) carried the blaKPC-2 gene. Interestingly, the rest of the six CZA- resistant isolates (CZHKP-03, CZHKP-05, CZHKP-06, CZHKP-07, CZHKP-08, and CZHKP-09) harbored a novel blaKPC gene with a point mutation (A to G) at nucleotide position 787 compared with the blaKPC-33 gene; this mutation resulted in the amino acid substitution of threonine to alanine (T263A), assigned by GenBank as blaKPC-145. In addition, the results of mCIM, carbapenemase inhibitor enhancement testing, and NG-Test CARBA 5 showed negative results for blaKPC-145-positive strains, which indicated that routine clinical detections have some defects on blaKPC-145-positive strains. Another immunochromatographic method, Goldstream Carbapenem-resistant K.N.I.V.O. Detection K-Set, which showed positive results in detecting blaKPC-145 strains, seems to have a better cost-to-benefit ratio. Moreover, molecular screening of GeneXpert for blaKPC-145 showed good sensitivity. Transferability blaKPC-2 and blaKPC-145 gene TABLE 1 Klebsiella pneumoniae drug susceptibility testing and carbapenemase testing results. Unfortunately, despite numerous attempts conducted by conjugation assays, the transfer of blaKPC-2 and blaKPC-145 genes failed in all 10 K. pneumoniae isolates. Electrotransformations were further performed, and 10 electrotransformants were successfully obtained. All of the electrotransformants exhibited resistance to all b-lactams, aminoglycosides, and fosfomycin. Whole-genome sequencing and bioinformatics analysis Genomic DNA of the selected isolates was extracted using a DNA extraction kit (Magen, Guangzhou, China), and 150 bp paired-end reads were obtained using an Illumina NextSeq 550 system (Illumina, San Diego, CA, USA). For each whole-genome sequencing (WGS) data, at least 100-fold coverage of original reads was obtained, and the assembly draft of the sequences was generated by SPAdes (Bankevich et al., 2012). Furthermore, a representative strain (CZHKP-07) was selected for further sequencing by using the MinION platform (Oxford Nanopore Technologies, Oxford, UK). On the 105th day of hospitalization, the anti-infection therapy was adjusted to tigecycline combined with meropenem. After over a week of treatment, the heat peak of the patient’s body temperature decreased and the drainage fluid decreased gradually. On the 113th day of hospitalization, CZHKP-10 isolated from abdominal drainage fluid culture recovered susceptibility to CZA (CZA MIC = 2 mg/mL, imipenem MIC ≥16 mg/mL). Through sufficient surgical drainage and reasonable anti-infection treatment, the patient’s condition ultimately improved and returned to the regular ward for further A hybrid assembly of both short Illumina NextSeq reads and long MinION reads was built using Unicycler with the Pilon option to modify the assembled readings (Walker et al., 2014; Wick et al., 2017) and annotated with RAST (Aziz et al., 2008) and Prokka (Seemann, 2014) for functionality. The PCR-based replicon typing (PBRT) and replicon sequencing typing (RST) methods were applied to 03 frontiersin.org Chen et al. 10.3389/fcimb.2023.1244511 TABLE 1 Klebsiella pneumoniae drug susceptibility testing and carbapenemase testing results. Strain MIC (mg/mL) mCIM Carbapenemase inhibitor enhancement NG-test CARBA 5 K.N.I.V.O. frontiersin.org Discussion A linear sequence comparison of the plasmids pCZHKP07-2, pHN7A8 (Accession No. JN232517), and p1068-KPC (Accession No. MF168402) was performed. The genetic structure suggested that pCZHKP07-2 possessed a similar backbone structure as F33:A −:B−plasmid pHN7A8, but a large segment of the conjugative transfer-associated genes (tra and trb) was absent (Figure 2A), which might be the reason for conjugation failure. Therefore, we speculate that the insertion of an extra copy of IS26 at target site TAACTGAA on the traI gene in the pHN7N8 plasmid, followed by homologous recombination between IS26 elements, may have led to the loss of the conjugative transfer regions. Meanwhile, two variable regions and a prophage region were embedded in the pCZHKP07-2 (Figure 2A). The first variable region (VR1) was 15,338 bp in length and was flanked at both ends by fragments of IS1 found in pCZHKP07-2, which consists of four different resistance genes, blaCTX-M-65, fosA3, blaTEM-1B, and rmtB, and abundant complete or truncated transposons. This structure was similar to the pHN7A8 except for the fosA3 resistance module with an opposite orientation, which was flanked by two intact copies of IS26 at both ends (Figure 2B). The second variable region (VR2) possessed blaKPC- 145 and blaSHV modules. The blaKPC-145 and blaSHV regions in pCZHKP07-2 were disrupted into two separate parts, which may be caused by homologous recombination after IS26 insertion. The genetic structure of blaKPC-145 was composed of Tn1722-based unit KPC-2-producing strains, especially those that belong to ST11, are the majority population of K. pneumoniae resistant to carbapenems and become one of the most urgent public health issues in China (Li et al., 2021b). Avibactam is a b-lactamase inhibitor with a wide range of therapeutic effects against serine b- lactamases, including KPC. Combined with ceftazidime, CZA has become an important therapeutic option for treating KPC- producing K. pneumoniae infections (Tumbarello et al., 2021). Unfortunately, the development of CZA resistance has increased since it was clinically approved. In the International Network for Optimal Resistance Monitor (INFORM) surveillance program (2015–2017), the susceptibility rate of CZA against CRE was 73.0% (Spiliopoulou et al., 2020). A recent study from the CHINET revealed that CZA exhibited potent activity against Enterobacterales (93.6% susceptible), while only 65.2% of CRE remained susceptible to CZA (Guo et al., 2022). WGS analysis and phylogenetic characteristics A total of 10 KPC-producing strains were subjected to WGS. The in silico analysis showed that these 10 strains belonged to ST11- Frontiers in Cellular and Infection Microbiology 04 frontiersin.org Chen et al. 10.3389/fcimb.2023.1244511 KL47 (associated with the wzi allele 209) and harbored 11 resistance genes, conferring resistance to aminoglycoside (aac3-IId, rmtB), fosfomycin (fosA3), quinolone (qnrS1), tetracycline (tetA), b-lactam (blaKPC-2 or blaKPC-145, blaCTX-M-64, blaTEM-1B, blaSHV-12), and trimethoprim/sulfamethoxazole (sulI, dfrA1). Furthermore, the representative K. pneumoniae strain (CZHKP-07) was sequenced using the MinION platform, followed by hybrid assembly. The CZHKP-07 chromosome is 5,448,939 bp in length, with a GC content of 57.44%, and harbors five plasmids with sizes of 230,198 bp (IncFIB and IncFII), 148,185 bp (IncFII), 55,161 bp, 11,970 bp (ColRNAI) and 5,596 bp (ColRNAI), named pCZHKP07-1 to pCZHKP07-5 (Supplementary Table S1). The pCZHKP07-2 was an F33:A−:B−plasmid and contained six resistance genes, including blaKPC-145. Meanwhile, the plasmid pCZHKP07-2 possessed addiction systems (pemKI, hok-sok-mok, ccdAB); these genes promote plasmid maintenance during vertical transmission. transposon including ISKpn27-blaKPC-145-ISKpn6-korC-klcA- DrepB, and the blaSHV module was flanked by two intact or truncated IS26, resulting the differed from p1068-KPC with the inversion of this segment (Figure 2C). Moreover, the prophage region (30,305 bp) of pCZHKP07-2, flanked by 8 bp ATCCTCCT direct repeats (DRs), was inserted into the plasmid pIR12183_unnamed1 ATPase gene and bound at both ends by IS26. A maximum likelihood phylogenetic tree and SNP matrix were constructed based on core- genome SNPs (cgSNPs) from the 10 genomes in this study. We identified four KPC-2-positive K. pneumoniae and six KPC-145- positive K. pneumoniae that were separated into two clades and shared 14 SNPs in total (Figure 3). Discussion In the present study, the first CZA resistance strain was isolated after treatment with CZA for 20 days, consistent with the previous reports that CZA resistance commonly appears after 10 to 19 days and sometimes after 33 days (Shi et al., 2020). Our study also showed that the blaKPC-145 gene-mediated CZA resistance was accompanied by a substantial decrease in carbapenem MICs. Moreover, when the therapy was subsequently adjusted to meropenem, it took only 4 days for the strains to change from FIGURE 1 History of the K. pneumoniae isolations and the clinical antimicrobial treatment course of a patient with KPC-Kp infection. FIGURE 1 History of the K. pneumoniae isolations and the clinical antimicrobial treatment course of a patient with KPC-Kp infection. 05 Frontiers in Cellular and Infection Microbiology Frontiers in Cellular and Infection Microbiology 05 frontiersin.org Chen et al. 10.3389/fcimb.2023.1244511 A B C FIGURE 2 Linear comparison of the KPC-145-positive plasmid pCZHKP07-2 identified in this study with plasmid pHN7A8, p1068-KPC, and pIR12183_unnamed1 (A) and two variable regions (B, C). The arrows indicate the positions and transcriptional directions of the ORFs, while homologous regions are represented in gray. A A B B C FIGURE 2 Linear comparison of the KPC-145-positive plasmid pCZHKP07-2 identified in this study with plasmid pHN7A8, p1068-KPC, and pIR12183_unnamed1 (A) and two variable regions (B, C). The arrows indicate the positions and transcriptional directions of the ORFs, while homologous regions are represented in gray. blaKPC-145 back to blaKPC-2. It was observed that the in vivo evolution of wild-type KPC-2 changed into KPC-145 and then reversed to its original wild-type KPC-2. Alternatively, the combination of CZA and carbapenems may be an ideal option, which has been proven to be effective in some clinical cases (Bianco et al., 2020; Shi et al., 2020; Li et al., 2021a). In addition, meropenem–vaboractam has been receiving increasing attention as a potential antibiotic for clinics, and there are no K. pneumoniae- harboring mutant blaKPC resistant to meropenem–vaborbactam (Wilson et al., 2019). lactamases, increased expression of KPC carbapenemases with porin mutations, and amino acid changes of KPC carbapenemases. Among them, the primary CZA resistance mechanism is a single mutation within the blaKPC gene allele, which is commonly observed in several pivotal regions, especially the omega loop (amino acid positions 164–179), the 240-loop (Galani et al., 2021) (aa 238–243) and 270-loop (aa 263–277) (Venditti et al., 2021). Frontiers in Cellular and Infection Microbiology Discussion The novel variant, KPC-145, with two amino acid substitutions (D179Y and T263A), was identified in our study. These two amino acid substitutions were observed in KPC-3, named KPC-70 (Carattoli et al., 2021). Our results revealed that KPC-145 mediated the CZA resistance and restored susceptibility to carbapenems, similar to KPC-70 in the So far, there are three principal CZA resistance mechanisms (Galani et al., 2021), including the production of metallo-b- Frontiers in Cellular and Infection Microbiology 06 frontiersin.org Chen et al. 10.3389/fcimb.2023.1244511 FIGURE 3 The maximum likelihood phylogenetic tree generated from the core-genome sequences of the 10 KPC-positive strains identified in this study. Each isolate is listed with its ST, capsular serotype, blaKPC allele, and antimicrobial susceptibility results. FIGURE 3 The maximum likelihood phylogenetic tree generated from the core-genome sequences of the 10 KPC-positive strains identified in this study. Each isolate is listed with its ST, capsular serotype, blaKPC allele, and antimicrobial susceptibility results. FIGURE 3 The maximum likelihood phylogenetic tree generated from the core-genome sequences of the 10 KPC-positive strains identified in this study. Each isolate is listed with its ST, capsular serotype, blaKPC allele, and antimicrobial susceptibility results. CG258, responsible for the global dissemination of CRKP. This underscores the importance of coordinated international action to surveillance and control the high-risk clone type. IncF is a narrow host plasmid that is widely found in Enterobacteriaceae. Numerous studies have shown that the IncF subtype IncFII plasmid carries a variety of ARGs and plays a significant role in the transmission of specific resistance genes, including the blaKPC gene (Shi et al., 2018). In our study, the untransferable IncFII plasmid carried by K. pneumoniae encodes the resistance genes blaKPC-145, blaTEM-1B, blaSHV-12, blaCTX-M-64, fosA3, and rmtB, which are associated with CZA, cephalosporins, fosfomycin, and aminoglycosides, suggesting that IncFII plasmid contribute to the transmission of resistant strains and ARGs. The mobile genetic elements were widely distributed in the bacterial genomes and played a crucial role in the dissemination of antibiotic resistance (Partridge et al., 2018). The diverse structure of variable regions may be caused by a series of molecular module rearrangements, acquisitions, or loss events mediated by insertion sequences such as IS26 by transposition or homologous recombination. It is also worth pointing out that IS26 not only mediated the inversion of the resistance modules but also caused the absence of large segments of the conjugative transfer region. previous report. Discussion The D179Y mutation within the omega loop may be the main mechanism for resistance change for the KPC-145 enzyme, which causes enhanced affinity to ceftazidime and restricts avibactam binding (Winkler et al., 2015). In addition, it is noteworthy that the number of KPC variants has been increasing rapidly in recent years; this situation poses a significant threat to public health. Therefore, rapid and accurate detection of KPC-subtype- producing strains plays an important role in clinical drug selection and patient prognosis. However, in our study, the results of mCIM, carbapenemase inhibitor enhancement testing, and NG- Test® CARBA 5 showed negative results for blaKPC-145-positive strains, which indicated deficiencies in routine clinical testing for blaKPC-145-positive strains. Both K.N.I.V.O. Detection K-Set and GeneXpert showed satisfactory diagnostic performance in detecting blaKPC-145, which was consistent with other studies on the detection ability of KPC-2 variants (Ding et al., 2021). The dissemination of resistance genes is also associated with sequence types and plasmid replicon types. Since the emergence of KPC-producing K. pneumoniae in 2001 (Yigit et al., 2001), ST11 has become the most prevalent CRKP clone in Asia, especially in China. In this study, we found that all isolates belonged to ST11-KL47, and the number of SNPs was 14. This implies that these blaKPC-positive K. pneumoniae isolates might originate from a single clone and long-term colonization in this patient during the 120-day therapy. Meanwhile, CZA resistance encoded by blaKPC variants has been increasingly reported in ST11 K. pneumoniae in China, including blaKPC-33 (Shi et al., 2020), blaKPC-51, blaKPC-52 (Sun et al., 2020), blaKPC-74 (Li et al., 2021b), and blaKPC-93 (Wu et al., 2022). Otherwise, ST11 is a single-locus variant of the globally popular high-risk clone ST258 through the acquisition of the tonB allele (Breurec et al., 2013). These two sequence types together with ST512 and a few other SNP variants comprising the clonal group References Aziz, R. K., Bartels, D., Best, A. A., Dejongh, M., Disz, T., Edwards, R. A., et al. (2008). The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9, 75. doi: 10.1186/1471-2164-9-75 Ding, L., Shi, Q., Han, R., Yin, D., Wu, S., Yang, Y., et al. (2021). Comparison of four carbapenemase detection methods for blaKPC-2 variants. Microbiol. Spectr. 9, e0095421. doi: 10.1128/Spectrum.00954-21 Galani, I., Karaiskos, I., and Giamarellou, H. (2021). Multidrug-resistant Klebsiella pneumoniae: mechanisms of resistance including updated data for novel beta-lactam- beta-lactamase inhibitor combinations. Expert Rev. Anti Infect. 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Frontiers in Cellular and Infection Microbiology 07 frontiersin.org Chen et al. 10.3389/fcimb.2023.1244511 Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2023.1244511/ full#supplementary-material This work was supported by the Guangdong Natural Science Foundation - General Program. Yili Chen is a grant recipient of the project (2023A1515011252). Author contributions YC, RY, QW, JH, and KL conceived and designed the experiments. YC, JD, and ZW collected clinical data. 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TO THE EDITOR: Professor Morley was correct in pointing out in our Perspective we focused on analysis of Poisson noise, a type of sampling error [1]. Indeed, it is sometimes useful to distinguish between errors in sampling cells from a sub-population and errors in sampling a sub-population from the entire population of cells. The underlying mechanisms for these two types of sampling errors could be similar. Although in our Perspective we did not dwell on spatial distribution of leukaemia cells, we now caution an uneven spatial distribution could also be due to Poisson noise, as exemplified by R. D. Clarke’s classic spatial analysis of the distribution of flying- bomb attacks in London during WWII [2]. It is also important to recognise any sampling error at the cell-count level cannot be salvaged by lysis of the sampled cells for subsequent nucleic acid analysis such as quantitative real-time polymerase chain reaction (RT-qPCR) or next-generation sequencing (NGS). Relapse-free survival (%) Some leukaemia treatment protocols do call for measurable residual disease (MRD)-testing at a time when collecting a large number of bone marrow cells is not always feasible [3, 4]. The ideal scenario is of course having a multi-parameter flow cytometry (MPFC)-based MRD-test declared positive only if ≥5 × 10E+5 cells are analysed and if ≥50 cells are positive, but oftentimes physicians need to make decisions under non-ideal conditions. Should consideration of sampling errors affect the treatment plan for a person? We believe it should, but not until more validation studies are conducted. We agree a global platform such as EuroMRD would be the right venue for advancing proper usage of MRD-tests. Years from initial treatment Fig. 1 Risk-stratification based on joint consideration of esti- mated relapse risk at diagnosis and MRDworst case on day 19 when MRDconventional on day 19 was <0:01%. The study cohort is as described previously [1]. opinion, it is crucial for an MRD researcher to clarify the impact of an MRD-testing result on the cumulative incidence of relapse itself. g g p p g Professor Morley argued that decisions based on the conven- tional MRD values will optimise treatment for the group as a whole. We disagree. The conventional MRD value is not the mean or median estimate of true MRD. Rather, when conventional MRD is zero, it is the optimist’s rosy estimate of true MRD assuming all such patients have near-zero leukaemia cell. Received: 23 October 2023 Revised: 31 October 2023 Accepted: 10 November 2023 Published online: 21 November 2023 TO THE EDITOR: Making decisions based on such false optimism would not optimise treatment for the group as a whole, not to mention some of the individual patients. Perhaps even a hospital administrator should consider including MRDworst case as one of her benchmarks for evaluating treatment efficacy as a whole. 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China. 2Tianjin Institutes of Health Science, Tianjin, China. ✉email: [email protected] Response to comment on Have we been qualifying measurable residual disease correctly? © The Author(s) 2023 Intermediate-risk & MRDworst_case high (49 cases) Low-risk & MRDworst_case high (127 cases) Intermediate-risk & MRDworst_case low (31 cases) Low-risk & MRDworst_case low (87 cases) ++ +++++ +++ + +++ + + ++ + +++ ++ + + + + + ++ +++ +++ + ++++++ +++ + + ++ + + + ++ ++ ++++++++++ ++++++++ +++ +++++ ++++++ ++ +++ +++ +++++++++ + +++++++++++++++ ++ +++ + + +++++ ++ ++ ++++ +++++++++++++++++++++++ ++ +++ + ++++++ ++ +++++++++ + Overall P < 0.001 65 70 75 80 85 90 95 100 0 2 4 6 1 3 5 Relapse-free survival (%) Years from initial treatment Fig. 1 Risk-stratification based on joint consideration of esti- mated relapse risk at diagnosis and MRDworst case on day 19 when MRDconventional on day 19 was <0:01%. The study cohort is as described previously [1]. Intermediate-risk & MRDworst_case high (49 cases) Low-risk & MRDworst_case high (127 cases) Intermediate-risk & MRDworst_case low (31 cases) Low-risk & MRDworst_case low (87 cases) ++ +++++ +++ + +++ + + ++ + +++ ++ + + + + + ++ +++ +++ + ++++++ +++ + + ++ + + + ++ ++ ++++++++++ ++++++++ +++ +++++ ++++++ ++ +++ +++ +++++++++ + +++++++++++++++ ++ +++ + + +++++ ++ ++ ++++ +++++++++++++++++++++++ ++ +++ + ++++++ ++ +++++++++ + Overall P < 0.001 65 70 75 80 85 90 95 100 0 2 4 6 1 3 5 Relapse-free survival (%) Years from initial treatment Leukemia (2024) 38:219–220; https://doi.org/10.1038/s41375-023- 02088-4 1. Feng Y, Qi S, Liu X, Zhang L, Hu Y, Shen Q, et al. Have we been qualifying measurable residual disease correctly? Leukemia 2023;37:2168–72. 2. Clarke RD. An application of the Poisson distribution. J Inst Actuaries 1946;72:481. 3. Jeha S, Pei D, Choi J, Cheng C, Sandlund JT, Coustan-Smith E, et al. Improved CNS control of childhood acute lymphoblastic leukemia without cranial irradiation: St Jude total therapy study 16. J Clin Oncol 2019;37:3377–91. CORRESPONDENCE OPEN ACUTE LYMPHOBLASTIC LEUKEMIA Response to comment on Have we been qualifying measurable residual disease correctly? CORRESPONDENCE OPE ACUTE LYMPHOBLASTIC LEUKEMIA Leukemia Leukemia www.nature.com/leu ADDITIONAL INFORMATION 4. Yang W, Cai J, Shen S, Gao J, Yu J, Hu S, et al. Pulse therapy with vincristine and dexamethasone for childhood acute lymphoblastic leukaemia (CCCG-ALL-2015): an open-label, multicentre, randomised, phase 3, non-inferiority trial. Lancet Oncol 2021;22:1322–32. Correspondence and requests for materials should be addressed to Junren Chen. Reprints and permission information is available at http://www.nature.com/ reprints 5. Walia A, Tuia J, Prasad V. Progression-free survival, disease-free survival and other composite end points in oncology: improved reporting is needed. Nat Rev Clin Oncol 2023;20:885–95. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ACKNOWLEDGEMENTS I acknowledge support from the Institute of Hematology, Chinese Academy of Medical Sciences (IHCAMS). I thank Yahui Feng, Wei Zhang and Saibing Qi for assistance in preparing this typescript. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/licenses/by/4.0/. REFERENCES Finally, in our cohort of children with acute lymphoblastic leukaemia treated on CCCG-ALL-2015, MRDworst case identified sub- groups of children with poorer relapse-free survival even though their conventional MRD-test results were nearly all zero (Fig. 1). Nonetheless, like others we avoid relying on composite end points that could have heterogeneous make-up of adverse events [5]. In our 1. Feng Y, Qi S, Liu X, Zhang L, Hu Y, Shen Q, et al. Have we been qualifying measurable residual disease correctly? Leukemia 2023;37:2168–72. 3. Jeha S, Pei D, Choi J, Cheng C, Sandlund JT, Coustan-Smith E, et al. Improved CNS control of childhood acute lymphoblastic leukemia without cranial irradiation: St Jude total therapy study 16. J Clin Oncol 2019;37:3377–91. Correspondence 220 AUTHOR CONTRIBUTIONS JC prepared the typescript and takes responsibility for the content. FUNDING FUNDING Supported, in part, by grants from the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2021-I2M-1–001 and 2022-I2M-2–003). COMPETING INTERESTS © The Author(s) 2023 The author declares no competing interests. Leukemia (2024) 38:219 – 220

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Människor och makter

Budkavlen

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118 118 13 Människor och makter: En introduktion till religionsve- tenskap 2008. Red. Jonas Svensson och Stefan Arvids- son. Halmstad: Högskolan i Halmstad. 91 sidor. https:// dspace.hh.se/dspace/handle/2082/2450 Människor och makter: En introduktion till religionsvetenskap Författarna är här Jan Hjärpe med ”Religion och politik”, Stefan Arvidsson med ”Klassifi kation och Jämförande religionshistoria”, Olav Hammer med ”Idéhistoria”, Catharina Raudvere med ”Antropologi”, Jonas Svensson med ”Könsperspektiv”, Karin Sjögren med ”Bildanalys” och Magnus Zetterholm med ”Bibeltolkning”. Artiklarnas rubriker beskriver i båda delarna tydligt innehållet, och varje författare har dess- utom strävat efter att ge konkreta exempel från sin egen forskning för att illustrera och klargöra. I slutet av varje artikel fi nns referenserna samlade, och i vissa fall förfat- tarens litteraturtips för vidare läsning. Viktigt att notera är att denna antologi är gratis: boken får lagras på hårddisk, spridas, tryckas och kopieras utan något tillstånd. Eftersom artiklarna är så många är det inte möjligt att presentera dem alla utförligt här, men jag vill gärna kort introducera Jonas Svenssons artikel ”Könsperspektiv” och Karin Sjögrens ”Bildanalys” eftersom deras ämnes- områden kanske inte alltid självklart förknippas med re- ligionsvetenskap. Svensson konstaterar att kombinationen kön och religion har blivit allt intressantare att studera bland religionsvetare. Också på religionens område har män innehaft den största makten och kvinnorna befunnit sig lägre i hierarkin. Utifrån detta presenteras feministisk teologi vars ursprung står att fi nna i feministisk kritik av religiösa traditioner med tro och praxis i historia och nutid. Det Svensson anser vara den mest betydelsefulla produk- ten av feministiskt tänkande kring religionsvetenskap är kravet på att kvinnors religiositet och bruk genom histo- rien ska synliggöras. Även religion, genus, könsroller och könsideologier som kulturella konstruktioner behandlas, t.ex. är det intressant att inom religionsvetenskapen se på ”hur religion som ideologi och som praxis formulerar, för- medlar och upprätthåller könsroller i ett samhälle” (Svens- son och Arvidsson 2008:76). Redaktörernas inledning till antologin lyfter fram fl era viktiga aspekter. Samhällsförändring med skiftande reli- giöst klimat är redan nämnt, och redaktörerna diskuterar även religionsvetenskapens och teologins roller och för- hållanden till varandra i den svenska högskolevärlden. De defi nierar religionsvetenskap som beskrivning av tradition och anhängare i religion genom tiderna samt att söka för- ståelse och förklaring av religiös tro och praktik i religion utifrån olika teorier och metoder, till skillnad från deras exempel Nationalencyklopedin som likställer religions- vetenskap med teologi. Eftersom svenska Högskoleverket betraktar religionsvetenskap som ett paraplybegrepp med bl.a. religionshistoria, religionsbeteendevetenskap och teologi som underavdelningar, vill också redaktörerna för- söka frigöra ämnet religionsvetenskap från de, enligt dem, vanligt förekommande associationerna med teologi eme- dan det begränsar och problematiserar. Människor och makter: En introduktion till religionsvetenskap Ett tillvägagångs- sätt skulle vara att tillämpa något de kallar ”metodologisk ateism”, d.v.s. ”att svar på vetenskapliga frågeställningar söks med det grundläggande antagandet att övernaturliga krafter inte har något förklaringsvärde” (Svensson och Ar- vidsson 2008:6). Sjögren åskådliggör i sin artikel bildens dubbelsidiga möjligheter: å ena sidan fungerar bilden som exakt doku- mentation av mänskliga samband, å andra sidan är bilden ett utmärkt redskap för manipulation och propaganda. Bilder kan tolkas olika beroende på kulturella preferenser och personliga erfarenheter och även bildens kontext, form och historia påverkar tolkningen. Dessutom bör risken för övertolkning beaktas. Sjögren belyser alltså olika aspekter av bildanalysen som bör tas i beaktande såväl i religions- vetenskapliga som i övriga sammanhang, och ger konkreta exempel från sin egen forskning. Som säkert framkommit framstod redaktörernas inled- ning till antologin i mitt tycke som intressant och relevant på grund av de viktiga frågor och problematiseringar de tog upp. Det var en utmärkt inledning med tanke på det breda ämnesområde artiklarna omfattar. Inledningen kan också upplevas som aktuell och fräsch medan artiklarna är relativt traditionella. Något helt nytt tycker jag egentligen inte att antologin bidrog med, men eftersom den har ett introducerande syfte tycker jag inte heller att den behöver göra det – istället är det bredden av ämnen inom religions- vetenskap som ska lyftas fram, och det har de lyckats med. Redaktörerna och artikelförfattarna har nämligen samman- ställt antologin i undervisningssyfte för att ge en bred in- troduktion till religionsvetenskap, och utöver detta skulle jag också vilja placera den i en forskningshistorisk kontext då fl era artiklar passar in i det sammanhanget. Kort sagt kan man använda olika delar av antologin enligt behov och tycke vilket jag också tror är redaktörernas avsikt. Något som säkert uppskattas är initiativet att göra boken tillgäng- lig och gratis för den som önskar läsa den – alla vet ju att studerandebudgeten inte alltid tillåter många bokköp. Vidare presenteras i inledningen grundläg- gande religionsdefi nitioner och förklaringar till varför dessa behövs. Religion som begrepp problematiseras och redaktörerna frågar sig varför ett sådant samlingsbegrepp överhuvudtaget bildats. Läsaren får en snabbguidning i begreppets historia från inomreligiös kristen kontext i tidigmodern tid via utomreligiös och fi losofi sk kontext, upplysningstidens religionskritik till dagens diskussioner kring religionsbegreppets för- och nackdelar. Även reli- gionsvetenskap som kritisk verksamhet behandlas var- med vikten av kritisk refl ektion lyfts fram. Människor och makter: En introduktion till religionsvetenskap doktor i islamologi och verksam som lektor i religionsve- tenskap vid Högskolan i Halmstad, och Stefan Arvidsson, docent i religionshistoria och verksam som lektor i reli- gionsvetenskap vid Växjö universitet. Antologin är sam- manställd i en svensk kontext och, enligt redaktörerna, för svenska behov. doktor i islamologi och verksam som lektor i religionsve- tenskap vid Högskolan i Halmstad, och Stefan Arvidsson, docent i religionshistoria och verksam som lektor i reli- gionsvetenskap vid Växjö universitet. Antologin är sam- manställd i en svensk kontext och, enligt redaktörerna, för svenska behov. Det svenska samhället har, liksom många andra väster- ländska sådana, genomgått en sekulariseringsprocess som innebär bl.a. minskat religiöst infl ytande i samhället och individer som söker sig från institutionaliserade religiösa former till alternativ religiositet såsom nyandlighet eller frikyrklighet. Hur påverkar då denna samhällsförändring religionsvetenskaplig undervisning och forskning vid svenska universitet och högskolor? Redaktörerna motive- rades av denna fråga när de lade grunderna till antologin Människor och makter13 med vilken de vill ge en intro- duktion till religionsvetenskapen och betona att den är en humanistisk och samhällsvetenskaplig disciplin av vikt. Ytterligare huvudsakligt syfte är att lyfta fram olika per- spektiv och förhållningssätt inom religionsvetenskapliga studier, och hur man praktiskt och teoretiskt kan nalkas ve- tenskapliga studier av religiösa trossystem och utövning. Redaktörerna bakom denna antologi är Jonas Svensson, Antologin består av 17 artiklar som presenterar lika många områden, och de är uppdelade i två delar. Den för- sta delen går under benämningen ”Att studera religion” och består av artiklar som introducerar ämnestraditioner samt behandling av material såsom insamling, värdering, systematisering och analysering. Skribenterna i den första delen är Christina Raudvere med ”Historia och historie- beskrivning”, Jonas Svensson med ”Källkritik”, Jonas Ot- terbeck med ”Diskursanalys”, Madeleine Sultán Sjöqvist med ”Religionssociologi”, Anton Geels med ”Grundad teori”, Anna Davidsson Bremborg med ”Fältarbete” och ”Intervjuer”, Curt Dahlgren med ”Statistisk analys och tolkning” samt Stefan Arvidsson med ”Religionsekologi”. ”A ä k k i li i ” ä d d d l 13 Människor och makter: En introduktion till religionsve- tenskap 2008. Red. Jonas Svensson och Stefan Arvids- son. Halmstad: Högskolan i Halmstad. 91 sidor. https:// dspace.hh.se/dspace/handle/2082/2450 ”Att tänka kring religion” är den andra delen vars tema är kritisk refl ektion kring religionsvetenskapliga teoretis- 119 ka perspektiv och frågeställningar. Människor och makter: En introduktion till religionsvetenskap Som övriga humanistiska och samhällsvetenskapliga ämnen bör även religionsvetenskapen fungera kritiskt, och Svensson och Arvidsson skriver att ”den kritiska religionsdimensionen i religionsvetenskap syftar bland annat till att belysa de maktförhållanden som har funnits inbäddade i kulturen genom historien – och som ännu fi nns där” (Svensson och Arvidsson 2008:8). Magdalena Udd

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There or not there? A multidisciplinary review and research agenda on the impact of transparent barriers on human perception, action, and social behavior

Frontiers in psychology

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Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Marquardt, G., Cross, E. S., De Sousa, A. A., Edelstein, E., Farne, A., Leszcynski, M., Patterson, M., & Quadflieg, S. (2015). There or not there? A multidisciplinary review and research agenda on the impact of transparent barriers on human perception, action, and social behavior. Frontiers in Psychology, 6(1381). https://doi.org/10.3389/fpsyg.2015.01381 There or not there? A multidisciplinary review and research agenda on the impact of transparent barriers on human perception, action, and social behavior Marquardt, Gesine; Cross, E.S.; De Sousa, Alexandra A.; Edelstein, Eve; Farne, Alessandro; Leszcynski, Marcin; Patterson, Miles; Quadflieg, Susanne Frontiers in Psychology There or not there? A multidisciplinary review and research agenda on the impact of transparent barriers on human perception, action, and social behavior Marquardt, Gesine; Cross, E.S.; De Sousa, Alexandra A.; Edelstein, Eve; Farne, Alessandro; Leszcynski, Marcin; Patterson, Miles; Quadflieg, Susanne Frontiers in Psychology PRIFYSGOL BANGOR / BANGOR UNIVERSITY PRIFYSGOL BANGOR / B DOI: 10.3389/fpsyg.2015.01381 Published: 15/09/2015 Publisher's PDF, also known as Version of record Cyswllt i'r cyhoeddiad / Link to publication Cyswllt i'r cyhoeddiad / Link to publication Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Marquardt, G., Cross, E. S., De Sousa, A. A., Edelstein, E., Farne, A., Leszcynski, M., Patterson, M., & Quadflieg, S. (2015). There or not there? A multidisciplinary review and research agenda on the impact of transparent barriers on human perception, action, and social behavior. Frontiers in Psychology, 6(1381). https://doi.org/10.3389/fpsyg.2015.01381 Hawliau Cyffredinol / General rights Hawliau Cyffredinol / General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. y g Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. Y t f th di t ib t th t i l it f fit ki ti it i l i study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy This Document is Protected by copyright and was first published by Frontiers. All rights reserved. it is reproduced with permission Take down policy This Document is Protected by copyright and was first published by Frontiers. All rights reserved. it is reproduced with permission T k d li Frontiers in Psychology Frontiers in Psychology DOI: 10.3389/fpsyg.2015.01381 Take down policy f Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. 24. Oct. 2024 REVIEW published: 15 September 2015 doi: 10.3389/fpsyg.2015.01381 REVIEW Edited by: Through advances in production and treatment technologies, transparent glass has become an increasingly versatile material and a global hallmark of modern architecture. In the shape of invisible barriers, it defines spaces while simultaneously shaping their lighting, noise, and climate conditions. Despite these unique architectural qualities, little is known regarding the human experience with glass barriers. Is a material that has been described as being simultaneously there and not there from an architectural perspective, actually there and/or not there from perceptual, behavioral, and social points of view? In this article, we review systematic observations and experimental studies that explore the impact of transparent barriers on human cognition and action. In doing so, the importance of empirical and multidisciplinary approaches to inform the use of glass in contemporary architecture is highlighted and key questions for future inquiry are identified. Reviewed by: Sergio Roncato, Università di Padova, Italy Elyssa Twedt, St. Lawrence University, USA Reviewed by: Sergio Roncato, Università di Padova, Italy Elyssa Twedt, St. Lawrence University, USA *Correspondence: Susanne Quadflieg, School of Experimental Psychology, University of Bristol, 12A Priory Road, Bristol, BS8 1TU, UK s.quadfl[email protected] Specialty section: This article was submitted to Cognitive Science, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Cognitive Science, a section of the journal Frontiers in Psychology Received: 12 December 2014 Accepted: 28 August 2015 Published: 15 September 2015 Keywords: extra-personal space, evidence-based design, translucency, transparency, multisensory integration Received: 12 December 2014 Accepted: 28 August 2015 Published: 15 September 2015 Gesine Marquardt1, Emily S. Cross2,3, Alexandra A. de Sousa4, Eve Edelstein5, Alessandro Farnè6, Marcin Leszczynski7, Miles Patterson8 and Susanne Quadflieg9,10* 1 Faculty of Architecture, TU Dresden, Dresden, Germany, 2 School of Psychology, Bangor University, Bangor, UK, 3 Department of Social and Cultural Psychology, Behavioural Science Institute, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, Netherlands, 4 Faculty of Psychology, School of Society, Enterprise, and Environment, Bath Spa University, Somerset, UK, 5 College of Architecture, Planning and Landscape Architecture, University of Arizona, Tucson, AZ, USA, 6 ImpAct Team, Lyon Neuroscience Research Center, INSERM U1028, CNRS UMR5292, University Claude Bernard Lyon I, Lyon, France, 7 Department of Epileptology, University Bonn, Bonn, Germany, 8 Department of Psychology, University of Missouri–St. Louis, St. Louis, MO, USA, 9 School of Experimental Psychology, University of Bristol, Bristol, UK, 10 Division of Psychology, New York University Abu Dhabi, Abu Dhabi, UAE There or not there? A multidisciplinary review and research agenda on the impact of transparent barriers on human perception, action, and social behavior Gesine Marquardt1, Emily S. Cross2,3, Alexandra A. de Sousa4, Eve Edelstein5, Alessandro Farnè6, Marcin Leszczynski7, Miles Patterson8 and Susanne Quadflieg9,10* Edited by: Isabella Pasqualini, Ecole Polytechnique Fédérale de Lausanne, Switzerland Reviewed by: Sergio Roncato, Università di Padova, Italy Elyssa Twedt, St. Lawrence University, USA *Correspondence: Susanne Quadflieg, School of Experimental Psychology, University of Bristol, 12A Priory Road, Bristol, BS8 1TU, UK s.quadfl[email protected] Edited by: Isabella Pasqualini, Ecole Polytechnique Fédérale de Lausanne, Switzerland Citation: Everyday experience attests that transparent barriers dominate modern architecture. As surfaces, apertures, windows, or walls, transparent panes of various sizes characterize numerous settings of significance, including airports, offices, schools, hospitals, restaurants, shops, homes, and exhibitions – to name just a few. According to renowned architect Richards (2006), the human fascination with erecting such barriers comes from the fact that they can be there and not there at the same time. In other words, while such barriers spatially separate and confine places (including their noise and climate conditions), they simultaneously keep these spaces visually connected. Thus, transparent barriers transmit light in a manner that enables us to see what is beyond them without allowing us to directly approach (or be approached by) what we see. In spite of these unique Marquardt G, Cross ES, de Sousa AA, Edelstein E, Farnè A, Leszczynski M, Patterson M and Quadflieg S (2015) There or not there? A multidisciplinary review and research agenda on the impact of transparent barriers on human perception, action, and social behavior. Front. Psychol. 6:1381. doi: 10.3389/fpsyg.2015.01381 September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 1 Transparent barriers Marquardt et al. cathedrals, churches, and cloisters). The architecture of common dwellings, by contrast, involved translucent filters, created from canvas or animal skins. architectural qualities, the impact of transparent barriers on human cognition and behavior has not yet attracted much scientific attention. This oversight is surprising given that the optimal architectural use of transparent structures depends not only on esthetic concerns, technological progress, and cost-efficiency, but also on the materials potential to serve setting-specific human needs and goals (cf. Steiner and Veel, 2015). In addressing the latter two issues, this article examines what is currently known, but also what remains to be studied, about human functioning in the presence of transparent barriers. More specifically, this review explores the extent to which barriers that are simultaneously there and not there from an architectural perspective, are actually there and/or not there from perceptual, behavioral, and social points of view. The reader is first introduced to the role of transparent barriers as architectural elements throughout the centuries. Subsequently, systematic observations and experimental studies that have begun to quantify the impact of transparent barriers on human functioning are summarized and discussed. Finally, the practical importance of empirical investigations on transparent barrier use and their real-world consequences is elucidated1. 1It should be noted that the scholarship on transparent barriers summarized in this article was identified by searching two prominent academic databases (i.e., Google Scholar and Scopus) for the terms “glass architecture,” “transparency perception,” “transparent barrier perception,” “transparent barrier detection,” “transparent barrier AND distance,” “transparent barrier AND privacy,” and “transparent barrier AND space.” Once relevant work was identified, its bibliography and citations were searched for further material of interest. Material was included in this review if its content (a) referred to human cognition and/or behavior, (b) elucidated architectural, perceptual, navigational, or social aspects of transparent barrier use, and (c) was representative of a major line of research on the topic (i.e., not every single paper on the role of reflections in transparency detection was included, but rather typical exemplars). Finally, reviewer suggestions were incorporated to optimize the manuscript. The Architectural Use of Transparent Barriers The use of transparent barriers for architectural purposes was originally intertwined with the human ability to produce glass (cf. Miodownik, 2013). Though the first intentional production of glass by humans took place in the form of beads around 3000 BC (Oppenheim et al., 1970), it was the advancement of a new architecture for cathedrals and churches in the Middle Ages (5th–14th century) that stimulated the production of transparent glass panes in Europe (Elkadi, 2006). During that time, glass began to serve as a decorative material that filled wall openings between load-bearing structures such as pillars, allowing daylight to penetrate buildings in an unprecedented manner (Marks, 1993). Although early glass panes varied in color and translucency, they were generally of modest size. To portray large images of Biblical scenes, several small panes would occasionally be joined together by metal frames. The creation of bigger and fully transparent panels, however, required new methods of glass production. Developed in the Baroque era (15th–18th century), these new production methods came at a significant expense, confining the use of generous glass elements mainly to palaces of the nobility and religious buildings (i.e., In 1914, the German architect and urban planner Bruno Taut constructed a structure from concrete and glass for the Cologne Werkbund Exhibition, known as the Glass Pavilion (Nielsen and Kumarasuriyar, 2014). The pavilion (for a detailed description see Haag Bletter, 1981) was financed by the German glass industry and aimed to illustrate the potential of different types of glass for architecture (Weston, 2004). During the same year, the German writer Paul Scheerbart dedicated an influential monograph to Taut, called Glass Architecture (Scheerbart, 1914). In this monograph, the author called for eliminating closed rooms by introducing large scale glass walls, rather than mere windows, in future buildings. In line with his suggestion, many leading European architects produced glass designs in the 1920s (cf. Korn, 1968; McQuire, 2003). Based on this progress, the founder of the Bauhaus, Walther Gropius, concluded that glass architecture had overcome its status as a poetic utopia and had turned into an unconstrained reality (Gropius, 1926). Rapid technological innovations, such as the discovery of crack-preventing laminations and coatings and the development of powerful adhesives that allowed connecting multiple glass panels almost seamlessly, further spurred the widespread use of transparent glass (Staib, 2008; Weller and Vogt, 2012). Citation: The modest style of everyday housing quickly changed in the increasingly wealthy European cities at the beginning of the 16th century (Staib, 2008). Especially in Dutch merchants’ houses, load-bearing structures of bricks were increasingly separated from walls that featured large glass openings, enabling passersby to glance at a buildings’ interior. This tendency toward publicizing the private life was further reinforced through the influence of the Calvinists in the 17th century who aimed to demonstrate their pious way of living to God as well as their neighbors (Vera, 1989). Similarly, in 18th century Britain, bow- windows became a popular feature of shops and houses, turning ‘looking in’ as well as ‘looking out’ into common pastimes (Lindsay, 2009). Despite these architectural advances, until the 19th century, glass was primarily used to cover wall openings. Though load-bearing building structures were continuously minimized, stone and clay remained the materials of choice to enclose the interior space. It was not until the development of cast and wrought iron that architects were able to construct magnificent buildings from steel and glass alone. At the end of the 1820s, the first large-scale use of sheet glass appeared in the Parisian Arcades (McQuire, 2003). In 1851, the English architect Joseph Paxton designed and built the Crystal Palace that hosted the Great Exhibition in London (McKean, 1994). The iconic, largely transparent building paved the way for a modern exploration of clear glass in architecture. Transparent Barriers: A Visual Challenge p Despite their global distribution, the use of transparent barriers in contemporary construction seems largely governed by esthetic, financial, legal, structural, and city planning concerns (e.g., Elkadi, 2006; Richards, 2006; Haldimann et al., 2008; Staib, 2008; Arbab and Finley, 2010). The human response to these structures, in contrast, suffers from a lack of consideration and systematic evaluation (cf. Sommer, 1974; Hamilton and Watkins, 2009). Yet, among rare advocates of a psychological approach, the indiscriminate endorsement of transparent architecture has caused skepticism (cf. Widrich, 2015). For instance, as early as in 1963, the architectural critic Colin Rowe and the artist Robert Slutzky distinguished between literal transparency (i.e., the “quality of a substance”, p. 46) and phenomenal transparency (i.e., “an intellectual imperative [. . .] for that which should be easily detected”, p. 45) in order to emphasize that physical and psychological states of transparency could, but would not necessarily have to, co-occur in glass buildings. In further support of their argument, the architectural historian Haag Bletter (1981) directly challenged the notion that transparent structures could somehow promote personal or societal progress. The contemporary architects Vidler (1992) and Colomina (2009), finally, went so far as to argue that transparent barriers could even pose psychological hazards by producing an uncanny loss of privacy. Given these concerns and the assumption that architectural structures are generally meant to benefit people, it must be asked whether and how transparent barriers actually impact human functioning and well-being (cf. Stone and Irvine, 1993). To answer this question in a systematic manner and to ultimately optimize the use of transparent barriers in their manifold manifestations, a thorough, data- driven understanding of the human response to such barriers is needed. Pivotal cues that facilitate the detection of transparent entities are related to their reflectance and transmittance properties (Brzezicki, 2013a). Whereas reflectance properties describe the manner in which light is reflected by a material, transmittance properties refer to the way that light passes through it. Interestingly, transparent materials tend to possess specific surface reflectance properties that can give away their presence (Beck and Prazdny, 1981; Blake and Bülthoff, 1990). In particular, glossiness and highlights (see Figure 1A) are known to draw perceivers’ attention to transparent structures (Motoyoshi, 2010; Sayim and Cavanagh, 2011). Equally effective at attracting people’s attention to transparency are failures of light transmittance. Transparent Barriers: A Visual Challenge development is intriguing, considering that in many geographic regions (such as the Gulf States) the use of glass leads to tremendous solar gains in a building’s interior, a circumstance that must be countered with energy-consuming air conditioning (Aboulnaga, 2006; Schittich, 2011). Despite this challenge, architects have continued to design and construct glass buildings worldwide (cf. Wigginton, 1996; Krampen and Schempp, 1999; Bell and Kim, 2009). Supported by the emergence of Computer Aided Design (CAD) software in the very early 21st century, the latest developments in glass architecture defy traditional constructional boundaries. Modern glass buildings can form amorphous structures of curvilinear, topographic design that lack right angles or symmetry, commonly referred to as Blob architecture (Lynn, 2009). Pivotal examples of contemporary Blob architecture include the National Centre for the Performing Arts in Bejing2 or the Great Glass House in the National Botanic Garden of Wales3. Aside from its role in large- scale architectural structures, glass has also penetrated modern domestic architecture, as illustrated by Ludwig Mies van der Rohe’s Farnsworth House4 or Philip Johnson’s Glass House5. Before being able to evaluate the consequences of transparent barriers on human behavior and wellbeing, it is of crucial importance to understand how people usually notice their presence. Although transparent materials are frequently detected on the basis of accidental cracks, traces of adhesives, or dirt in everyday life, humans are surprisingly skilled at seeing transparent structures even in the absence of such opaque markers. However, the visual perception of transparent barriers seems largely guided by perceivers’ experience-based expectations of where to find them (e.g., embedded in window frames, Sayim and Cavanagh, 2011). In addition, a person’s situation-specific monitoring for such panes (maybe resulting from a previous collision) and the detection of visual cues that signal the presence of an invisible structure are of essential importance (Awh et al., 2012). The cues that “give rise to the perception of two surfaces, one of which is seen through the other” (Metelli, 1974a, p. 95) have puzzled artists (e.g., Albers, 1963) and vision scientists (e.g., Helmholtz, 1867) for decades. On the basis of their work, it is now assumed that transparency, unlike other visual features such as shape or color, is detected through a combination of informative cues (Wolfe et al., 2005). Phrased differently, the human visual system must integrate various pieces of information to decipher that something see-through (i.e., non-opaque) is present (Kersten et al., 1992). The Architectural Use of Transparent Barriers The architecture critics Johnson and Hitchcock (1932) described the cross-cultural dissemination of glass architecture as a signal of globalized minimalistic and functionalist architectural tendencies. To date, countries around the world use transparent glass in large scale building projects to demonstrate their progress, modernity, and wealth (Kulterman, 1999; Dawson, 2005). This September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 2 Marquardt et al. Transparent barriers Transparent Barriers: A Visual Challenge Copyright 2011 American Psychological Association; reprinted by permission the impression that some parts were seen directly, whereas others were seen through a transparent layer (see Figure 2A). In addition to these so-called photometric cues, geometric cues play a pivotal role in transparency detection. The configuration of contour junctions, for instance, frequently signals the relation between two surfaces. Whereas T-junctions typically imply occlusion (Figure 3A; Rubin, 2001; Hillstrom et al., 2013), contours aligned in the way of an X-junction (see Figure 3B) indicate transparency (Metelli, 1974b; Watanabe and Cavanagh, 1993; Rubin, 2001; Hillstrom et al., 2013). In a similar way, edge assignment can affect transparency perception (Nakayama et al., 1989; Qiu and von der Heydt, 2007). As illustrated in Figure 4A, the ellipse marks a border that is considered part of the contour of a small light-gray square. In Figure 4B, in contrast, the same visual input (but in a different context) is perceived as the overlapping edge between a horizontal transparent bar lying on top and a darker vertical bar underneath. Yet, when the corners of the light and dark squares are rounded off(Figure 4C), the ellipse again appears to mark the border of the small light square. These examples illustrate that numerous visual cues can induce the impression of seeing two surfaces along the same line of sight but at different levels of depths (Beck et al., 1984; Beck and Ivry, 1988; Singh and Anderson, 2002). Phrased differently, these cues enable perceivers to segregate or ‘scission’ visual input into several components, those that constitute a transparent pane and those that constitute opaque entities located behind it (Koffka, 1935; Robilotto et al., 2002; Delogu et al., 2010). FIGURE 2 | Illustration of the role of photometric cues in transparency detection. (A) A set of luminance relations (depicted as light > dark) signaling that the lighter square is seen as transparent and in front of the darker square on the farther surface. (B) A set of luminance relations failing to signal which square is seen as transparent and in front of the other. FIGURE 2 | Illustration of the role of photometric cues in transparency detection. (A) A set of luminance relations (depicted as light > dark) signaling that the lighter square is seen as transparent and in front of the darker square on the farther surface. Transparent Barriers: A Visual Challenge Although transparent barriers, per definition, transmit the light that falls upon them, the quality of this transmission can vary based on their thickness and unevenness. Specifically, processes of diffusion (i.e., the spreading of light) or refraction (i.e., the bending of light) can result in image distortions that perceivers readily use to infer the presence of a transparent structure (see Figure 1B; Fleming et al., 2011). In a closely related manner, the detection of transparent barriers can be aided by their coloring (i.e., due to glazing). Under such conditions, even transparent barriers can absorb some light, thereby producing systematic luminance differences for entities that are seen through them (i.e., affecting their lightness, brightness, and contrast appearance; Masin, 2006; Kingdom, 2011). 2http://www.chncpa.org/ens/ 3www.gardenofwales.org.uk 4www.farnsworthhouse.org 5http://theglasshouse.org/ The impact of luminance cues on transparency perception was originally explored by the Italian psychologist Metelli (1970, 1974a,b). Metelli demonstrated that specific luminance differences between neighboring parts of a surface would induce September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 3 Marquardt et al. Transparent barriers FIGURE 1 | A computer-generated image that conveys the impression of a smooth, pebble-shaped, transparent blob inside a textured box. Transparency is conveyed by (A) reflections of light (e.g., highlights) as well as by (B) refractions of light (e.g., image distortions such as changes in texture and misalignments of borders due to bending of light). Source: From Fleming et al. (2011). Copyright 2011 American Psychological Association; reprinted by permission. mooth, pebble-shaped, transparent blob inside a textured box. refractions of light (e.g., image distortions such as changes in texture and ). Copyright 2011 American Psychological Association; reprinted by permission. FIGURE 2 | Illustration of the role of photometric cues in transparency detection. (A) A set of luminance relations (depicted as light > dark) signaling that the lighter square is seen as transparent and in front of the darker square on the farther surface. (B) A set of luminance relations failing to signal which square is seen as transparent and in front of the other. FIGURE 1 | A computer-generated image that conveys the impression of a smooth, pebble-shaped, transparent blob inside a textured box. Transparency is conveyed by (A) reflections of light (e.g., highlights) as well as by (B) refractions of light (e.g., image distortions such as changes in texture and misalignments of borders due to bending of light). Source: From Fleming et al. (2011). Transparent Barriers: A Visual Challenge (A) Opaque square and circle, with T-junctions marked by dashed circles. (B) Transparent square and circle, with X-junctions marked by dashed circles. Source: From Hillstrom et al. (2013). Copyright 2013 American Psychological Association; reprinted by permission. FIGURE 4 | Illustration of the interplay between border ownership assignment and transparency detection. The same edge (identified by an identical luminance difference throughout) is perceived as (A) part of the contour of the small square, (B) part of a black vertical bar that lies underneath a transparent horizontal bar, (C) again part of the contour of the small square. Source: From Qiu and von der Heydt (2007). Copyright 2007 Nature Publishing Group; reprinted by permission. FIGURE 3 | Illustration of the role of geometric cues in transparency detection. (A) Opaque square and circle, with T-junctions marked by dashed circles. (B) Transparent square and circle, with X-junctions marked by dashed circles. Source: From Hillstrom et al. (2013). Copyright 2013 American Psychological Association; reprinted by permission. FIGURE 3 | Illustration of the role of geometric cues in transparency detection. (A) Opaque square and circle, with T-junctions marked by dashed circles. (B) Transparent square and circle, with X-junctions marked by dashed circles. Source: From Hillstrom et al. (2013). Copyright 2013 American Psychological Association; reprinted by permission. FIGURE 4 | Illustration of the interplay between border ownership assignment and transparency detection. The same edge (identified by an identical luminance difference throughout) is perceived as (A) part of the contour of the small square, (B) part of a black vertical bar that lies underneath a transparent horizontal bar, (C) again part of the contour of the small square. Source: From Qiu and von der Heydt (2007). Copyright 2007 Nature Publishing Group; reprinted by permission. FIGURE 4 | Illustration of the interplay between border ownership assignment and transparency detection. The same edge (identified by an identical luminance difference throughout) is perceived as (A) part of the contour of the small square, (B) part of a black vertical bar that lies underneath a transparent horizontal bar, (C) again part of the contour of the small square. Source: From Qiu and von der Heydt (2007). Copyright 2007 Nature Publishing Group; reprinted by permission. Transparent Barriers: A Visual Challenge barrier can confine an individual to a limited physical space (i.e., the person’s sense of touch identifies an impenetrable barrier), while simultaneously providing visual access to a larger space (i.e., the person’s sense of vision fails to identify a barrier). The experience of such divergent sensory information is noteworthy because humans usually create representations of the space closely surrounding their bodies by integrating both tactile and visual information into one multisensory representation (Làdavas and Farnè, 2004; Spence et al., 2004). Compared to mere unisensory analyses, multisensory representations usually result in a more accurate perception of one’s environment (Ernst and Bülthoff, 2004), fostering individuals’ rapid and adaptive responses to their surroundings (Calvert et al., 2004). But what happens when the two sensory systems convey conflicting information, as is commonly the case in the presence of transparent barriers? the ability to perceive transparency seems to be actively acquired during the first months of one’s life (Johnson and Aslin, 2000). Exactly how and when the visual system establishes expertise with transparent structures or certain informative cues signaling their presence, however, is an issue of ongoing debate (cf. Otsuka et al., 2006; Kavšek, 2009). Equally unclear is whether frequent exposure to transparent barriers may facilitate a perceiver’s ability to spontaneously detect relevant visual cues. It also remains to be studied whether and to which extent distraction interferes with a person’s capacity to perceive transparent materials, for instance, by impairing the integration of several diagnostic cues. Given that undetected transparent barriers pose the risk of unintentional collisions, finding answers to the above questions is of pivotal relevance for their architectural use. Transparent Barriers: A Visual Challenge (B) A set of luminance relations failing to signal which square is seen as transparent and in front of the other. structures’ borders and edges remain inaccessible, making their detection particularly difficult (Brzezicki, 2014). Unsurprisingly, undetected transparent barriers can pose a serious health and safety risk. Accidents due to collisions with glass doors or walls have been reported for both children and adults (e.g., Gur et al., 2001; Algaze et al., 2012). Their occurrence has elicited attempts to monitor and standardize the architectural use of glass barriers. In the UK, for instance, health and safety regulations for workplaces require the conspicuous marking of windows and glass doors with warning stickers (United Kingdom Health and Safety Executive, 1992, Regulation 14). In Australia, it has been dictated that glass doors must be made from laminated or toughened glass to reduce the likelihood of serious injuries upon collision (see standards AS 1288 & AS2208; see Kimia et al., 2009; AGGA, 2011). In addition, architects worldwide have been urged to remember that very clear, smooth, faultless, and frameless panes of glass are most likely to result in detection failures (Brzezicki, 2013b). Unfortunately, this segregation process does not always succeed. For example, only specific combinations of luminance relations create the perception of transparency in a unique depth order (Kitaoka, 2005; Koenderink et al., 2010). Alternatively (see Figure 2B), perceptions of transparency with an ambiguous depth order due to non-diagnostic luminance relations are possible (creating the impression of so-called bistable transparency; Adelson and Anandan, 1990; Anderson, 1997; Delogu et al., 2010; Fukiage et al., 2014). In addition, if the shape of a transparent pane coincides with the shape of the background, no transparency may be seen (Arnheim, 1971). Such correspondence is not uncommon in architectural settings in which large glass panels fill a person’s entire field of view. Under such conditions, visual information about the transparent Although the use of transparent structures in architecture is nowadays expected to be accompanied by a careful assessment of their visibility from different viewpoints and under different lighting conditions (daytime, nighttime, backlighting; Brzezicki, 2014), several important questions regarding the topic remain unanswered. For instance, rather than reflecting an inborn talent, September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 4 Marquardt et al. Transparent barriers FIGURE 3 | Illustration of the role of geometric cues in transparency detection. Transparent Barriers: A Multi-Sensory Challenge Importantly, so-called defensive withdrawal also occurs toward images of objects that rapidly grow in size, even if those images are merely projected on a screen. In other words, under conditions in which objects growing in size seem to approach an individual but actually have no chance of touching him or her, perceivers are still likely to show rapid startle and withdrawal responses toward these objects (King et al., 1992). These findings suggest that, given the appropriate visual input, awareness that one’s body is shielded from events occurring behind a transparent barrier may not suffice to suppress physical arousal and motor reflexes. Indeed, the ineffectiveness of transparent barriers to interfere with basic reflexes is regularly taken advantage of in medical examinations. For instance, the so-called blink reflex (i.e., a rapid closure of the eyelids to a quickly approaching object such as a thrown ball) is generally tested by separating patients from approaching objects with a piece of plexiglass. Under these circumstances, the transparent barrier not only protects patients from the impact of an impending collision, but also enhances the p Under such conditions, the relevant finger simultaneously exists within a person’s peripersonal space (based on its visual properties), but also outside of it (due to being located behind the barrier). Results indicate that even when a wiggling finger is presented behind a transparent barrier, it triggers tactile extinction in these patients, to the same extent as when no barrier is interposed (Farnè et al., 2003). In other words, the patients’ automatic representation of their peripersonal space is unaffected by the presence of the barrier. Thus, the mere knowledge that actual touch is impossible does not modulate the observed extinction effect. A similar observation has been reported for healthy adults (Kitagawa and Spence, 2005). Adopting a so- called cross-modal congruency task, participants received tactile stimulation to either upper or lower portions of the left or right hand. They then had to indicate as quickly and accurately as possible the place of stimulation while simultaneously seeing one of two visual distractor lights near their hands (see Figure 6). Again, the presence of a transparent barrier placed between the tactile stimulation and the visual distractors did not impact participants’ performance, indicating that visual–tactile interactions in peripersonal space are unaffected by the presence of transparent barriers. Transparent Barriers: A Multi-Sensory Challenge Initial evidence indicates that when faced with conflicting information coming from tactile and visual sensors, humans rely more strongly on the latter, especially when the sensory contradictions arise within a person’s peripersonal space (cf. Làdavas et al., 2000). The term peripersonal space denotes the Not only our sense of sight but also our sense of touch can signal the presence of transparent barriers. Consequently, transparent September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 5 Marquardt et al. Transparent barriers test’s diagnostic value by shielding them from drafts caused by the motion of the object which may trigger the reflex in a non- visual manner (van Hof-van Duin and Mohn, 1986; Guzetta et al., 2001). Further evidence for a lack of interference of transparent barriers with basic reflexes comes from two behavioral studies exploring the occurrence of so-called visual–tactile interactions (Farnè et al., 2003; Kitagawa and Spence, 2005). The first of the two studies examined a group of tactile extinction patients (Farnè et al., 2003). Such patients tend to display an intriguing deficit: a visual sensation experienced in close proximity to one side of their body (e.g., a finger moving down close to their right hand, signaling the potential for touch) inhibits their ability to experience a simultaneously applied tactile sensation to their other side (e.g., a tap applied to the left hand). Crucially, this tactile extinction is less likely to occur the farther the wiggling finger is located from the patient’s hand (i.e., the farther it is located beyond a perceiver’s peripersonal space and the less likely it is to result in physical contact). This pattern of results signals an integrated visual–tactile system coding of peripersonal space in humans. But how is such coding affected by the introduction of a transparent barrier in close proximity to a person’s hand? space closely surrounding a person’s body (see Figure 5; Previc, 1998; Caggiano et al., 2009; Brozzoli et al., 2014). Conflicting tactile and visual information occurring within this space can lead to intriguing effects. In many species, including humans, avoidance reactions such as head or hand withdrawals are easily triggered by quickly approaching objects that threaten to collide with individuals by entering their peripersonal space (Dunkeld and Bower, 1980; Makin et al., 2009; Serino et al., 2009). Transparent Barriers: A Multi-Sensory Challenge For instance, for short, untraveled distances (i.e., when people merely look at a space, but do not walk around in it), transparent barriers seem to elicit larger (rather than smaller) distance overestimation effects than opaque barriers (Sherman et al., 1979). Moreover, transparent barriers may primarily affect observers’ spatial representations when the barriers are placed between them and a target. Initial research on egocentric distance-to-target judgments suggests that such judgments are more accurate when perceivers are exposed to a continuous, homogenous texture ground surface than to a surface that contains a gap or an opaque barrier (Sinai et al., 1998; He et al., 2004; see Figure 7). Whether transparent barriers actually result in accurate or biased judgments is undetermined. An additional issue for further research on transparent barriers is whether distance-to-target judgments tend to be more accurate when taken outdoors (i.e., on a lawn) rather than indoors (i.e., in a hallway or lobby; Lappin et al., 2006). The effect appears to be mediated by the amount and kind of space that is visible beyond an actual target (so-called vista space, Witt et al., 2007). Yet again, due to a scarcity of empirical investigations, the effect of transparent barriers located in vista space on target-to-distance estimates is unclear. In summary, the effects of transparent barriers on spatial representations remain poorly understood. This lack of empirical insight is particularly worrisome as it undermines the ability to predict how people orient themselves in glass environments, a limitation that poses far-reaching health and safety concerns (i.e., in emergency flight situations; Piller and Sebrechts, 2003; Abu-Safieh, 2011). FIGURE 6 | Schematic illustration of the experimental set-up to study visuo–tactile interactions in healthy adults. In the transparent barrier condition, the participants’ hands were covered by a transparent Perspex occluder. Source: From Kitagawa and Spence (2005). Copyright 2005 Springer Publishing Group; reprinted by permission. transparent barriers does it generally take until habituation is noticeable (i.e., is it a rapid or time-consuming process)? (b) Do some people habituate more or less quickly when shielded by a transparent barrier than others and if so, why? (c) Can habituation effects be transferred across different settings (i.e., would a person that has worked behind a transparent barrier in one setting more quickly habituate when placed behind a transparent barrier in another setting)? Transparent Barriers: A Multi-Sensory Challenge FIGURE 5 | The peripersonal space, mainly based on the integration of tactile and visual information coming from the body and the space directly surrounding the body, constitutes a privileged interface for the body to interact with nearby objects. This figure depicts the body schema, head and hand-centered space, and the peripersonal/arm-centered reaching space. Source: From Cardinali et al. (2010). Copyright 2010 Elsevier Publishing Group; reprinted by permission. The above work suggests that people can be fully aware of being protected from collisions or touch by a transparent wall, yet simultaneously show rapid responses to approaching objects or people as if no wall was present. What remains to be studied is whether frequent exposure to and experience with transparent barriers may alter mechanisms of multisensory integration and/or people’s spontaneous responses (cf. Wesslein et al., 2015). The recurrent perception of objects or people behind a transparent barrier may lead to habituation, such that the strength and/or likelihood of reflexive responses toward entities on the other side of a transparent shield may decline over time. If such habituation can be observed, several related questions deserve scientific consideration: (a) How much exposure to FIGURE 5 | The peripersonal space, mainly based on the integration of tactile and visual information coming from the body and the space directly surrounding the body, constitutes a privileged interface for the body to interact with nearby objects. This figure depicts the body schema, head and hand-centered space, and the peripersonal/arm-centered reaching space. Source: From Cardinali et al. (2010). Copyright 2010 Elsevier Publishing Group; reprinted by permission. September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 6 Transparent barriers Marquardt et al. school-aged children overestimated distances between objects separated by transparent barriers (Herman et al., 1987). The absence of a spatial overestimation effect for transparent barriers in viewers familiar with their occurrence indicates that these barriers are not spontaneously used as spatial delineators. FIGURE 6 | Schematic illustration of the experimental set-up to study visuo–tactile interactions in healthy adults. In the transparent barrier condition, the participants’ hands were covered by a transparent Perspex occluder. Source: From Kitagawa and Spence (2005). Copyright 2005 Springer Publishing Group; reprinted by permission. y Alternatively, the space-delineating properties of transparent barriers may only arise under specific circumstances. Transparent Barriers: A Multi-Sensory Challenge Finally, (d) would the habituation of reflexes behind a transparent barrier ultimately compromise a person’s rapid responses in settings that lack such barriers? These concerns are perpetuated by the observation that humans must actively learn to treat transparent structures as physical barriers. Such learning is most notable in young infants who show a prevalent tendency for reaching or crawling into impermeable transparent surfaces. Butterworth (1977), for instance, tested 9-month-olds search abilities by hiding attractive Frontiers in Psychology | www.frontiersin.org Navigating Transparent Barriers This response pattern does not seem to result from an inability to see the barrier, but rather from a flawed assumption that transparent barriers can be physically penetrated (Johnson and Aslin, 2000; Shinskey and Munakata, 2001). Thus, once this assumption is rectified (e.g., by allowing 9-month-olds to play with transparent covers before testing them) infants systematically remove opaque as well as see- through covers before they reach for a toy of interest (Yates and Bremner, 1988). reflects a reduced ability to use cues of transparency for adequate motor planning or whether colliding with transparent instead of opaque barriers may seem less severe to a naïve perceiver (cf. Schmuckler, 1996). Regardless of the underlying mechanism(s), however, these data signal that humans must actively learn to resist the alleged penetrability of transparent barriers. Seminal work on the visual clifffurther demonstrates the idea that transparent surfaces initially appear immaterial. In this work, the psychologists Gibson and Walk (1960) tested infants’ response to perceived downward depth using a horizontal transparent barrier that covered a cloth with a checkerboard pattern. While the transparent barrier sat directly on the cloth on one side of the apparatus, the cloth was dropped about four feet on its other side. In doing so, the researchers created an apparent cliffcovered by a transparent pane (see Figure 9). In this setting, infants capable of crawling typically hesitated to cross the surface, despite encouragement from a parent on the other side of the cliff. The effect prevailed even when children were allowed to establish through touch that the surface was rigid and when they witnessed that a hard ball was able to bounce offthe surface (Gibson and Schmuckler, 1989). Though it seems obvious that humans improve their ability to think of transparent barriers as impenetrable entities as they grow older, how this improvement takes place and to what extent is less clear. What seems evident is that even adults occasionally question the rigidity and durability of transparent barriers as they explore their environment. These data demonstrate that negotiating transparent materials is developmentally dependent on interacting with them. The term social ontologies has been introduced to describe how human capabilities – both in terms of physical and cognitive abilities – are frequently forged through interactions with the human-made material world (Gosden, 2008). Research on barrier crossing provides further evidence that navigating transparent obstacles is an acquired skill. Navigating Transparent Barriers Further evidence that people are easily inclined to disregard the presence of transparent barriers comes from research on spatial perception and navigation. Navigational research with opaque barriers suggests that humans frequently rely on the presence of walls or fences to orient in space (Acredolo and Boulter, 1984; Herman et al., 1987; Han and Becker, 2013; Buckley et al., 2014). Although such a strategy benefits learning the location of meaningful objects and spatial layouts, it can also distort the representation of spatial information: Distances between entities in different subdivisions of space are judged larger than the same distances between objects located within the same subdivision (Allen, 1981; Maki, 1981, 1982; Newcombe and Liben, 1982). In the case of transparent barriers, however, such spatial overestimation effects seem to be absent (Montello, 2005). The first study on distance estimation, for instance, revealed that preschoolers exaggerated distances between objects separated by both opaque as well as transparent barriers, whereas adults only exaggerated distances across opaque barriers (Kosslyn et al., 1974). A subsequent study demonstrated that neither adults, nor FIGURE 7 | Schematic illustration of the experimental set-up to study distance-to-target judgments in healthy adults. FIGURE 7 | Schematic illustration of the experimental set-up to study distance-to-target judgments in healthy adults. FIGURE 7 | Schematic illustration of the experimental set-up to study distance-to-target judgments in healthy adults. September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 7 Marquardt et al. Transparent barriers toys behind opaque or transparent covers. Along similar lines, Diamond and Gilbert (1989) examined 7-11-months-olds’ reach strategies by putting toys in opaque and transparent boxes (see Figure 8). In both cases, children younger than 10 months of age tried to directly reach for toys “through” transparent surfaces, even though they readily reached around opaque barriers. Similar observations have also been made regarding young infants’ detour abilities. Lockman (1984), for example, used opaque and transparent barriers to investigate 8-months-olds’ ability to select a path to a goal. This work revealed that, up to 10-months of age, infants displayed significant difficulty in detour ability when obstructed by a transparent barrier. Specifically, they hesitated or refused to select an alternative path to a goal when their current path was blocked ‘merely’ by something transparent (cf. Lockman and Adams, 2001; Noland, 2008). Navigating Transparent Barriers Decades of social–psychological research suggest that people’s physical surroundings fundamentally shape social processes related to privacy, crowding, interpersonal involvement, and territoriality. Privacy is best understood as a dialectic process through which people strive for some momentary optimal level of contact with others (Altman, 1975). A person’s temporary need for privacy may be violated by crowding. Crowding refers to a negative affective response elicited by a high density of people in a specific location (Stokols, 1972). To regulate privacy under conditions of crowding, people may adjust their interpersonal involvement with others, that is, they may avoid close interpersonal distance, mutual gaze or touch, facial expressions of approachability, and so on (Patterson, 2011, 2013). Alternatively, people may try to own and/or control access to specific physical locations, thus showing territorial behavior (Altman, 1975; Bell et al., 2001; Brown, 2009; Scannell and Gifford, 2010). Social discomfort resulting from privacy violations, crowding, unwanted interpersonal involvement, and territorial intrusions is a common experience in everyday life. Its occurrence is particularly likely when strangers share a common presence, as is the case in many public settings, such as restaurants, waiting rooms, elevators, public transportation, open plan offices, or checkout/ATM lines (e.g., Camperio and Malaman, 2002; Manzo, 2005; Evans and Wener, 2007; Li and Li, 2007). In managing negative experiences around privacy, crowding, interpersonal involvement, and territoriality people not only adjust their own non-verbal behavior relative to others, but also use and manipulate elements in their physical environment. FIGURE 9 | A mother urging her child from across the deep side of the visual cliff. Despite a transparent surface covering the cliff, the child hesitates to move forward. Source: From Gibson and Walk (1960). Copyright 1960 Nature Publishing Group; reprinted by permission. contact-experience that signals the surface’s impenetrability. This phenomenon further confirms that in response to conflicting tactile and visual experiences humans are inclined to rely more strongly on the latter. Importantly, overcoming this overreliance on visual information in the presence of transparent barriers does not only seem to require extensive practice, but also consistent awareness of the problem at hand. People suffering from cognitive decline, such as Alzheimer patients, have been found to cope poorly with transparent environments. Navigating Transparent Barriers In studies on the topic, young children were asked to step over barriers that varied in height. Subsequently, thresholds for successful barrier crossing (i.e., crossings that do not result in damaging the obstacle) were compared for opaque and transparent barriers. Both 12- and 18-month-olds were found to successfully cross opaque barriers at larger thresholds than transparent barriers, suggesting that a child’s ability to perform an adequate motor response suffers when faced with transparency (Schmuckler, 1996). Additional research is necessary to determine if the observed difference This phenomenon is nicely illustrated by widespread responses toward glass walkways as erected over natural cliffs (e.g., in the Grand Canyon6; in the Tianmen Mountain7) or on top of modern buildings (e.g., the Transparent Observatory at the Oriental Pearl Radio and TV tower8). When entering such walkways, many visitors hesitate to step forward, despite witnessing other people standing or moving around on the same structure9 (Morse, 2011). Crucially, this hesitation tends to linger throughout a person’s initial steps and despite a 6http://www.grandcanyonwest.com/skywalktour.php 7http://www.tourismchina.org 8http://www.orientalpearltower.com/en/index.html 9https://www.youtube.com/watch?v$=$Z8WeJD2lc30 a childs ability to perform an adequate motor response suffers when faced with transparency (Schmuckler, 1996). Additional research is necessary to determine if the observed difference 7http://www.tourismchina.org 8http://www.orientalpearltower.com/en/index.html 9https://www.youtube.com/watch?v$=$Z8WeJD2lc30 FIGURE 8 | Experimental set-up to study infants’ toy retrieval in the presence of a transparent barrier. The arrows signal the required reaching behavior based on whether the transparent barrier (A) blocks or (B) fails to block direct retrieval. Source: From Diamond and Gilbert (1989). Copyright 1989 Elsevier Publishing Group; reprinted by permission. FIGURE 8 | Experimental set-up to study infants’ toy retrieval in the presence of a transparent barrier. The arrows signal the required reaching behavior based on whether the transparent barrier (A) blocks or (B) fails to block direct retrieval. Source: From Diamond and Gilbert (1989). Copyright 1989 Elsevier Publishing Group; reprinted by permission. September 2015 | Volume 6 | Article 1381 8 Frontiers in Psychology | www.frontiersin.org Marquardt et al. Transparent barriers types of situations, the link between social interactions and their physical environments is considered more broadly. FIGURE 9 | A mother urging her child from across the deep side of the visual cliff. Despite a transparent surface covering the cliff, the child hesitates to move forward. Source: From Gibson and Walk (1960). Copyright 1960 Nature Publishing Group; reprinted by permission. Navigating Transparent Barriers Their struggle to open glass doors (rather than trying to directly pass through them) or to look for alternative paths around transparent barriers suggests that they have lost the ability to overcome the visual illusion of penetrability caused by such structures (Passini et al., 2000). Barriers, both permanent (e.g., walls, columns, partitions) and/or movable (e.g., furniture or plants), play a pivotal role in the regulation of social interactions between strangers (Levitt and Weber, 1989; Manzo, 2005; Robson, 2008). These barriers, collectively termed anchors (Robson, 2002), can limit spatial access to a person and provide temporary screening from the sight, sound, or proximity of others (Robson, 2008). What remains uncertain is whether people also treat transparent barriers as architectural elements with anchoring qualities. Transparent barriers form unique kinds of barriers, given that they separate space physically, but not visually. Toddlers as young as 14 months of age understand that, unlike an opaque barrier, a transparent barrier fails to block another person’s line of sight (Dunphy-Lelii and Wellman, 2004). Thus, both children and adults assume that a physical separation through a transparent barrier does not interfere with the transmission of visual information. Given that the exchange of visual information remains unimpaired, to which extent does a separation of strangers by a transparent barrier impact social behavior? The Impact of Transparent Barriers on Social Behavior Interpersonal distance violations can precipitate flight from a setting or, at least, compensatory non-verbal behavior, such as turning away or avoiding gaze to reduce the effect of a stranger’s close presence (Sommer, 1969; Patterson et al., 1971; Patterson, 1973; Koneˇcni et al., 1975). It remains unclear, however, whether people’s need for controlling their involvement with others depends on the presence or absence of transparent barriers between them. On one hand, it could be argued that transparent barriers should not alter interpersonal distance preferences because visual information from others remains unobstructed. Alternatively, because transparent barriers interfere with tactile, auditory, and olfactory input (Crusco and Wetzel, 1984; Haans and IJsselsteijn, 2006), responses to the perceived proximity of others may change in their presence. By enabling the visual processing of others while preventing a physical interaction, transparent barriers may alter how we respond to the proximity of strangers (see Figure 10). under such conditions, there may be less discomfort resulting from being looked at in the presence of a transparent barrier compared to a no barrier arrangement. Social interactions may also change their course depending on whether they are taking place in a space surrounded by transparent barriers. Experimental studies have demonstrated, for instance, that close spatial proximity to strangers produces less discomfort in open than in confined spaces (Cochran et al., 1984). Along similar lines, encounters with members of social outgroups (e.g., people perceived as having a different racial background than a perceiver) elicit associations related to a fight when they occur in a small booth, but to flight when they occur in an open field (Cesario et al., 2010). In other words, confined spaces defined by non-transparent barriers seem to encourage aggression, rather than withdrawal, during stressful social encounters. Whether people consider spaces largely defined by transparent barriers as confined or open, however, remains an issue of debate. Given that transparent barriers can be as impenetrable as their non-transparent counterparts, they can clearly be understood as physically confining. Their visual permeability, however, may reduce a person’s sense of confinement (Stamps, 2010). As a result, the same density level may reduce feelings of crowding in spaces largely defined by transparent barriers than in spaces defined by non-transparent barriers. The effect of perceived spaciousness, in turn, may translate into a decreased readiness to aggress and an enhanced willingness to consider withdrawal during perceived social threat. The Impact of Transparent Barriers on Social Behavior Aside from investigating people’s visual, tactile, and spatial representations of transparent barriers in their environment, the barriers’ impact on people’s social functioning has attracted initial scientific attention (Procter, 1970). To understand the consequences of transparent barriers on social interactions more fully, the interplay between these structures and the types of social behavior they may foster and/or hinder must be considered (cf. Drew, 1971; Knapp et al., 2014; Patterson and Quadflieg, 2015). The current section focuses therefore on two types of social situations in which transparent barriers may impact social behavior: situations in which people are separated from each other by transparent barriers and situations in which people are surrounded by transparent barriers. Before addressing these two Though architects have long claimed that the visual and acoustic permeability of barriers affects social encounters (Zeisel, 1981), empirical investigations on this topic remain rare. In consequence, the impact of transparent barriers on social behavior is poorly understood. Consider, for instance, the observation that most people try to maintain a minimal interpersonal distance from unfamiliar others (Hall, 1959; September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 9 Marquardt et al. Transparent barriers Sommer, 1959). When a stranger initiates an inappropriately close approach, arousal, and discomfort result (Sommer, 1959; McBride et al., 1965; Middlemist et al., 1976; Patterson, 1976; Hayduk, 1983). Interpersonal distance violations can precipitate flight from a setting or, at least, compensatory non-verbal behavior, such as turning away or avoiding gaze to reduce the effect of a stranger’s close presence (Sommer, 1969; Patterson et al., 1971; Patterson, 1973; Koneˇcni et al., 1975). It remains unclear, however, whether people’s need for controlling their involvement with others depends on the presence or absence of transparent barriers between them. On one hand, it could be argued that transparent barriers should not alter interpersonal distance preferences because visual information from others remains unobstructed. Alternatively, because transparent barriers interfere with tactile, auditory, and olfactory input (Crusco and Wetzel, 1984; Haans and IJsselsteijn, 2006), responses to the perceived proximity of others may change in their presence. By enabling the visual processing of others while preventing a physical interaction, transparent barriers may alter how we respond to the proximity of strangers (see Figure 10). Sommer, 1959). When a stranger initiates an inappropriately close approach, arousal, and discomfort result (Sommer, 1959; McBride et al., 1965; Middlemist et al., 1976; Patterson, 1976; Hayduk, 1983). The Impact of Transparent Barriers on Social Behavior Are spaces bounded by transparent barriers sufficient to fulfill privacy needs? To what degree can transparent barriers define a territory that is recognized by others? Finally, can spaces defined by transparent barriers influence social interactions via changes in environmental features, such as lighting conditions and the level of visual stimulation? Initial evidence suggests, for instance, that in work settings people prefer rooms with sunlight and outdoor views (Wang and Boubekri, 2010; Aries et al., 2015). But can such preferences shape the course and quality of social interactions? Changes in light conditions over the course of a day can certainly entrain circadian rhythms and modulate physiological states, such as a person’s endocrine levels and heart rate (Edelstein et al., 2007). Further evidence suggests that lighting conditions around transparent barriers may even impact people’s social behavior. Brighter rooms, for instance, seem to facilitate social inhibition (Hirsh et al., 2011), an effect that can reduce anti-social behavior (such as aggression or dishonesty, see Page and Moss, 1976; Prentice- Dunn and Rogers, 1980; Zhong et al., 2010) but also prosocial behavior (such as the willingness to collaborate, see Steidle et al., 2013). Most importantly, these rare examples of experimental research show that the effects of transparent barriers (beyond corresponding changes in lighting conditions) on social exchange deserve empirical attention in order to understand these barriers’ impact on our everyday life. g p Differential preferences for transparency between architects and users may contribute to lethargy and difficulties concentrating as discussed in the context of the so-called sick building syndrome (Apter et al., 1994; Sahlberg, 2012). Although the potential contribution of transparent boundaries to this syndrome has yet to be studied, their role deserves particular scientific attention due to the profound perceptual, behavioral, and social repercussions as described in this article. At the same time, the use of transparent materials also merits consideration in the context of healing architecture. In contrast to the sick building syndrome, the concept of healing architecture refers to design features that promote human health and wellbeing. The term is usually applied in the context of healthcare buildings, where it denotes the capacity of architectural design to promote healing processes in the people it accommodates. A seminal article in this field “View through a window may influence recovery from surgery” was published in Science magazine (Ulrich, 1984). The authors analyzed outcomes of patients with gall bladder surgery. The Impact of Transparent Barriers on Social Behavior In other words, the requirement to respond to and navigate transparent barriers has emerged only recently in human phylogeny. So what goals do architects typically have when using transparent design features and what is the evidence that these goals are met? nature versus built environments; Kahn et al., 2008; Bratman et al., 2015). With increased globalization and migration, many contemporary societies are characterized by frequent encounters between strangers from different ethnic, cultural, and/or religious backgrounds. A large body of work indicates that such encounters often elicit mutual discomfort and anxiety (Stephan and Stephan, 1985; Smith and Mackie, 2010). If design features could enhance the safety and comfort of such interactions, architects should consider their regulatory impact when designing public spaces that welcome human diversity. As frequently discussed in architectural circles “the quality, or state of being transparent is both a material condition [. . .and. . .] an intellectual imperative” (Rowe and Slutzky, 1963). This twofold meaning of transparency has turned glass architecture into a marketing tool used to symbolize accessibility (e.g., in financial or governmental institutions; Whiteley, 2003) and democratic information exchange (Barnstone, 2005). Indeed, initial observations suggest that openness and transparency in workplace settings can facilitate productivity and innovation by enhancing the exchange of knowledge and skills between individuals (e.g., Hascher et al., 2002). At the same time, however, employees’ preferences and demands for privacy and defensible territories may interfere with architectural ideals of transparency (Kim and de Dear, 2013). Anecdotal evidence reveals that in modern buildings plants, posters, and other non-transparent items are often strategically placed to cover facades and interior walls made of glass. The well-known artist Wassily Kandinski, for instance, was once observed to cover a transparent glass wall of a Bauhaus building in white paint in order to avoid being constantly looked at by passersby (cf. Whiteley, 2003). These user-driven changes to built environments frequently signal contrasting preferences between architects, who aim to dematerialize spatial boundaries by using transparent glass, and building occupants who strive to re-establish them. y The lack of empirical data addressing the above issues is particularly unfortunate because many closely related questions of importance remain equally unaddressed. For example, can beneficial consequences of social proximity, such as the inhibition of stress hormones in the presence of social ingroup members, occur across transparent barriers (cf. Millidine et al., 2009; Beckes and Coan, 2011)? The Impact of Transparent Barriers on Social Behavior In contrast, feelings of crowding may occur even in low density spaces if there is a high-density environment on the other side of a transparent barrier. In other words, humans may not be affected by perceived spaciousness per se, but by the actual content of the views through their surrounding transparent barriers (Kaplan, 2001). Thus, a person’s readiness to aggress against social threats encountered indoors may get reduced or amplified, depending on whether transparent barriers afford stress-reducing or stress-inducing views (e.g., exposure to g Similarly unresolved is the issue of how transparent barriers affect tacit norms of looking behavior. Establishing eye contact with another person frequently serves as a signal to initiate (verbal or non-verbal) communication (Kleinke, 1986). Thus, when such communication is not sought, people tend to respect others’ privacy by not looking at each other (Argyle and Dean, 1965; Laidlaw et al., 2011). But how are looking norms affected by the presence of transparent barriers? Are people inclined to stare longer at others through transparent barriers, knowing that under these conditions they can gather social information without the obligation to engage in any further exchange? Equally important is the questions of whether targets of prolonged looks may be more comfortable with attracting someone’s gaze through a transparent barrier than when no barrier is present. Because neither a spatial intrusion, nor a verbal approach is likely to follow FIGURE 10 | Schematic depiction of two people waiting at a bus stop illustrating an uninvestigated question of social relevance: Would the close presence of a stranger be more comfortable when occurring (A) across a transparent barrier or (B) without such a barrier? [Images of humans were downloaded from www.shutterstock.com and are reproduced in this manuscript in adherence with the company’s standard license terms of service (http://www.shutterstock.com/licensing.mhtml)]. FIGURE 10 | Schematic depiction of two people waiting at a bus stop illustrating an uninvestigated question of social relevance: Would the close presence of a stranger be more comfortable when occurring (A) across a transparent barrier or (B) without such a barrier? [Images of humans were downloaded from www.shutterstock.com and are reproduced in this manuscript in adherence with the company’s standard license terms of service (http://www.shutterstock.com/licensing.mhtml)]. September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 10 Marquardt et al. Transparent barriers from an evolutionary point of view (Brzezicki, 2013a). The Impact of Transparent Barriers on Social Behavior After undergoing the procedure, patients were accommodated in rooms on the second and third floors of a three-story wing of a hospital building. Windows of the patient rooms on one side of the wing looked out on either a grove of trees or on a brown brick wall. The results showed faster, less painful recovery for surgical patients with a windowed view to a natural setting than for those with a view of a brick wall. The authors noted, however, that many physical attributes in addition to view itself, such as the quality of light may have influenced the obtained results. Frontiers in Psychology | www.frontiersin.org Concluding Remarks As interest in understanding and predicting how people respond to built environments begins to grow, the need for systematic research on the affective, cognitive, and behavioral responses to varied environments increases. The widespread, and occasionally undifferentiated, use of transparent barriers in modern construction poses a pivotal example of how architectural decisions could benefit from valid and reliable data in order to ensure a space’s functionality and user-friendliness. Through focusing on how transparency is experienced visually, haptically, and socially, the present paper integrated a number of seemingly disparate domains in a multidisciplinary manner. In doing so, it revealed the impact of transparent surfaces on various important aspects of human behavior. It was shown that seeing transparent barriers requires the detection and integration of several visual features, a circumstance posing a unique challenge to the visual system. Failure to detect transparent barriers, irrespective of their protective or restraining function, is common and can result in unintentional collisions. Further evidence suggests that such collisions are possible even upon the detection of a transparent barrier. That is, very young children and individuals suffering cognitive decline struggle with navigating transparent structures in their environments. These observations signal that the human mind has to actively construe the presence of a transparent entity, a process that requires experience with the material as well as the capacity to retrieve these experiences. Healing, or at least beneficial, effects of transparent barriers may be particularly likely in settings that require individuals to simultaneously connect with others, but also to protect their privacy. In this regard, open plan offices (i.e., offices that are not fully enclosed by internal walls) provide an interesting starting point for effective transparent barrier use. Open plan designs generally aim to optimize communication and information flow across individuals. Yet, their practical implementation is frequently accompanied by complaints about privacy violations (cf. De Croon et al., 2005; Kim and de Dear, 2013; De Been and Beijer, 2014). These complaints partially arise from intrusions caused by noise pollution from neighboring work stations (Lee and Brand, 2005; Veitch et al., 2007; Kaarlela-Tuomaala et al., 2009; Jahncke et al., 2011). Erecting transparent barriers between work stations may therefore allow architects to keep spaces visually connected, yet acoustically separated. Practical Implications A pivotal methodology, so-called evidence-based design, promotes the integration of traditional, predominantly intuition- driven architectural design with evidence-based decision-making (Rosswurm and Larrabee, 1999; Brown and Ecoff, 2011). The approach involves systematically tracking, comparing, and evaluating the consequences of architectural decisions on human health and wellbeing, so that the obtained findings can be applied to the design of new buildings (Lohr, 2004). The idea of evidence- based design is particularly relevant when it comes to the use of transparent barriers. Not only are such barriers unique in their dual nature (i.e., they are simultaneously absent and present), but they also form a novel type of environmental structure September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 11 Marquardt et al. Transparent barriers Finally, people not only select settings, but settings also select people (Wicker, 1979). That is, humans are rarely in a particular environment by chance. For example, behavioral scientists are likely to be found in university classrooms and research labs, but less frequently in corporate boardrooms or machine shops. The combined selection by individuals and settings increases the likelihood that people in a particular setting are more similar to one another than are people randomly sampled from a range of different settings. The process of structural constraints acting in concert with self- and setting-selection processes, social norms, and shared goals to limit behavioral options and to increase coordination in a given setting has been termed synomorphy (Wicker, 1979). The extent to which synomorphic mechanisms arise from environments with transparent barriers is another matter of speculation. It seems worthy of investigation, for instance, whether people with chronically low privacy needs, reduced impression management concerns, low territoriality claims, and/or claustrophobic tendencies experience greater satisfaction in surroundings with transparent barriers than people with contrasting needs and motives (e.g., people with high privacy needs and/or chronic fears of social evaluation by others). In a related manner, the use of glass features in architecture might be most welcome by occupants from cultures that are willing to publicize their everyday lives (Kükelhaus, 1973; Vera, 1989; Abel, 1997; Rieger-Jandl, 2006). Despite its ambiguity, this landmark study inspired further studies to investigate the relationship between architectural design and positive health outcomes (for a review see Lawson, 2010). Practical Implications Though it is generally agreed upon that environmental modifications can hardly ensure recovery from injury or disease, healthcare professionals increasingly recognize that architectural design can act as therapeutic assets by affecting occupants’ mood and social interaction patterns (Sommer, 1974; Kahn et al., 2008; Sternberg, 2009). g, ) Building on this work, architects to date must strive to understand in which kinds of environments transparent barriers are likely to act as stressors or healers. In addition, they should explore the versatile cognitive and behavioral responses that humans adopt upon encountering and inhabiting transparent environments. A particular focus should lie on identifying human responses that arise specifically from adapting toward these rather novel environments. To systematically study the richness of human responses toward transparent barriers, the concept of behavior settings as originally introduced in ecological psychology may prove helpful (Barker, 1968). A behavior setting is a bounded geographical area in which human and environmental components interact in a coordinated fashion to facilitate an ordered series of events over a period of time (Wicker, 1979). Examples of a behavior setting include an office meeting, lunch at a restaurant, a church service, or a university lecture. Importantly, transparent barriers may have strikingly different consequences on human functioning across different behavior settings. A lecture room separated from a busy corridor by a transparent barrier, for instance, might interfere with students’ attention to the lecturer. In contrast, a transparent wall separating two administrative assistants working on collaborative tasks, but needing acoustic screening, may facilitate their effectiveness. Considering these examples, the decision to erect transparent barriers, either in order to replace non-transparent ones or to subdivide previously open spaces, should always entail evaluating potential changes in the series of events that characterize a specific setting. References Arnheim, R. (1971). Art and Visual Perception: A Psychology Of The Creative Eye. Berkeley, CA: University of California Press. Awh, E., Belopolsky, A. V., and Theeuwes, J. (2012). Top-down versus bottom-up attentional control: a failed theoretical dichotomy. Trends Cogn. Sci. (Regul. Ed.) 16, 437–443. doi: 10.1016/j.tics.2012.06.010 Abel, C. (1997). 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Furthermore, although plenty of evidence suggests that a cognitive understanding of transparent barriers as solid separators of space is acquired at an early developmental stage, September 2015 | Volume 6 | Article 1381 Frontiers in Psychology | www.frontiersin.org 12 Transparent barriers Marquardt et al. even healthy adults occasionally treat transparent barriers as if they were not there. Both, tests of reflexive responding around such barriers as well as high-stake transactions with the material (e.g., requiring to pass a high cliffon a transparent walkway) reveal a human tendency to respond to their environments predominantly on the basis of incoming visual information. As a result, the unique property of transparent barriers – their visual penetrability – can override a person’s tactile experience and/or explicit knowledge that such barriers form solid separators of space. This tendency to consider transparent barriers as not there is also seen in studies on distance estimation. Several studies failed to detect spatial bias around glass barriers. This observed lack of bias may signal that transparent barriers are less likely to aid a perceiver’s ability to remember and navigate a space’s layout than opaque barriers, a possibility that requires further empirical examination. Similarly pressing is the question of how transparent barriers affect issues of privacy, crowding, territoriality, and interpersonal involvement. Most importantly, the existing work indicates that building transparent structures to serve complex human needs and goals requires a design process grounded in more than “intuition.” That is, the impact of transparent structures on human perception, cognition, and behavior needs to be systematically researched, combining the expertise of architects, designers, and behavioral scientists. 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Copyright © 2015 Marquardt, Cross, de Sousa, Edelstein, Farnè, Leszczynski, Patterson and Quadflieg. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2015 Marquardt, Cross, de Sousa, Edelstein, Farnè, Leszczynski, Patterson and Quadflieg. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. 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